Method of quantitative analysis of cell number of alive bacterium of interest with applying rrna as target

FIELD: medicine.

SUBSTANCE: offered invention can be used for the quantitative determination of alive bacteria. The offered method involves PCR-based product amplification with using the primers able to specific hybridisation with rRNA of a bacterium of interest. It is followed with the analysis of cell number of the alive bacterium of interest taking into account PCR cycle number.

EFFECT: invention allows precise determination of cell number of the alive bacterium of interest in a test sample.

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The technical field

The invention relates to a method of quantitative analysis or detection of microorganisms, especially in a live state, with the use of rRNA as a target.

Background of invention

As the method of quantitative determination of the microorganism has been traditionally used chiefly method including obtaining a culture of the microorganism in the pre-evaluated selective medium and measuring the amount of microbial cells, the process comprising culturing microorganisms in liquid selective medium and measuring the optical density or absorption. The following methods were also used for identification of the microorganism necessary for detection of a microorganism in the sample: for example, a process involving the identification through morphological study, staining Gram and microbiological characteristics, such as oxygen demand, the ability to assimilate sugar and conditions for growth in the medium; the method including determination of the microorganism with the help of test DNA-DNA homology; and a detection method using monoclonal antibodies to surface antigen of microbial cells. However, these methods require time and skill and, therefore, was a problem from the standpoint of quick who you and simplicity.

In recent years, methods of gene amplification, including PCR method was applied in a wide range of areas as methods for the determination of trace amounts of nucleic acids. These methods have the advantages that can lead to the acceleration and simplification, including the lack of mandatory requirements for cultivation of the microorganism contained in the sample, and the possibility of direct use of the sample as the sample. Thus, methods were investigated for application in quantitative analysis and detection of the organism.

As an example, in which the PCR method was used for analysis of the microorganism, a method for the quantitative determination of bacteria by PCR method, which uses the preparation of total DNA as a target sequence and universal primers (Patent document 1). Also were obtained methods using 16S rDNA as a target. Known examples include a method of quantitative analysis by PCR using 16S rDNA as the target sequence (Patent document 2), a method for determining intestinal bacteria by PCR method using 16S rDNA as the target sequence (Patent document 3) and a method of detecting bacteria bacterial strain of the genus Lactobacillus, calling the nd turbidity of beer (Patent document 4). However, these methods were problematic in that they cannot be used as an alternative to the conventional method, which has traditionally been applied, since the sensitivity of detection reaches the value which is obtained by using the culture method. As an example, the implementation of the method of quantitative analysis, as it is disclosed in Patent document 2 requires a large amount of template DNA, corresponding microbial number 105/μl or more, which makes the method impractical. Low detection sensitivity is probably connected with the low number of copies (number of matrices) total DNA or 16S rDNA provided as template for PCR in the microorganism. Since it is known that DNA is maintained even after the death of the organism, these methods can only be performed quantitative analysis and discover the dead and live bacteria together, that also causes the problem, which is that they are complex for accurate quantitative analysis and detection of living microorganisms (non-Patent document 1).

As examples of the application of the PCR method for the analysis of microorganisms have also been attempts to use the methods with the use of mRNA as a target sequence; well-known examples of these methods include the quantities of the config analysis of lactobacilli in the faeces, using the mRNA as a target sequence (non-Patent document 2). There are also known methods for determining cancer cells, which are used as sequences of target mRNA specific to the cancer cells in the samples (Patent documents 5 and 6). However, even these methods do not provide the sensitivity of the determination within that can be compared with the traditional way as methods of quantitative analysis. In particular, the limit of detection for the quantitative analysis, as shown in Patent document 2, is only 103,5or more cells/g faeces; the method of analysis could not be used as an alternative to the conventional culture method in connection with the detection sensitivity. In addition, these methods are aimed at the mRNA of genes that are unique to microorganisms, and were unsuitable for the detection of microorganisms in a test sample that contains many microorganisms, in connection with such problems as the difficulty of designing primers and reduced specificity.

Accordingly, the expected development of the method, which provides the sensitivity of the determination to the same extent as conventional methods of determining, as a quick way, using PCR or the like, and which additionally allows for precise provincialization analysis and to detect the presence of living organisms.

To increase the sensitivity may change the structure of the target so that the target could be presented in a more stable state, or in a greater relative number of cells. However, such a stable target is probably undesirable to detect only the living organism, given that it is expected to stay long in the dead cells. Thus, it is not easy to achieve definition of living cells and a sufficiently high detection sensitivity.

It is also known that rRNA is approximately 85% of the total RNA in the cell in the form of many copies and that rRNA is stable compared to the mRNA, because it forms a complex with the protein. It is also reported that rRNA is determined within a time period of approximately 48 hours after microbial death (non-Patent document 3) and are therefore generally considered to be unsuitable for the detection of living microorganisms (non-Patent document 1).

Patent document 1: Published by the Japan patent No. 2002-238585

Patent document 2: Published by the Japan patent No. 2003-259879

Patent document 3: Published by the Japan patent No. 2001-112485

Patent document 4: Published by the Japan patent No. 10-210980

Patent document 5: Published by the Japan patent No. 10-248600

Patent documents is 6: international publication WO 00/17395 directory

Non-patent document 1: J Food Prot, vol. 67, No. 4: 823-832 (2004)

Non-patent document 2: FEMS Microbiology Letters, vol. 231: 125-130 (2004)

Non-patent document 3: Appl. Environ. Environ., vol. 64, No. 11: 4264-4268 (1998)

The aim of the present invention is to provide a method of quantitative analysis of microorganisms, which can achieve the detection sensitivity within such limits that allow you to replace the conventional culture method, and more accurate detection of living microorganisms.

Disclosure of inventions

As a result of intensive studies, the authors of the present invention found that rRNA (i.e. 5S, 16S and 23S in bacteria and 5S, 18S, 26S or 28S in eukaryotic cells), which was considered to be unsuitable for the determination of living organisms from the point of view of stability, may be used as targets for accurate quantitative analysis and determine the number of live microbial cells without the inclusion of dead cells and, moreover, that the use of PCR method in quantitative analysis and determination can achieve such detection sensitivity, which allows you to replace the conventional method, and thus, the present invention has achieved its purpose.

Thus, the present invention provides a method of quantitative analysis of interest microorganis the s using as an indicator the number of rRNA of the microorganism in the test sample.

The present invention also provides a method of determining the interest of microorganisms using as an indicator of the presence of rRNA of the microorganism in the test sample.

The present invention also provides a fragment of the nucleic acid used in the above method, where the fragment is a fragment of a nucleic acid containing a sequence of bases as described in one of SEQ ID NO: 2, 3, and 5-28, or she complementary sequence of bases, or a fragment of a nucleic acid that contains a sequence of bases homologous or functionally equivalent.

The present invention additionally provides a kit for the above-described method.

The detection method using as a target rRNA in accordance with the present invention can be applied to achieve high detection sensitivity in comparison with the method using the conventional target, due to the presence of targets in a large number, if also more precise definition and quantitative analysis of living organisms. The PCR method can also be applied when determining to achieve this detection sensitivity, which allows you to replace the conventional culture method. Furthermore, the method and the PR with the application of the PCR method can provide a noticeable speed and simplicity compared to conventional methods, such as cultural way. In other words, the method according to the present invention can be used to simultaneously provide high detection sensitivity, more accurate quantitative analysis and/or determination of living organisms and speed and simplicity. Thus, the method according to the present invention can be applied in practice, when it is required to detect and/or quantify microorganism, as is done in the study of intestinal flora, and detecting and/or quantifying microorganisms living in the sample of the food product or organism.

Brief description of drawings

Figure 1 is a series of graphs showing the correlation between the growth of different microorganisms and value of rRNA transcription.

Figure 2 is a series of graphs, each of which shows a standard curve obtained by a quantitative method RT-PCR, and comparison of the range of this method and the method of quantitative PCR.

Figure 3 is a graph showing the limit of detection of P. aeruginosa from the feces of a person.

Figure 4 is a graph showing the comparison of quantitative values for enterobacteria in faeces of a person when determining the method of quantitative RT-PCR and culture : the BOM.

Figure 5 is a series of graphs showing the detection sensitivity of E. coli, S. aureus and B. cereus in cow's milk.

Figure 6 is a series of graphs showing the detection sensitivity of P. aeruginosa and S. aureus in the blood.

Figure 7 is a graph showing the detection sensitivity of E. coli in fermented dairy product.

The best ways of carrying out the invention

Method of quantitative analysis or determination of interest microorganisms in accordance with the present invention is characterized by the use of index numbers or the presence of rRNA of the microorganism in the test sample.

Under rRNA interest microorganism means rRNA, which may be a microorganism which should be subjected to quantitative analysis and definition. Examples include rRNA prokaryotic 5S, 16S and 23S rRNA and eukaryotic 5S, 5.8S,, 18S, 26S and 28S rRNA; 16S, 23S, 18S and 26S rRNA are especially preferred because they are generally used as reliable indicators in the modern classification of microorganisms. Under interest microorganism refers to a microorganism which should be detected and subjected to quantitative analysis, and is not limited in any way. Examples of casimirarosario include microorganisms of the family Enterobacteriaceae and the genera Enterococcus, Lactobacillus, Streptococcus, Staphylococcus, Veillonella, Pseudomonas, Clostridium, Bacteroides, Bifidobacterium, Eubacterium, Prevotella, Ruminococcus, Fusobacterim, Propionibacterium, Peptostreptococcus, Vibrio, Bacillus, Campylobacter, Acinetobacter, Lactococcus, Pediococcus, Weissella, Leuconostoc, Oenococcus, Helicobacter, Neisseria, Listeria, Haemophillus, Mycobacterium, Gardnerella, Legionella spp., Aeromonas, Moraxella and Candida, and microorganisms, as described in Tables 2 and 3, which will be referred to hereinafter. Interest microorganism in accordance with the present invention is a concept including not only the one microorganism strain, but also one group, genus and family, which consists of a population of 2 or more strains with certain properties.

The test sample refers to the object that should be investigated for the presence of, quantity of the microorganism or the like. Examples of test samples include samples from a biological source, such as a swab of conjunctiva of the eye, Tartar, plaque, sputum, swab from the throat, saliva, nasal discharge, bronchoalveolar wash, pleural effusion, gastric juice, erosion of the stomach, urine, cervical mucus, vaginal discharge, lots of damage to the skin, faeces, blood, ascitic fluid, tissue, cerebrospinal fluid, synovial fluid and flush with a damage site; and objects, potentially containing microorganisms, such as food, pharmaceuticals, cosmetics, pharmacy, the economic means, polupolirovannye food products (semi-finished products), pharmaceuticals and cosmetics, microbial broth, plants, soil, activated sludge and drainage water. Sample the sample for testing refers to a sample taken or obtained from the sample for testing, and not specifically limited, provided that the sample is able to reflect the presence or number of microorganisms in the sample. Examples include mixtures containing nucleotides, and mixtures containing RNA contained in the sample for testing; preferred from the point of view of application of the PCR method is a mixture containing RNA present in a sample for testing.

Sample the sample for testing can be appropriately obtained, for example, of the whole or part of the sample for testing, using a known method, if necessary, after pre-treatment with application of methods of extraction, separation and purification. As an example, a mixture containing RNA can be obtained, for example, by extraction with application of a universal method, such as method of ultracentrifugation in an environment containing chloride guanidine-cesium”, “the method including processing a sour mixture of guanidine-phenol chloroform (AGPC)”, “way of the magnetic beads and the way silicon columns, if necessary, after prior is sustained fashion processing, including the known method, such as filtration, centrifugation and chromatography; also this can be used a commercial kit (e.g., QIAGEN RNeasy Kit, TRIZOL).

Applied Swatch samples for testing preferably represents RNA in a stable condition in the organism, to prevent its collapse to maintain a high detection sensitivity. Stabilization may be performed using, for example, commercial stabilizing agents (for example, RNAprotect Bacterial Reagent, RNAlater). Stabilization is preferably carried out immediately after sampling, in order to avoid changing the amount of RNA in the organism.

In quantitative analysis of interest of the microorganism in accordance with the present invention is used as an indicator of the amount of RNA of the microorganism in the sample for testing. Here the amount of RNA of interest of the microorganism in the sample for testing can be determined, for example, by (1) obtaining the amount of product amplified using PCR with the use of fragments of nucleic acid, capable of specific hybridization with rRNA interest of the microorganism, and the sample of the sample, (2) implementation of effective hybridization between fragments of nucleic KIS is the notes, capable of specific hybridization with rRNA interest of the microorganism, and the sample of the sample or (3) application of a quantitative method using another known method.

Here, in the case of (1) the application of the method of PCR, fragments of nucleic acid, capable of specific hybridization with rRNA interest microorganism” can be constructed by comparing the base sequence of the microorganism with sequences of other microorganisms to select sequences that are specific for rRNA, which may be of interest microorganism. In this description, the sequence of rRNA, which may be a microorganism can be obtained, for example, by checking for compliance with the database (DDBJ, GenBank, etc.). Also sequences can be aligned using the software (e.g., Clustal X) for detection of specific sequences of visual or in any other way. Sequence-specific interest of the microorganism, preferably chosen, taking into account the widths of the borders, which enters the microorganism(s)subjected to quantitative analysis. In particular, for example, if the strain must be accurately quantified, prepact the tion is chosen sequence, specific to strain; if must be conducted quantitative analysis of the genus, preferably choose a sequence specific for the genus. The choice may suitably be carried out using a known method.

In addition to sequences, designed fragments of the nucleic acids capable of hybridizing to rRNA of interest microorganism, can be each properly established on the basis of common principles in the field; the sequence of bases complementary to the above-described base sequence, the sequence of bases homologous to it, as well suited for the quantitative analysis of interest microorganism, and such sequences can also be applied. Examples of homologous sequences include a fragment of a nucleic acid that contains (a) the above sequence of bases, which further comprises a substitution, insertion or deletion of one or more, preferably from 1 to 10 bases, (b) the sequence of bases identical to the above-described base sequence 90% or more, preferably 95% or more, more preferably 99% or more, or (C) the sequence of bases capable of hybridization or the under stringent conditions with a DNA containing the sequence of bases complementary to the above-described base sequence.

The fragment of the nucleic acid may also be a part of the fragment of the nucleic acid to which one or both ends, preferably, from 5'-end, added preferably 100 bases, more preferably 20 bases, more preferably 10 bases or less.

The length of the fragment of the nucleic acid is not specifically limited; however, the fragment preferably contains from 5 to 50, more preferably from 12 to 35 bases.

Fragment of nucleic acid, so constructed, may be synthesized artificially, for example, on a DNA synthesizer in accordance with its sequence of bases. The fragment is preferably such that its specificity was verified. Here, the specificity can be tested, for example, by evidence that the use of interest rRNA as a matrix provides a amplified using PCR product, when compared to a suitable control.

Examples of the fragment of the nucleic acids include nucleic acid fragments containing the base sequence described in SEQ ID nos: 1-30, or a base sequence complementary to them, or nucleic acid fragments, operasie base sequence, homologous or functionally equivalent. In this description, examples of fragments of nucleic acid containing a base sequence homologous or functionally equivalent, include fragments of the nucleic acid, as shown in paragraphs (a)to(C) below, which can be used for quantitative analysis and determination of rRNA interest of the organism.

(a) a Fragment of a nucleic acid that contains a sequence of bases represented by one of SEQ ID nos: 1-30, or a sequence of bases complementary to it, where the fragment contains a deletion, substitution or insertion of one or more bases.

(b) a Fragment of a nucleic acid sequence which is 90% or more, preferably 95% or more, more preferably 99% or more identical to the base sequence represented by one of SEQ ID nos: 1-30, or a sequence of bases complementary to it.

(c) a Fragment of a nucleic acid that contains a sequence of bases capable of hybridizing in stringent conditions with a DNA containing the sequence of bases represented by one of SEQ ID nos: 1-30, or a sequence of bases complementary to it.

In this document the identity of the sequence of bases is calculated using the program search homolo the AI GENETYX (R).

“Stringent conditions” include, for example, hybridization at 42°C for 16 to 24 hours in a solution containing 50% formamide, 5×SSC, 5×solution of Denhard and 250 mg/ml DNA salmon sperm.

The fragment of the nucleic acid suitable for quantitative analysis and detection of rRNA interest of the microorganism can be obtained, for example, through implementation of the PCR method for the selection of the fragment of the nucleic acid, which produces a product of amplification, if the matrix is applied rRNA of this organism, but does not give the product when used as a matrix to another target, for example, rRNA another microorganism or mRNA.

Then, (1) the fragment of the nucleic acid containing the sequence of bases as described in SEQ ID NO: 1 or 2, or a sequence of bases complementary to it, or a fragment of a nucleic acid that contains a sequence of bases homologous to her and functionally it is equivalent, may be applied to specific quantitative analysis and detection of Bacillus cereus; (2) a fragment of a nucleic acid that contains a sequence of bases as described in SEQ ID NO: 3 or 4, or a sequence of bases complementary to it, or a fragment of a nucleic acid that contains a sequence of bases homologous to her and options the online her equivalent, can be used for specific quantitative analysis and detection of Clostridium perfringens; (3) a fragment of a nucleic acid that contains a sequence of bases as described in SEQ ID NO:5, or 6, or a sequence of bases complementary to it, or a fragment of a nucleic acid that contains a sequence of bases homologous to her and functionally it is equivalent, can be used for specific quantitative analysis and detection of Enterobacteriaceae; (4) a fragment of a nucleic acid that contains a sequence of bases as described in SEQ ID NO: 7, or 8, or a sequence of bases complementary to it, or a fragment of a nucleic acid containing a sequence bases homologous to her and functionally it is equivalent, may be applied to specific quantitative analysis and detection of Staphylococcus; (5) a fragment of a nucleic acid that contains a sequence of bases as described in SEQ ID NO: 9, or 10, or a sequence of bases complementary to it, or a fragment of a nucleic acid that contains a sequence of bases homologous to her and functionally it is equivalent, may be applied to specific quantitative analysis and detection of the genus Pseudomonas; (6) a fragment of a nucleic acid that contains a sequence of bases as described in SEQ ID NO: 11 or 12, Il is a sequence reason complementary to it, or a fragment of a nucleic acid that contains a sequence of bases homologous to her and functionally it is equivalent, may be applied to specific quantitative analysis and identification of the genus Enterococcus; (7) a fragment of a nucleic acid that contains a sequence of bases as described in SEQ ID NO: 13, or 14, or a sequence of bases complementary to it, or a fragment of a nucleic acid that contains a sequence of bases homologous to her and functionally it is equivalent, may be applied to specific quantitative analysis and identification of subgroups of Lactobacillus acidophilus; (8) a fragment of a nucleic acid that contains a sequence of bases as described in SEQ ID NO: 15, or 16, or a sequence of bases complementary to it, or a fragment of a nucleic acid that contains a sequence of bases homologous to her and functionally it is equivalent, may be applied to specific quantitative analysis and identification of subgroups of Lactobacillus ruminis; (9) a fragment of a nucleic acid that contains a sequence of bases as described in SEQ ID NO: 17, or 18, or a sequence of bases complementary to it, or a fragment of a nucleic acid that contains a sequence of bases homologous to her and functionally it is equivalent, may be p Kenen for specific quantitative analysis and detection subgroups of Lactobacillus plantarum; (10) a fragment of a nucleic acid that contains a sequence of bases as described in SEQ ID NO: 19, or 20, or a sequence of bases complementary to it, or a fragment of a nucleic acid that contains a sequence of bases homologous to her and functionally it is equivalent, may be applied to specific quantitative analysis and identification of subgroups of Lactobacillus reuteri; (11) a fragment of a nucleic acid that contains a sequence of bases as described in SEQ ID NO: 21, or 22, or a sequence of bases complementary to it, or a fragment of a nucleic acid that contains a sequence of bases homologous to her and functionally it is equivalent to can be applied to specific quantitative analysis and identification of subgroups of Lactobacillus sakei; (12) a fragment of a nucleic acid that contains a sequence of bases as described in SEQ ID NO: 23, or 24, or a sequence of bases complementary to it, or a fragment of a nucleic acid that contains a sequence of bases homologous to her and functionally it is equivalent, may be applied to specific quantitative analysis and identification of subgroups of Lactobacillus casei; (13) a fragment of a nucleic acid that contains a sequence of bases as described in SEQ ID NO: 25 or 26, or a sequence of bases, complementary is her or a fragment of a nucleic acid that contains a sequence of bases homologous to her and functionally it is equivalent, may be applied to specific quantitative analysis and identification of Lactobacillus brevis; (14) a fragment of a nucleic acid that contains a sequence of bases as described in SEQ ID NO: 27 or 28, or a sequence of bases complementary to it, or a fragment of a nucleic acid that contains a sequence of bases homologous to her and functionally it is equivalent, may be applied to specific quantitative analysis and detection of Lactobacillus fructivorans; and (15) a fragment of a nucleic acid that contains a sequence of bases as described in SEQ ID NO: 29, or 30, or a sequence of bases complementary to it, or a fragment of a nucleic acid that contains a sequence of bases homologous to her and functionally it is equivalent, may be applied to specific quantitative analysis and detection of Lactobacillus fermentum.

In this description, the fragment of the nucleic acid containing the sequence of bases in SEQ ID NO: 1, is a known fragment of the nucleic acid, as described in FEMS Microbiology Letters, vol. 202: 209-213 (2001). The fragment of the nucleic acid containing the sequence of bases in SEQ ID NO: 4, is a known fragment of the nucleic acid, the AK is described in Environ. Immunol., vol. 46, No. 5: 353-358 (2002). The fragment of the nucleic acid containing the sequence of bases in SEQ ID NO: 29 or 30, is a well-known fragment of the nucleic acid, as described in the Published Japan patent No. 11-151097. In contrast, fragments of nucleic acid containing a base sequence described in SEQ ID NO: 2, 3, and 5-28, are fragments of nucleic acids, found by the authors of the present invention.

The PCR method with the use of fragments of nucleic acid, thus obtained, and specimen samples for testing can be carried out via PCR in a reaction system containing the sample, with the use of nucleic acid fragments as primers and rRNA interest of the microorganism as a matrix. The PCR method is not specifically limited, provided that the reaction is specifically amplificates nucleotide fragment derived from rRNA interest of the microorganism. Preferred is a process comprising the step of using rRNA interest of the microorganism as a matrix to obtain cDNA using the enzyme, preferably, the reverse transcriptase or the like. More preferred is a process comprising, in addition to the step described above, the stage of use of the cDNA, the scientists thus, as a matrix for amplification of nucleotide fragment. The PCR method can be performed using, for example, known RT-PCR. In this description RT-PCR may be performed using a known method such as two-step RT-PCR and one-step RT-PCR; however, one-step RT-PCR is preferred because it is particularly simple and prevents cross contamination.

Method one-step RT-PCR may be performed using, for example, a commercial kit (e.g., a set QIAGEN One-Step RT-PCR). The enzyme having transcriptional activity, which can be used in the RT reaction can be any of various reverse transcriptase inhibitors, such as reverse transcriptase M-MHV. The DNA polymerase used in the amplification of DNA using PCR, preferably, is thermally stable at a temperature of 90°C and above.

PCR can be conducted through one or more cycles of the reaction temperature denaturation for converting double-stranded DNA into single DNA reaction annealing for hybridization of primers to the matrix cDNA and reaction completion (lengthening) to provide opportunities to act DNA polymerase at a temperature of from 90 to 98°C, from 37 to 72°C and from 50 to 75°C, respectively. A preferred example of the reaction conditions is thermal de is acurate at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension (elongation) at 72°C for 60 seconds.

For PCR, two types of primers, preferably, used as a set. In this description it is necessary to make two primers to form a combination of the leading chains and trailing chain. Fragments of the nucleic acid provided by the present invention, each composed in such a way as to have an approximately constant temperature annealing in RT-PCR, which allows the nucleic acid fragments of many microorganisms to be studied simultaneously. The fragment of the nucleic acids of the present invention can also be used as a probe and can also be used in combination with other known universal primer, the oligonucleotide or the like.

Sample the sample for testing, containing rRNA, providing a matrix for RT-PCR, preferably has a total content of RNA from 1 PG to 1 μg, more preferably from 10 PG to 0.1 µg.

When PCR is carried out properly, there is usually a correlation between the amount of product amplified using PCR”, “number of PCR cycles and the number of matrix for PCR”. Thus, the number of rRNA interest of the microorganism can be determined if the count is properly taking into account the number is tion of the amplified product, formed as a result thus PCR and the number of cycles of PCR.

As shown in the Figure 1 Example, which will be described, it has been shown that there is also a clear correlation between the number of rRNA interest microorganism defined thus, “the number of cells of interest of the organism”. The number of cells of interest of the microorganism can therefore be determined if the calculation is performed taking into account “the number of rRNA interest microorganism”, defined in this way. Even without the process of computing the number of rRNA interest of the microorganism, the number of cells of the microorganism can be determined by an appropriate calculation with regard to the amount of the amplified product resulting from PCR and the number of cycles of PCR, obtained in the manner described above.

The number of amplified in the PCR product and the number of PCR cycles can be find out by any method without particular limitation, for example, by determining the number of PCR cycles at which the amount of DNA reaches an arbitrarily chosen level. The determination may be performed, for example, by using PCR method, including the subsequent labelling of PCR product, in combination with the PCR method, comprising measuring the amount of label in time” to determine the number of PCR cycles to reach a certain selected fluorescence intensity. In this description, a certain intensity of fluorescence is preferably selected within a range of values, of which the intensity can be achieved when the amount of amplification product increases logarithmically, as a reflection of the corresponding correlation between them. The range of values can properly be understood with the application of a known method. In this specification, examples of labeling includes labeling with a fluorescent dye; examples of measuring the amount of label include measurement of fluorescence intensity. Here, examples of the labeling fluorescent dye include tagging intercalating fluorescent dye. Examples of intercalating fluorescent dyes include SYBR(R) Green I. Intercalating dye has the property, according to which the intensity of fluorescence increases when it is turned on in the double-stranded nucleic acid, leading, thus, to the emission of a fluorescence having an intensity that reflects the amount of amplified in the PCR product. The labeling fluorescent dye can also be carried out using samples TaqMan, Molecular beacon or t the th like, labeled with a fluorescent dye. Sample TaqMan or Molecular beacon are samples in which a fluorescent dye and a quencher linked to the oligonucleotide homologous internal phase sequence, amplified using PCR, and used thanks to the possibility of coexistence in the PCR system. The interaction of a fluorescent dye and quencher associated with breakdown, makes possible the emission of fluorescence in response to reaction amplification with PCR, allowing, thus, the monitoring of the amount of amplification product in PCR as measuring the intensity of fluorescence at each stage of PCR. However, the TaqMan probe, Molecular beacon or the like necessitates the selection of the specific microbe complementary sequences suitable for holding samples that may be difficult, depending on the object.

The number of rRNA can be determined by considering the number of amplified in the PCR product and the number of cycles of PCR, researched thus, the results of appropriate comparative experiment. In particular, the number of rRNA interest of the microorganism can be calculated using known methods, for example, by incorporating the results of a comparative experiment carried out with application of rRNA, the number of known” for proper matching with this “number of the amplified PCR product and the number of cycles of PCR, defined as described above.

Then the number of cells of interest of the microorganism can be determined by considering “the amount of rRNA of the microorganism, is designed in such a way, and the results of appropriate comparative experiment. In particular, the number of cells of interest of the microorganism can be calculated using known methods, for example, by incorporating the results of the comparative experiment, carried out with the use of specimen samples for testing, in which the number of cells of the corresponding microorganism known for proper matching with the number of rRNA interest microorganism”calculated in this way. On the contrary, for simplicity, preferably use a standard curve, which shows the correlation between the number of cells of interest microorganism”is used as template for PCR, and the number of cycles of PCR, when reaching a certain amount amplified in the PCR product (hereinafter sometimes called the CT value). A standard curve usually get through the m building plot of the CT values from the number of cells of the microorganism target (see Figure 2). The microorganism used for the standard curve, can be a known strain, such as its typical strain.

Without the implementation of specific process of counting the number of rRNA, the number of cells of interest of the microorganism can also be directly calculated through proper matching of the results of the comparative experiment conducted using sample samples for testing, in which a known number of cells of the corresponding microorganism” with “the amount of amplified PCR product and the number of PCR cycles”, was investigated as described above. In particular, the CT value obtained from the sample the sample for testing, can be used for the above standard curve.

As described above, the number of rRNA interest of the microorganism in the sample for testing can also be defined, for example, by (2) determine the effectiveness of hybridization between the fragment of the nucleic acid, capable of specific hybridization with rRNA interest of the microorganism, and the sample tested sample.

In this description, the fragment of the nucleic acid, capable of specific hybridization with rRNA interest microorganism, which can be applied, is the way the th, for example, fragment, prepared and obtained as described above. Fragment of nucleic acid, preferably, is labeled fragment of the nucleic acid. In the present description, examples of labels include an enzyme, a paramagnetic ion, Biotin, a fluorescent dye, a chromophore, a heavy metal and radioisotope; more preferable examples of the label include the enzyme. In this specification, examples of the enzyme include horseradish peroxidase and alkaline phosphatase. Tagging can be performed by a known method.

The number of rRNA interest of the microorganism in the test sample and/or the number of cells of the microorganism may be studied using well-known transformation method by measuring the degree of hybridization between the sample the sample for testing and the fragment of the nucleic acid. The method of measuring the degree of hybridization is not definitely limited and can be performed in accordance with the known method; for example, it can be performed by measuring the amount of label added to the fragment of the nucleic acid. In particular, for example, the method can be performed by measuring the fluorescence intensity using a fragment of the nucleic acid labeled with a fluorescent dye. The measurement is preferably performed is parallel with the measurement with the use of proper control. In this specification, examples of suitable controls include a sample known that he is not hybridized used with a fragment of nucleic acid”, “sample derived from the test sample, where the sample contains a known number of cells of interest of the microorganism and the sample taken or obtained from the sample for testing, where the sample contains a known amount of rRNA of interest microorganism”. When compared with the control number rRNA or the number of cells of interest of the microorganism can be obtained by using known conversion method. The number of cells of interest of the microorganism can also be studied using well-known methods based on the number of rRNA interest of the microorganism, thus, calculated, and results appropriate comparative experiment.

The way to discover the interest of the microorganism in accordance with the present invention uses as an indicator of the presence of rRNA of the microorganism in the sample of the sample for testing. In this description, the term “detection of microorganism” includes the identification of the microorganism. The term also includes determining whether detected microorganism in the sample or absence detective the alignment of the microorganism in the sample.

To determine whether rRNA interest of the microorganism in the sample for testing, using the detection method of the present invention, for example, can be applied detection method described below in (1), (2) or (3).

(1) Detection of the product amplified by PCR with the use of the fragment of the nucleic acid, capable of specific hybridization with rRNA interest of the microorganism, and sample the sample for testing.

(2) Detecting hybridization between the fragment of the nucleic acid and the sample.

(3) Detection of rRNA of the microorganism using other known method.

Methods (1)to(3) can easily be carried out taking into account the previously described methods. The presence of rRNA interest microorganism indicates that the microorganism was present in the tested sample, which makes possible the detection of the microorganism. However, detection of, preferably, is performed by comparison with a suitable control, as may occur non-specific amplification of PCR product and non-specific hybridization.

As shown in the following Examples, it was demonstrated that a high detection sensitivity can be achieved using the method of quantitative analysis using the number of rRNA in the quality of the solid fuel indicator and the detection method using the presence of rRNA as an indicator for comparison with the index of the conventional ways, using as an indicator the number of rDNA. As shown in examples which will be described, it was also demonstrated that the method using the number of rRNA as an indicator can accurately quantify microorganisms in live view, not taking into account the present dead cells.

Thus, the application of the method of quantitative analysis or detection of the present invention (hereinafter referred to here is also called the method according to the present invention”) makes it possible to perform specific quantitative analysis and detection of microorganisms with a higher detection sensitivity than the sensitivity of the determination in the conventional ways, and even in a live state. Therefore, the method according to the present invention can be used, for example, in the application forms described in paragraphs (1)to(4) below.

(1) Application, which represents the interest of the microorganism contained in a sample for testing, is subjected to quantitative analysis and detection in living condition with a higher detection sensitivity than conventional methods.

(2) Application, in which the number of dead cells of the microorganism contained in a sample for testing, quantitatively determined and detected with a higher detection sensitivity, is eaten by traditional methods.

(3) Application, in which the ratio of quantities of dead and live cells of microorganism is measured with a higher detection sensitivity than conventional methods.

(4) Application in which the presence or abundance of living organisms “shall be determined with higher sensitivity than conventional methods.

In this description, the definition includes, for example, (a) quantitative analysis and detection to establish the presence or abundance of living organisms, when the number of cells of living organisms should be set more accurately, and (b) if the number of cells in a living organism” was calculated in a different experimental system, the determination to study the accuracy of the experiment and the accuracy of the calculated numerical values. In this regard, when the number of dead cells, the measurement of the total number of dead cells and living cells, preferably, is with this in mind, for example, in a known manner to detect dead cells together with living cells. The number of dead cells can be determined by subtracting the number of live cells was calculated using the method of the present invention, the total number of cells.

The method according to the present invention can also be applied in ka is este method for quantitative analysis or detection of the microorganism, which is difficult to measure using conventional methods, such as a microorganism, unable to form colonies, and the organism is unable to grow in liquid medium.

As shown in the following Examples, it was demonstrated that the application of the PCR method for the quantitative analysis and detection can achieve the same detection sensitivity as when the culture method. Thus, the method according to the present invention can also be used as a method of quantitative analysis or determination of the microorganism with the same or greater than the detection sensitivity, as with the culture method, i.e, when the detection sensitivity of 100cells or more per gram of sample or 100cells or more per ml of sample.

With the method of quantitative PCR analysis or detection of the microorganism may also be carried out very quickly and easily, compared with the culture method. In addition, in accordance with the method using the PCR method, the process from the extraction of RNA from the sample prior to quantitative analysis and determination of the microorganism can be completed within approximately 6 hours. Thus, the method according to the present invention can also be applied as a method suitable for determining microorganism over to the short period of time (within 6 hours).

The application of the method using the PCR method, in accordance with the present invention can provide high detection sensitivity, more accurate quantitative analysis and detection of a living organism, and the quickness and ease. Thus, the method according to the present invention can be used, for example, in the application of the study of infectious and harmful bacteria, pathogens or the like in the field of medicine and food industry, where rapid sensitive quantitative analysis or detection is required in particular.

The method according to the present invention can also be performed with the use of the kit for the complete method. In this description and the examples set for performing the method includes a set containing (1) fragments of nucleic acid, capable of specific hybridization with rRNA interest of the microorganism, (2) a Protocol that describes how the implementation of, and/or (3) the reagent used for the extraction of RNA, RNA stabilization and/or PCR. However, the kit of the present invention is not limited to this, and refers to the entirety of the whole or parts of the necessary elements for the implementation of all or part of the steps of the method. In this description of “the necessary elements for the implementation of phases” may respectively pony is Atisa considering the above descriptions in this specification.

EXAMPLES

The content of the present invention is described below more specifically by using Examples. However, it is not intended to limit the invention to these examples.

Example 1: Obtain primers

For different bacterial strains, DNA sequence of the 16S rRNA and 23S were obtained from the DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp/Welcome-j.html). These sequences were aligned using the program Clustal W with the subsequent receipt of the phylogenetic tree. The strains were classified by families, genera, and subgroups on the basis of the phylogenetic tree; the sequences of primers were designed for each classification. The obtained sequences of the primers and of interest types of rRNA are shown in Table 1. References that describe the sequence shown in the column “references” in Table 1. If column is blank, this indicates that the sequence is a new sequence found by the authors of the present invention. In this regard, non-Patent document 4 is Environ. Immunol., vol. 46, No. 5: 353-358 (2002); non-Patent document 5 is FEMS Microbiology Letters, vol. 202, 209-213 (2001); and Patent document 7 is Published by the Japan Patent No. 11-151097.

Example 2: determination of the specificity of the primers

For the distribution, whether the primers of Example 1 have the specificity or not, they were examined for specificity against different bacteria. Specifically, 50 μl of each of the different bacterial cultures, as shown in table 2 (57 species from 28 genera) and Table 3 (60 species from 18 genera), was added to 2-fold volume of RNAprotect Bacterial Reagent (QIAGEN) and incubated at room temperature for 5 minutes. The suspension is then centrifuged at 5000 g for 10 minutes and supernatant was removed. There was added to 450 μl of bacteriolytic buffer (346,5 ál buffer RLT (QIAGEN), and 3.5 μl of β-mercaptoethanol, 100 μl of TE buffer) and 300 mg of glass beads (0.1 mm in diameter), which is then strongly stirred using a FastPrep FP120 (Bio 101) at 5000 rpm for one minute to destroy bacterial cells. To the resulting solution was added 500 μl of water-saturated phenol and then incubated at 60°C for 10 minutes. There was added 100 μl of chloroform/isoamyl alcohol (CIA), was stirred and then subjected to centrifugation at 12000 rpm for 5 minutes at 4°C. To the extracted supernatant was added an equal volume of water saturated phenol/chloroform, then mixed and centrifuged in the same conditions. To the extracted supernatant was added an equal volume of CIA, then shook and again subjected to centrifugation under the same conditions. K µl of the extracted supernatant was added an equal volume of isopropyl alcohol and 1/10 volume of 3M sodium acetate, then mixed by inversion and centrifuged at 15000 rpm for 10 minutes at 4°C. the Supernatant was removed, to the precipitate was added to 500 ál of 75%ethanol, mixed by flip and centrifuged the mixture at 15,000 rpm for 2 minutes at 4°C. After removing the supernatant and drying in air content test tubes, the precipitate was dissolved in 50 ál of water, free of ribonuclease, to get the total extract RNA. Quantitative RT-PCR was performed with a set QIAGEN One-Step RT-PCR Kit (QIAGEN). The composition of the reaction mixture (total volume: 25 µl) was as follows: 2 μl of a solution of total RNA (equivalent to 2×105SOME); and a single buffer QIAGEN One-Step RT-PCR Buffer, 0.5 mm dNTP mixture, 1/25 volume of QIAGEN One-Step RT-PCR Enzyme Mix, 1/100000 volume SYBR(R) Green I (Molecular Probes) and 0.75 μm (each) of primers (described in Table 1), which was adjusted so that the appropriate number formed the final concentration. The amount of RNA, equivalent to 2×105SOME were used as template in RT-PCR. The solution for the reaction is first subjected to the reaction of reverse transcription at 50°C for 30 minutes and then warmed up at 95°C for 15 minutes to inactivate the reverse transcriptase. After this was performed from 40 to 45 cycles of 94°C for 20 seconds, 55°C or 60°C for 20 seconds and 72°C for 50 seconds, to measure if the esta product of amplification by the fluorescence intensity of SYBR(R) Green I for each cycle. These series of reactions was carried out using the system ABI PRISM(R) 7900HT system (Applied Biosystems).

As a result, as shown in Table 2, it was shown that only interest a bacterial genus or strain can be specifically determined using primers En-lsu 3F/3'R (Enterobacteriaceae), g-Staph-F/R (genus Staphylococcus), PSD7F/R (genus Pseudomonas), s-Clper-F/ClPER-R (Clostridium perfringens), S-S-Bc-200-a-S-18/Bc2R (Bacillus cereus) or g-Encoc F/R (genus Enterococcus). Moreover, as summarized in Table 3, it was shown that only interest subgroup or strain can be specifically defined by the primer sg-Laci-F/R (subgroup Lactobacills acidophilus), sg-Lsak-F/R (subgroup Lactobacillus sakei), sg-Lcas-F/R (subgroup Lactobacillus casei), sg-Lrum-F/R (subgroup Lactobacillus ruminis), sg-Lreu-F/R (subgroup Lactobacillus reuteri), sg-Lpla-F/R (subgroup Lactobacillus plantarum), s-Lbre-F/R (Lactobacillus brevis), s-Lfru-F/R (Lactobacillus fructivorans) or LFer-1/2 (Lactobacillus fermentum). In tables 2 and 3 + indicates that the specific detection can be achieved (the value of CTfrom 1 to 30); - indicates that the value of CTis 31 or more, and that the amplification product was not received.

Example 3: a Study of the relationship between the status of growth of various microorganisms and the level of transcription of rRNA

Using cells of Escherichia coli, S. aureus and P. aeruginosa at different phases of growth, the cult of the market investigated the ratio of the number of living bacterial cells, measured using the culture method, the number of bacterial cells capable of forming colonies, derived from the level of transcription of rRNA measured using a quantitative method RT-PCR. In particular, after the start of the aerobic cultivation of a culture of each bacterium with shaking at 37°C in an environment BHI, bacterial culture were collected over time, with the subsequent use of crops for measuring the amount of bacterial cells by culture method using agrarian environment BHI (37°C, 24 hours). On the other hand, from just samples RNA was extracted and subjected to analysis of quantitative RT-PCR. The number of bacterial cells in each sample was calculated using a standard curve, obtained as described in Example 4, using RNA extracted from bacterial strain in late logarithmic growth phase, the number of cells in which it is known. In this regard, the extraction of total RNA and quantitative RT-PCR was performed as described in Example 2. The results are shown in Figure 1. In Figure 1 the black circle (•) indicates the number of bacterial cells, calculated on the level of RNA transcription, and the white circle (○) shows the number of bacterial cells, certain cultural way. For all bacterial strains were subjected to the s analysis, from the logarithmic phase of growth to the death phase, observed a strict correlation between the curves of the number of living bacterial cells, a particular cultural way in the solution containing the bacteria, and the number of bacterial cells based on the level of transcription of rRNA. It showed that the number of cells of living organisms can be determined under any conditions by measuring the level of transcription of rRNA.

Example 4: generation of a standard curve and compared with the quantitative PCR method

Standard curves were obtained by the method according to the present invention (a quantitative method RT-PCR) using grown in cell cultures of P. aeruginosa YIT6108T(typical strain) and S. aureus YIT6075T(typical strain), in the logarithmic growth phase. Standard curves were obtained by quantitative PCR method for comparison with standard curves obtained by the method according to the present invention. Exericise cells of each strain grown in the medium BHI, was divided in such a way as to ensure the number of cells 105, 104, 103, 102, 101and 100, and subjected to RNA extraction as described in Example 2. Each extract was subjected to quantitative RT-PCR in accordance with Example 2, using Primero is, as described in Table 1. Investigated the correlation between the received value CT and the number of cells defined by culture method described in Example 3. Using the method described below, each of the DNA obtained from the same samples were also investigated for the quantitative analysis of the PCR method with the use of rDNA as a target sequence. In particular, 1 ml PBS was added to each bacterial solutions separated thereby to provide a number of cells equal to 105, 104, 103, 102, 101and 100each of them was mixed and then centrifuged at 15000 rpm for 5 minutes at 4°C, followed by removal of supernatant. The procedure was repeated twice, with 1 ml of PBS was added to the precipitate, and then mixed, centrifuged and supernatant was removed. To the precipitate obtained was added 300 μl of bacteriolytic buffer (100 mm Tris-HCl, 40 mm EDTA, 1% SDS, pH: 9,0), 500 ál of TE-saturated phenol and 300 mg of glass beads (0.1 mm in diameter), which then was badly shaken using a FastPrep FP120 at 5000 rpm for 30 seconds for the destruction of bacterial cells. The solution with the destruction of the bacterial cells was centrifuged at 15000 rpm at 4°C for 5 minutes, then dump the supernatant. To the supernatant was added phenol (the E-rich)/chloroform/isoamyl alcohol, much shaken in FastPrep FP120 at 4000 revolutions / minute for 45 seconds and then subjected to the procedure of centrifugation at 15000 rpm, 4°C for 5 minutes. The alcohol precipitation was carried out from separated and stored supernatant, followed by dissolving the precipitate in 50 μl of THE buffer for receiving the DNA solution. Subsequently, PCR was performed using the obtained DNA solution as a matrix. PCR was performed in a reaction mixture of a total volume of 25 µl containing 2 µl of DNA solution and 10 mm Tris-HCl (pH 8,3), 50 mm KCl, 2.5 mm MgCl2, 0,45% Triton X-100, 200 μm dNTP mixture, 1/100000 volume SYBR(R) Green I, 11 ng/ál of TaqStart antibody(R) (ClonTech), Of 0.05 U/µl DNA polymerase Taq (Takara) and 0.25 μm (each) primer (PSD7F/R or g-Staph-F/R) in the form of final concentration. The reaction solution warmed up at 94°C for 5 minutes, then subjected to 40 cycles of 94°C for 20 seconds, 60°C for 20 seconds and 72°C for 50 seconds, and then kept at 72°C for 10 minutes. The amount of amplification product was measured at each cycle, the fluorescence intensity of SYBR(R) Green I. These series of reactions were performed using ABI PRISM(R) 7900HT. For this reaction were subjected to 1/25 extracted quantity of each RNA and DNA.

As a result, as shown in Figure 2, both methods showed extremely good correlation between logarithmic number of bacterial glue is OK, and the value of C T. In Figure 2 the value of CTdisplays the dependence of the number of cells/extract, measured by the culture method for each bacterial strain, which serves as a sample. Black circle (•) indicates data quantitative RT-PCR, and the white circle (○) shows the data of the quantitative PCR. On the approximated curve obtained using a quantitative method RT-PCR, the correlation coefficient values (R2) is 0,9955 for P. aeruginosa and 0,9961 for S. aureus. It shows that the standard curves allow to count the number of bacterial cells on the basis of the values of CT. In addition, a quantitative method RT-PCR can detect 100bacterial cells in samples, showing that the method has a detection sensitivity comparable to conventional culture method. This demonstrates that the method can be used for quantitative analysis and detection of microorganisms as an alternative culture method. The method according to the present invention has a detection sensitivity of approximately 1000 times greater than the PCR method with the use of rRNA as a target sequence, demonstrating that he has a noticeable detection sensitivity compared to previous by means of quantitative analysis of the microorganism used the eat method of gene amplification.

Example 5: Quantitative determination of bacteria in the faeces

Different concentrations of P. aeruginosa was added in the faeces of man to compare the detection limits of the method for quantitative PCR with the method according to the present invention. Samples of faeces with the addition of P. aeruginosa was obtained with the number of cells in each of them equal to 101, 102, 103, 104, 105, 106, 107or 108cells on 20 mg of faeces human. Total RNA was extracted from each sample of faeces with the addition of P. aeruginosa and was used as matrix for the implementation of quantitative RT-PCR of the present invention. From each sample were extracted DNA was used as template to perform quantitative PCR. In addition, in the same samples was measured by the number of bacterial cells using the culture method. Extraction of total RNA and the method of quantitative RT-PCR was performed as described in Example 2; the culture method as in Example 3; and extraction of the DNA and the method of quantitative PCR as in Example 4. In this sense, 1/2500 quantities obtained total RNA, and total DNA was subjected to quantitative RT-PCR and quantitative PCR, respectively.

As a result, as shown in Figure 3, the method according to the present invention showed a linear approximated curve, is received through measurements in the range 10 2,9-1010cells / g faeces samples of faeces with the addition of P. aeruginosa. In Figure 3 the level of CTreflects the dependence of the number of cells per g faeces measured by culture method for P. aeruginosa, serving as a sample. Black circle (•) indicates data quantitative RT-PCR and white circle (○) shows the data of the quantitative PCR. In the faeces human quantitative limit of the method according to the present invention was 102,9cells or more g of faeces was approximately comparable to the limit for the culture method, which is 102cells or more on g faeces. The culture method takes one day, while the method according to the present invention is, from the stabilization of the RNA to the quantitative analysis, for a time of approximately 6 hours. On the other hand, in the analysis method for quantitative PCR, was observed linearity of the approximated curve obtained from measurements in the range 105,8-1010cells / g of faeces, and the detection limit is approximately 1000 times lower than the detection limit of the method for quantitative RT-PCR.

Example 6: a Study of enterobacteria in faeces of people using quantitative RT-PCR and culture method

Flora in the faeces of humans was investigated by quantitative RT-PCR is a specific label for enterobacteria primers En-lsu 3F/3'R. Collected fresh feces in 38 adults and diluted in the ratio of 1/10 under anaerobic conditions of transport medium (10% glycerol, 5% cysteine, 1% lab lemco powder, 0,045% NaCl, 0,0225% KH2PO4, 0,0225% K2HPO4, 0,0225% (NH4)2SO4, 0,00225% CaCl2, 0,00225% MgSO4). 200 ál aliquots (20 mg faeces) were taken from the solution and subjected to extraction of total RNA using the method of quantitative RT-PCR. Quantitative RT-PCR was performed using 1/2500 amount of total RNA as template. An aliquot of the same solution also have been subjected to quantitative analysis through SOME culture method (selective environment DHL). Stabilization of RNA extraction total RNA and quantitative RT-PCR corresponded to Example 2, and the culture method was conducted in accordance with generally accepted way. Total RNA extracted from E. coli YIT 6044T(type strain), was used to obtain the standard curve to calculate the number of bacterial cells by quantitative RT-PCR.

As a result, as shown in Figure 4, it was demonstrated that the method of quantitative RT-PCR using as a target rRNA in accordance with the present invention and the culture method show a very strong correlation (correlation coefficient 0,9255). In Figure 4, the ordinate represents the results to castenago analysis, conducted cultural way, and the abscissa represents the results of the quantitative analysis obtained by the method of the present invention. For the culture method requires 2 days to perform all operations, while for the method of the present invention, all operations complete within about 6 hours.

Example 7: the Study of microorganisms in cow's milk

Different concentrations of E. coli, S. aureus and B. cereus were added to the plant cow's milk for comparison of quantitative evaluation method of cultivation in a layer of agar on plates with the method according to the present invention. E. coli or S. aureus add to the plant cow's milk in such a way as to ensure the microbial number equal to 100, 101, 102, 103, 104, 105and 106per ml, to obtain samples. 1 ml of each sample were subjected to extraction of total RNA and 1 ml was subjected to the method of cultivation in agar plate (E. coli: Wednesday desoxycholate agar, S. aureus and B. cereus: traditional agrarian environment, 37°C, 20±2 hours). All extracted RNA was investigated by the method of quantitative RT-PCR using primers as described in Table 1, for detecting the correlation between the received value CT and the number of microbial cells obtained using the method of cultivation in agar plate. In connection with the Tim, extraction of total RNA and the method of quantitative RT-PCR was carried out as described in Example 2; 1/25 the number of all extracted RNA was subjected to quantitative RT-PCR.

As a result, as shown in Figure 5, the value of CTcorrelated with the number of microbial cells in the range 100-106cells per ml of milk for any strain. In Figure 5 the value of CTdepends on the number of cells per ml of milk, measured by the method of cultivation in agar plate for the quantitative analysis of E. coli (top left in Figure 5), S. aureus (top right in Figure 5) and B. cereus (bottom left in Figure 5)serving as a sample. The quantitative limit of the method according to the present invention is 100cells or more per ml of milk and compare with the quantitative limit of the method of cultivation in agar plate. This shows that the method according to the present invention can provide an alternative method of cultivation in agar plate, using the common media for culturing (Wednesday desoxycholate agar or generally accepted agricultural environment), as described in Government law concerning the standard of composition, etc. for milk and milk products. Furthermore, the method of cultivation in agar on cups a day, whereas the method according to the present invention is, from one hundred the waste utilization technologies RNA sample prior to quantitative analysis, for about 6 hours.

Example 8: Determination of bacteria in the blood

Various concentrations of S. aureus or P. aeruginosa was added to human blood for comparing the quantitative values of method of cultivation in agar plate (method of cultivation of blood) with the quantitative value of the method according to the present invention. S. aureus or P. aeruginosa was added to ensure that the bacterial count of 100, 101, 102, 103, 104and 105on ml, human blood to which an anticoagulant is added 1/10 volume of 3.8% solution of sodium citrate to obtain samples. 0.5 ml of each sample were subjected to extraction of total RNA and 0.5 ml was subjected to the method of cultivation in agar plate (agar medium BHI). Dedicated total RNA was investigated by the method of quantitative RT-PCR to determine the correlation between the obtained CT value and the number of bacterial cells obtained using the method of cultivation in agar plate. Extraction of total RNA and the method of quantitative RT-PCR was performed by the method described in Example 2. In this regard, 1/25 the number of all extracted RNA was subjected to quantitative RT-PCR.

As a result, as shown in Figure 6, the number of bacterial cells was correlated with a value of CTin the range of 100-105cells is and 0.5 ml for each strain. In Figure 6 the value of CTdepending on the number of cells per 0.5 ml of blood, measured by the method of cultivation in agar plate for the quantitative analysis of P. aeruginosa (on the left in Figure 6) or S. aureus (right in Figure 6), serving as a sample. The quantitative limit of the method according to the present invention is 100cells or more than 0.5 ml of blood and compare with the quantitative limit of the method of cultivation in agar plate. This shows that the method according to the present invention could provide an alternative method of cultivation in agar plate. Furthermore, the method of cultivation in agar on cups a day, whereas the method according to the present invention is, from the stabilization of the RNA sample prior to quantitative analysis, for about 6 hours.

Example 9: Determination of E. coli in fermented dairy product

E. coli was added to the factory product Yakult (Yakult Honsha Co., Ltd.) thus, to ensure bacterial number 100, 101, 102, 103, 104and 105per ml to obtain samples. 1 ml of each sample was subjected to extraction of total RNA and 1 ml was subjected to the method of cultivation in agar plate using desoxycholate agrarian environment (37°C, 20±2 hours). Total extracted RNA was investigated by means of pic is BA quantitative RT-PCR using specific enterobacteria primers En-lsu 3F/3'R to determine the correlation between the obtained value of C Tand the number of microbial cells obtained using the method of cultivation in agar plate. Extraction of total RNA was performed as described in Example 2, except for the destruction of bacterial cells by adding glass beads, and a method of quantitative RT-PCR was performed as described in Example 2. In this regard, 1/25 the number of all selected RNA was subjected to quantitative RT-PCR.

As a result, as shown in Figure 7, the value of CTcorrelated with the number of bacterial cells in the range 100-105cells / ml In Figure 7 the value of CTdepends on log10cells per ml Yakult, measured by the method of cultivation in agar plate for the quantitative analysis of E. coli, which serves as a sample. The quantitative limit of the method according to the present invention is 100cells or more per ml Yakult and compare with the quantitative limit of the method of cultivation in agar plate. This shows that the method according to the present invention could provide an alternative method of cultivation in agar plate, using the common media for culturing (Wednesday desoxycholate agar)as described in Government law concerning the standard of composition, etc. for milk and milk products. Furthermore, the method of cultivation in agar plate C which takes one day, while the method according to the present invention is, from the stabilization of the RNA sample prior to quantitative analysis, for about 6 hours.

Example 10: Identification of lactobacilli and enterococci in faeces of man by quantitative RT-PCR and culture method

The number of cells of bacteria of the genera Lactobacillus and Enterococcus in the faeces of man compared by the method of quantitative RT-PCR using primers as described in Table 1 and by the culture method. Collected fresh feces in 48 healthy adults, were processed using the method described in Example 6, and subjected to stabilize RNA extraction total RNA and quantitative RT-PCR by the methods described in Example 2. In this regard, from 1/2000 to 1/200000 number of all obtained RNA was subjected to quantitative RT-PCR. An aliquot of the same solution of faeces was also subjected to quantitative analysis through SOME cultural way (genus Lactobacillus: Wednesday LBS, genus Enterococcus: Wednesday COBA, at 37°C for 48 hours in both cases). The culture method was performed in accordance with generally accepted way; emerging colonies were subjected to species identification of bacteria by biochemical tests properties (staining Gram, index test, API Strep). The number of cells of bacteria of the genus Lactobacillus ways through which and quantitative RT-PCR was calculated by summing the number of bacteria cells obtained by means of quantitative RT-PCR using primers sg-Laci-F/R (subgroup Lactobacills acidophilus), sg-Lsak-F/R (subgroup Lactobacillus sakei), sg-Lcas-F/R (subgroup Lactobacillus casei), sg-Lrum-F/R (subgroup Lactobacillus ruminis), sg-Lreu-F/R (subgroup Lactobacillus reuteri), sg-Lpla-F/R (subgroup Lactobacillus plantarum), s-Lbre-F/R (Lactobacillus brevis), s-Lfru-F/R (Lactobacillus fructivorans) and LFer-1/2 (Lactobacillus fermentum).

As a result, as shown in Table 4, the number of cells of bacteria of the genus Lactobacills and the genus Enterococcus in the faeces of man is roughly comparable to the method according to the present invention and cultural way. In contrast, the frequency of detection is high for both genera for the method of the present invention compared with the culture method. Apparently, this is due to the following reasons: (a) attended bacteria belonging to the genus Lactobacills or Enterococcus and targeted, but not growing as a selective medium has a higher selectivity than is necessary; or (b) weak selectivity applied selective environment has led to growth in the environment genera of bacteria represented in greater numbers than the target, which makes impossible the determination of bacterial genera, which are the targets. The above results suggest that the method according to the present invention not only allows you to retrieve the number of bacterial cells, comparable with the cult of the actual way, but also allows you to detect or quantitatively determines bacteria which were previously impossible to detect by culture method. In addition, the completion of all operations, including the identification of bacterial species, culture method takes 7 days, while for the method of the present invention, all the operations are completed for approximately 20 hours.

Table 4
RodThe method of quantitative RT-PCRThe culture method
log10cell/g of faecesFrequency (%)log10CFU/g of faecesFrequency (%)
Lactobacillus5,2±1,244/46 (96)5,5±1,437/46 (80)
Enterococcus6,2±1,046/46 (100)6,2±1,923/46 (50)

1. Method of quantitative analysis of the number of cells of interest bacteria in a live state with application in quality is TBE rate of rRNA interest bacteria in a sample for testing, includes measuring the amount of product amplified by PCR performed using primers capable of specific hybridization with rRNA interest bacteria, and specimen samples for testing, and
in which the number of cells of interest bacteria in a live state is determined taking into account the number of cycles of PCR.

2. The method according to claim 1, wherein measuring the amount of the amplified product includes determining the number of cycles of PCR, which achieved a certain amount of the amplified product.

3. The method according to claim 1 or 2, comprising measuring the amount of the amplified product in time.

4. The method according to claim 1, in which a sample for testing is obtained from faeces, food or body.

5. The method according to claim 1, in which rRNA interest bacteria in the sample the sample for testing stabilize in bacteria.

6. The method according to claim 1, wherein the primers are capable of specific hybridization with rRNA interest bacteria represent a fragment of nucleic acid, each containing a sequence of bases as described in one of SEQ ID NO: 2, 3, and 5-28, or a sequence of bases complementary to it, or a fragment of a nucleic acid that contains a sequence of bases capable of hybridization in jesd the x terms with DNA containing the sequence of bases represented by one of SEQ ID NO: 2, 3, and 5-28, or a sequence of bases complementary to it.

7. The primer used in the method according to any one of claims 1 to 6, where the primer is a fragment of a nucleic acid containing a sequence of bases as described in one of SEQ ID NO: 2, 3, and 5-28, or a sequence of bases complementary to it; or
primer contains a sequence of bases capable of hybridizing in stringent conditions with a DNA containing the sequence of bases represented by one of SEQ ID NO: 2, 3, and 5-28, or a sequence of bases complementary to it.

8. Set for the implementation of the method according to any one of claims 1 to 6, including
(1) primers capable of specific hybridization with rRNA interest bacteria,
(2) dNTP,
(3) DNA polymerase,
(4) the reaction buffer and/or
(5) the reagent used for the extraction of RNA and/or stabilization of RNA.



 

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FIELD: medicine.

SUBSTANCE: elaborated is method of obtaining factor, which takes part in process of control of appetite and/or body weight. Also described are genes, obtained by said method, polypeptides, coded by said genes, intended for treatment, control or diagnostics of diseases, associated with eating disorders and/or control of body weight. Invention also relates to substances, which inhibit activity of said genes or said polypeptides, intended for treatment, control or diagnostics of diseases, associated with process of appetite and/or body weight control.

EFFECT: using tiasolidindions, possessing PPARγ-agonistic activity, it is possible to obtain genes and polypeptides, involved into regulation of appetite and/or body weight reduction.

27 cl, 41 dwg, 35 ex

FIELD: medicine.

SUBSTANCE: RNA is recovered from peripheral blood or synovial liquid. Further, cytokine balance is evaluated by quantitative analysis of interleukin-2 (IL-2), interleukin-4 (IL-4) and interleukin-10 (IL-10) or interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-17 (IL-17), interleukin-1p (IL-1p) cytokines mRNA genes expression, as well as by quantitative analysis of interferon-gamma (IFNG) and a tumour necrosis factor (TNF) by reverse transcription and polymerase chain reaction with recording the accumulation of reaction products by direct fluorescence. The direct fluorescence is used to evaluate the cytokine balance. Further, a pair balance of expression of various cytokines is calculated on the basis a functional interrelation.

EFFECT: use of the invention reduces an evaluation error related to specific properties of the control gene expression.

4 dwg, 6 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: offered is a method and a set of primers for determining a haplotype of a DNA-target containing two heterozygous polymorphic sites. The method according to the invention provides a) taking a genome DNA sample and amplifying the area on the 3'-side of which there is a first polymorphic site, and on the 5'-side of which the second polymorphic site is found; b) conducting a PCR of said specific area with using one of two primers allele-specific relatively to the first polymorphic site and a downstream primer which is located so that to include the second polymorphic site in an amplicon; c) hybridising the prepared PCR product with two probes one of which is specific to a mutant type sequence, and another one - to a wild type sequence within the second polymorphism and d) determining a haplotype where the first polymorphism is described by a type of an extended upstream primer, and the second polymorphism - by a type of the probe hybridised with the PCR product.

EFFECT: use of the invention provides relatively simple and high precise diagnostic system.

4 cl, 5 dwg, 5 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to synthetic oligonucleotide primers, complementary to high conservative VP60 gene region of a genome of rabbit viral hemorrhagic disease virus, to a method for identifying rabbit viral hemorrhagic disease virus and to a test system for identifying RNA of rabbit viral hemorrhagic disease virus. The offered invention can be used in veterinary virology. The method for identifying rabbit viral hemorrhagic disease virus involves sample preparation, RNA recovery from the biological material. It is followed with conducting a polymerase chain reaction with using primers 5'-caa cgt get cca gtt ttg gta cg-3', 5'-att ctg tct ggt tgg ggc gtg t-3'. Further, viral RNA is amplified. Then, the reaction is assessed by agarose gel electrophoresis, with a reaction result considered as positive if the PCR product corresponds to the size of 398 base pairs.

EFFECT: invention allows higher sensitivity of the method, as well as reduced time of diagnostic manipulations with organ and blood samples of the infected animals.

3 cl, 4 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, namely to synthetic oligonucleotide primers complementary to a conservative S-segment region of a genome of sheep Nairobi disease virus, to a method for identifying sheep Nairobi disease virus and to a test system for identifying DNA of sheep Nairobi disease virus. The offered invention can be used in veterinary virology. The method for identifying sheep Nairobi disease virus involves RNA recovery from the biological material. It is followed with conducting a polymerase chain reaction with using primers NSD F1 5'TATGCTTCTGCCTTGGTTG-3' NSD R1 5'-ATCCGATTGGC AGTGAAG-3', NSD F2 5'-AGAGCACATTGACTGGGC-3', NSD R2 5'-GCCTTCCAAAGCCAGTAG-3' Thereafter, virus RNA is amplified. Then, the reaction is assessed by agarose gel electrophoresis, with a reaction result considered as positive if the PCR product corresponds to the size of 360 base pairs.

EFFECT: offered invention allows identifying genome RNA of sheep Nairobi disease virus by means of a nidicolous version of RT-PCR with using two synthesized pairs of oligonucleotide primers complementary to conservative gene region of core protein N.

3 cl, 4 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: sequence typing is used to differentiate Yersinia pestis strains. The technique provides recovery of chromosomal DNA of the investigated strain, polymerase chain reaction (PCR) with amplification of rhaS, araC, metB, asp A and thiH gene fragments to be analysed for nucleotide sequences. A genotype of the investigated strain is stated by nucleotides being in the positions 482, 494, 671 reHarhaS, in the position 773 of the gene rhaS, in the positions 988 and 989 of the gene metB, in the positions 1087-1089 of the gene aspA and in the position 552 of the gene thiH. A sequence type (ST) of the Y. pestis strain is specified by rhaS, araC, metB, aspA and thiH gene alleles, while the subspecies differentiation is enabled by comparing to the sequence types of the major and minor subspecies. The sequence type (ST) is specified for each subspecies.

EFFECT: use of the method provides fast, reliable and effective differentiation of Yersinia pestis subspecies.

2 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention refers to a synthetic oligonucleotide kit for identifying DNA of human monocytic ehrlichiosis (HME) agent - pathogenic representatives of Ehrlichia genus by a polymerase chain reaction. The offered invention can be used for the diagnostic purposes for detecting monocytic ehrlichiosis Ehrlichia spp. by the real-time polymerase chain reaction. Said kit includes primers as follows: 5'- GGG GAA AGA TTT ATC GCT ATT AG -3', 5'- CGG CAT AGC TGG ATC AGG CT -3' and a sample: (BHQl)-5'- CCC ACT GCT GCC (FdT)CC CGT AGG AGT CTG G - 3'P, where BHQ1 means a dark fluorescence killer attached to 5'-terminal nucleotide, while FdT is a fluorescent dye FAM attached to nucleotide T.

EFFECT: invention allows reliable identification of Ehrlichia representatives in the biological material.

1 ex

FIELD: medicine.

SUBSTANCE: live attenuated Pasteurella multocida bacterium is modified by introduing a mutation into the gene Orf-15. The mutation represents insertion and/or deletion in the gene Orf-15. As a result of modification, the bacterium is not able to express the functional protein Orf-15 that leads to its attenuation. Also, disclosed is the use of such bacterium for preparing a related vaccine for human or animal protection against Pasteurella multocida bacteria infection or pathogenic effects of the infection.

EFFECT: increased efficacy of applying the composition.

16 cl, 1 dwg, 3 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: new natural version of ghrelin predecessor resulted from an alternative gene splicing is described. A recombinant DNA method has been used to produce an active protein coded by said splicing version and showed ability to reduce body weight and food intake level after been analysed for functional properties on animal models.

EFFECT: using a new version of ghrelin and its active fragments for the purpose of preparing drugs for obesity or diabetes is offered.

7 cl, 28 dwg, 7 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, and represents an application of siRNA able to inhibit 11 beta-steroid dehydrogenase 1 (11beta-HSDl) expression for preparing a drug for treating an eye disease characterised by intraocular pressure (IOP) variations. Also, a method of treating such eye disease, as well as a recovered siRNA compound able to inhibit 11beta-HSDl expression, are presented.

EFFECT: invention can be used in treating the eye disease characterised by intraocular pressure variations.

21 cl, 10 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention describes method of constructing recombinant bacteria, belonging to genus Pantoea, method of constructing recombinant bacterium belonging to genus Pantoea - L-amino acid producent, bacterium belonging to genus Pantoea, obtained by said method, as well as method of obtaining L-amino acid with application of said bacterium.

EFFECT: invention makes it possible to increase production of L-amino acids.

12 cl, 7 dwg, 5 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a chemically modified molecule of a double stranded nucleic acid, which includes a sense chain and an antisense chain: , as well as a composition which contains such a molecule of the double strand nucleic acid.

EFFECT: nucleic acid molecule enables mediation of RNA-target activity through RNA interference and has high stability and activity in vivo.

5 cl, 43 dwg, 8 tbl, 16 ex

FIELD: medicine.

SUBSTANCE: invention presents an eukaryotic host cell for producing a gamma-carboxylated protein, containing an expression vector containing a nucleic acid molecule coding the protein requiring gamma-carboxylation, and associated expression control sequences, and the expression vector containing a nucleic acid molecule coding vitamin K-epoxide reductase and the associated expression control sequences. Besides, the host cell contains the nucleic acid molecule coding γ-glutamyl carboxylase and the associated expression control sequences. The given invention also relates to a method for producing gamma-carboxylated protein and to a method for preparing a pharmaceutical composition effective for blood coagulation induction or hypercoagulability or hypocoagulability stimulation, based on the produced gamma-carboxylated protein.

EFFECT: invention allows high effective production of the gamma-carboxylated protein.

20 cl, 2 dwg, 9 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention represents novel endo-(1-4)-β-D-xylanases of mycelial fungus Penicillium canescens. Invention also related to method of obtaining endo-(1-4)-β-D-xylanases with application of eukaryotic cells, transformed by fragment of DNA, coding said xylanases from Penicillium canescens.

EFFECT: invention makes it possible to extend arsenal of known endoxylanases.

12 cl, 6 dwg, 4 tbl, 7 ex

New virus of plants // 2411290

FIELD: agriculture.

SUBSTANCE: first plant or its part is exposed to infection dose of ToTV. Then plants with no or slight symptoms of disease are identified.

EFFECT: plants identified in such a manner as resistant to virus are used as donor ones to cross with recipient plants, and plants resistant to ToTV are chosen from descendant plants.

8 cl, 7 dwg, 5 tbl, 2 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and medicine and specifically to direct thrombin inhibitors. The invention discloses a modified aptamer oligodeoxyribonucleotide of general formula d R-GTGACGTANGGTTGGTGTGGTTGGGGCGTCAC-R where d is deoxyribose (oligonucleotide contains deoxyribose in all positions), R denotes chemical substitutes. The substitute used is aminohexanol (NH2 group), which esterifies the phosphate group on the 5' or 3' end and PEG which is bonded on the 5' and/or 3' end. At least one of the substitutes bonded on the 5' or 3' end is polyethylene glycol (PEG). The disclosed modified DNA aptamer inhibits thrombin activity in human blood plasma by prolonging thrombin clotting time, prothrombin time and activated partial thromboplastin time and inhibits thrombin-stimulated aggregation of thrombocytes. During intravenous administration to an experimental animal, the modified DNA aptamer circulates in the blood longer than the basic aptamer RE31.

EFFECT: said DNA aptamer can serve as the base for antithrombotic medicinal agents.

6 dwg, 3 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to genetic engineering. Disclosed are expression DNA structures which include a T-shaped DNA structure with a fragment (19-23 bases)of the target gene sequence subject to transcription with formation of siRNA, which provide specific inhibition of said gene in a cell via RNA interference. Described is a method of obtaining the disclosed DNA structure, which does not involve PCR steps and is a simple three-step procedure which is performed in a single reaction vessel.

EFFECT: invention can be used for practical purposes for inhibiting production of target proteins in cells.

14 cl, 7 dwg, 1 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to molecular biology and gene therapy. disclosed is a group siNA (siRNA) aimed at certain gene areas of the P2X7 (P2RX7) receptor and which efficiently inhibit expression of said receptor.

EFFECT: invention discloses use of siRNA directly, as well as a component of medicinal agents for treating diseases characterised by high expression and/or activity of the P2X7 receptor such as neural degeneration, Alzheimer disease, inflammatory diseases, certain types of malignant tumours etc.

26 cl, 5 dwg, 3 ex

FIELD: biotechnology, molecular biology, biochemistry.

SUBSTANCE: invention relates to regulatory sequences. Method involves isolation of DNA molecule with nucleotide sequence SEQIDNO:2 or SEQIDNO:3 that is necessary for expression of the required encoding sequence. Then vector comprising any of indicated sequences and the required sequence is constructed followed by transformation a plant with the prepared vector. Invention provides preparing transgenic plants with regulating expression of the required gene.

EFFECT: improved preparing method.

19 cl, 1 tbl, 6 ex

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