Method of microorganism detection in biological sample
SUBSTANCE: method involves application of a diagnostic multiplex panel (DMP) designed for simultaneous identification of a set of possible microorganisms which can be found in a biological sample, with applying primer extension reaction to high conserved nucleotide sequences in sampled microorganisms. The biological sample is immobilised either on, or in a solid substratum in a first position, then transferred to the other position and extracted on the solid substratum so that to extract DNA of any microorganism which is found in the sample. It is followed by amplification of nucleic acid on the extracted DNA of microorganisms, then target sequences are mixed with primers with using the DMP. It results in genotyping of any microorganisms found and identifying of the required microorganisms. For implementation of the method, the diagnostic multiplex panel (DMP) applicable for genotyping of pathogenic microorganisms, and a testing kit for microorganisms is used.
EFFECT: use of the invention allows reliable selection of the biological samples and their testing, providing simultaneous testing of a set of microorganisms.
22 cl, 2 dwg, 3 tbl, 1 ex
The SCOPE TO WHICH the INVENTION RELATES.
The invention relates to high-performance multiplex testing for microorganisms that may be present in a biological sample, using enzymatic methods based on nucleic acids. More specifically, the invention relates to the identification of pathogenic microorganisms.
BACKGROUND of INVENTION
Since, as in the nineteenth century, the microorganisms were identified as the main cause of morbidity and mortality, continued attempts to monitor and control the spread of communicable diseases. The earliest attempts were made by the young pioneers of the science of epidemiology, such as Dr. John Snow (John snow). In the twentieth century, the advent of antibiotics, mass vaccination and antiviral drugs have provided an unprecedented level of control over the spread of such diseases, at least in developed countries. Nevertheless, worldwide, infectious diseases are still one of the main causes of death, and it seems that every year, inevitably appears a continuous series of "new" germs killer. MRSA (Staphylococcus aureus, methicillin-resistant), SARS (severe acute respiratory syndrome (SARS), SARS), avian influenza, HIV and malaria are only small hours of the completion of many infectious pathogens, causing fear and anxiety all over the world.
The international organization responsible for health, such as the UN and the who, have repeatedly expressed concern about the unlimited use of antibiotics, leading to higher levels of resistance of many bacteria to antibiotics. In addition, currently, in the world of sexually transmitted infections (STIs STIs)are one of the major problems associated with infectious diseases, and in some regions, particularly in Africa and in the former Soviet Union are at an epidemic level. According to one study (Adler, M., 2005, " Why sexually transmitted infections are important. In: ABC of Sexually Transmitted Infections. 5thed. BMJ Books) confirmed the number of infected in Western Europe is estimated at 17 million, in the United States 15 million in Africa 70 million, and just in the world, 400 million people.
Combating sexually transmitted infections, and HIV/AIDS remains a major government programs for health in the world, and the need for greater availability of health facilities and in the development and provision of diagnostic services available to all who need them, is obvious. Many STI are asymptomatic and may be diagnosed only by means of tests, however, the standard screening program is extremely rare, public prejudice the iroko common funding is inadequate, and the understanding of the population is limited.
The impact of infectious diseases is not limited to the human population, large economic losses bring outbreaks of livestock and cultivated plants. The plague epidemic in pigs, the virus of foot and mouth disease in cattle and virus bird flu spread quickly and cause great damage to agricultural production and the economy. In the UK 2001 epidemic of foot and mouth disease led to the loss of more than seven million head of cattle and sheep, as well as to the actual "closing" of the agricultural land.
The modern system of screening for the presence of microorganisms in the sample obtained from the owner (such as a patient or animal source vegetable source), are characterized by low productivity. In clinical conditions usually in the same sample is tested only one microorganism, if there is no medical evidence that the owner may suffer several diseases caused by microorganisms. This means that in one test it is possible to detect only a single infection that greatly affects the cost and time of testing. In addition, the owner with the asymptomatic nature of the disease is often difficult to decide on what organism should wire the t test to the patient. Negative results on two or three infectious agents can create a false sense of security.
In the latest systems of detection (discovery), used in clinical practice for the detection of infection by microorganisms use analyses based on DNA. The most common methods based on polymerase chain reaction (PCR, PCR), ligase chain reaction, amplification preemptive chain, transcription amplification, self-sustained replication sequences (first NASBA) and some others. These methods are also low-productivity, time consuming, require a lot of manual labor and difficult to automate. In addition, there is still no reliable method that could be used for detection of multiple pathogens in a single test on a single sample taken from the host body.
Therefore, it would be desirable to create a reliable and cheap way to test a variety of microorganisms that may be present in the biological sample. In particular, it would be desirable to create a method of exercise testing, which would allow reliable screening of biological samples (samples) at home or in the field.
In General, the present invention solves the above problems of the previous prior art by providing methods for the s and devices for high-performance testing of biological samples, which may contain or not to contain microorganisms. Methods include the use of diagnostic multiplex panel (DMP, DMP), specially created for the simultaneous determination of many probable types of microorganisms that may be present, or absent, in a biological sample.
In the first aspect of the invention includes a method of determining whether one or more specified (precisely defined) of microorganisms is a biological sample that may contain microorganisms, including:
(a) immobilization of the biological sample and/or in a solid substrate (rigid substrate) in one place;
(b) the transfer of the immobilized biological sample, at least, in another place and the implementation phase extraction on a solid substrate so that was extracted DNA of any microorganism immobilized on and/or in a solid substrate;
(C) implementation stage nucleic acid amplification on DNA of microorganisms extracted at the stage (b), and stage amplification refers to the amplification of at least one highly conserved sequence of one or more specific (concrete, specified) microorganisms and amplificatoare sequence referred to as the target sequence;
(g) see the solution (Association) of the target sequences with the sequences of a number of primers, published on diagnostic multiplex panel (DMP), and the sequence of each primer helps genotyping target sequence;
(d) carrying out a primer extension reaction using join (mixture) of target sequences and DMP present on stage (g), thereby obtaining the reaction product with DMP; and
(e) analysis of the reaction product in order to determine the genotype of any present target sequence, and correlation of the genotype of the target sequences present in the reaction product, with the identification of specific microorganisms in a biological sample.
In the second aspect of the invention includes a DMP is suitable for use in the genotyping of pathogens known to cause at least one infectious disease that may be present in the biological sample, and DMP contains a number primernih sequences that aim to identify at least two or more SNP (single nucleotide polymorphisms) in the highly conserved allele at least one microorganism, known as calling an infectious disease, when used in a primer extension reaction.
In the third aspect of the invention includes a kit for testing microorganisms suitable for the North-South Expressway is the first use by the user, located in the first place, and the set contains surface testing, located in the pressurized chamber, the surface of the testing further comprises a solid substrate (the substrate), can mobilitat biological sample or in or on its surface, and when the biological sample is deposited on the surface of the test, the sealed space (camera) around the surface of the test so that the test set can be sent in second place for analysis in order to determine whether the biological sample is one or more microorganisms.
In the fourth aspect of the invention includes a method of treating an animal, including man, presumably, which is a carrier of one or more infectious organisms, which consists in obtaining a biological sample of an animal, testing a biological sample in accordance with the methods according to this description, diagnosis as a result of this, whether the animal is infected by one or more infectious organisms, and treatment of the animal, and this treatment is carried out (available) subject information regarding the type (types) of infectious microorganisms found in the biological sample. Optional treatment optional organization is : with regard to the information concerning the status of the resistance of one or more infectious organisms to antibiotics detected in the biological sample. These and other applications, features and advantages of the invention should be obvious to experts in the art from the disclosure of this specification.
Description of the DRAWINGS
Figure 1 schematically presents a highly conserved consensus sequence, a DNA consensus sequence obtained by comparing multiple strains of the same species - shows the locations where the primers for amplification reactions or elongation of the primers selected for use in the DMP according to the invention. For each primer first two letters identify the body, the next letter identifies the target sequence (1-3); 1stPCRP denotes a first PCR primer; 2ndPCRP refers to the second PCR primer; E1 or E2 denotes a primer for elongation; in the case of Ureaplasma has two sites, which are designed primers (UU1 and UU2). The identity of the primers are presented in Table 1. Includes the following (micro)organisms: (a) the Candida albicans (Candida albicans); (b) Chlamydia specific antibody in the sample (Chlamydia trachomatis); (C) Gardnerella vaginalis (Gardnerella vaginalis); (d) Mycoplasma of genitalium (Mycoplasma genitalium); (d) Mycoplasma, hominis (Mycoplasma hominis); (e) Neisseria gonorrhoea (Neisseria gonorrhoea); (g) Pale Treponema (Treponema pallidum) (C) Trichomonas vaginalis (Trichomonas vaginalis); (and) the Ureaplasma urealyticum (Ureaplasma urealyticum).
DETAILED description of the INVENTION
In the description of the invention provides a number of definitions that will help to understand the invention. For the avoidance of doubt, all cited in this description reference materials are introduced by reference in its entirety. If not stated otherwise, all technical and scientific terms used herein have the same meaning as known to the ordinary skilled in the art to which this invention relates. In the description of conventional methods of molecular biology, it is assumed that the specialist in the art knows these methods, for example, from standard textbooks such as Sambrook J. et al., (2001) Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.
It is assumed that the term "certain (specific specified) microorganisms" in this description refers to one or more specific species of microorganism, which may or may not be present in the biological sample. Certain microorganisms are microorganisms of the corresponding viral, bacterial, fungal (including single-celled yeast) and/or protozoa (protozoa, including Plasmodium) origin. Usually some microorganisms are pathogenic against the owner of the animal in some periods of its jesnen the th cycle. However, the present invention is suitable for testing species of microorganisms, which are in a state of deep rest, called symbiotic infection or subclinical infection.
It is assumed that the term "biological sample" in this description covers samples that contain one or more of certain microorganisms tested in accordance with this invention. Depending on the destination DMP biological sample can be obtained from human, non-human animal, plant or food product. In the latter case, it is assumed that the DMP is designed to determine the contamination of food by pathogens that transmit the disease through nutritional. Samples may include, for example, urine; faeces; vaginal, nasal or oral swabs, blood, saliva, and/or sputum; semen; vaginal or urethral discharge and strokes; tears (for example, selection of the lacrimal glands); tissue biopsies; and swabs (swabs from surfaces that may Deposit the above allocation and substances. Although the biological sample of the present invention may contain cells, tissue and/or DNA of the host body, for example, a man by the content of the invention is tested for the presence of microorganisms in the host - not testing their own bottoms the owner.
The solid substrate (rigid substrate) according to the invention is chosen appropriately from absorbing (hygroscopic) of fibrous material impregnated with one or two reagents that facilitate immobilization and inactivation of all microorganisms in a biological sample, or, even simpler, cause immobilization of nucleic acids in microorganisms. Such reagents can include detergents: anionic or cationic detergents, chelating agents (such as EDTA), urea and/or uric acid. Himself a solid substrate may include an absorbent material selected from cellulose paper (such as paper for blotting); membrane microfiber; fiber; material from polymeric fibers such as nylon membrane filter); woven fabric and non-woven material. Suitable solid substrates (substrate holders) are described, for example, in U.S. patents№ 549562, 5756126, 5807537, 5939259, 5972386, 5985327, 6168922, 6746841 and 6750059. In specific embodiments of the present invention, the solid substrate (wafer carrier) includes filter paper treated with the reagent Whatman FTA®or Whatman FTA Elute®such as paper Whatman FTA®or Whatman FTA Elute®.
A notable advantage of the method according to the present invention is that the solid substrate is treated with the reagent, such as Rea is UNT Whatman FTA Elute ®that person may not apply in a clinical setting. For example, consider that the initial phase of sampling (samples) of the method according to the invention can be implemented, individually purchase a set and just wetting of solid sales substrate, for example, the breakdown (sample) urine or saliva. Solid substrate immobilize any microorganisms in the urine, or saliva, so infectious pathogens become non-infectious and the sample (the sample) can be transferred to the test site (it does not require expensive manipulation, namely, cooling or additional fixing chemical reagents), for example, by regular mail. DNA immobilized microorganism can be easily removed from the solid substrate in the test site, for example, a simple heating and elution with water. Thus, the invention includes a system in which the selection of biological samples (samples) can be done even at home or in the field, samples can be stored indefinitely, and test spend later. The advantages of this procedure is significant, because up to now, most diagnostic tests are difficult to conduct due to the need to take samples in a clinical setting, which reduces the availability for the population as a whole.
In accordance with a variant of the present invention suitable set for testing at home with the holding sealing chamber (case), which includes a surface for testing. The kit includes instructions for use, which indicate to the user where on the surface of the test to put the biological sample (sample), such as a drop of urine. The surface of the test comprises a solid substrate (wafer carrier) of the type indicated above. After the sample is placed on the surface of the testing, resealable Luggage (case) can be closed so that she has encapsulated and protected the surface from further testing of the impact. In a tightly closed set remains protected from external impact or pollution, and it can be sent to a testing center that is located at a distance from the place of residence of the user. In the test center resealable Luggage can be opened (if necessary, opening the camera), giving access to the surface of the test for analytical purposes in accordance with the method according to the invention.
"Nucleotide sequence" ("nucleic acid sequence") represents a single or double-stranded linked by covalent bonds a nucleotide sequence, where the 3' and 5' ends of each nucleotide is linked phosphodieterase links. Nucleotide sequences are usually polynucleotide, which can consist of which of deoxyribonucleotide units or ribonucleotidic links. Polynucleotide include DNA and RNA and can be synthetic methods in vitro or to stand out from natural sources. The size of the nucleotide sequences are usually expressed as the number of base pairs (BP, b..) for the double-stranded polynucleotides, or, in the case of single-stranded polynucleotides, as the number of nucleotides (nt). One thousand BP (b..) or nt equals kilobase (KBP called). Polynucleotide, having a length of less than about 40 nucleotides, usually referred to as "oligonucleotides". Sequence of primers (primernye sequence)used in the present invention for the stages of nucleic acid amplification and extension primers are single-stranded oligonucleotides.
The expression "reaction amplification of nucleotides (nucleic acids)" in this description refers to any of a number of related enzymatic methods, which uses a thermostable DNA polymerase for amplification of a specific DNA sequence during periodic (repetitive) cycles of primer extension, denaturation and hybridization. Typically, PCR is the preferred reaction amplification of nucleotides used in the method according to the present invention.
It is assumed that the term "reaction primer extension" refers to a reaction in which the nucleotide primers is ostautsya thus, to gibridizatsiya with the target sequence and biocatalytic (using enzymes) to be extended by adding one or more nucleotides to the 3'-end of the primer. The primers hybridize on the position in the target sequence that is directly 5' to or a few bases "above" (5') from the position of polymorphisms such as single nucleotide polymorphism. In embodiments of the invention, in which the primer hybridizes on the basis of the above (upstream, 5') known SNP site, the elongation of the primer by one base affects hybrids primer target sequence by adding a chain-terminating nucleotide, often dideoxynucleotide. Only one of the four nucleotides that extend primer is a single nucleotide, which is complementary sequences on the target strands (chains, heavy). The identity of the added nucleotide is determined in the analysis phase of the method according to the invention, described in more detail below.
The term "polymorphic allele" is used in this description to designate one or more alternative forms of a genetic sequence, occupying the same chromosomal locus and controlling (regulating) the same genetic trait. Allelic variant in nature occurs by mutation and may result in resultaten phenotypic polymorphism within populations, or may lead to conservative (non - phenotypic) polymorphism. Mutations in genes usually result in the modified nucleotide sequence. The phenomenon of allelic polymorphism in this description is used in relation to single nucleotide polymorphisms (SNPS), insertions, divisions, inversions and substitutions, all they can occur in genes that are highly conserved in this species. SNP is a polymorphism, in which alleles are replaced (replacement/substitution) of a single nucleotide in the DNA sequence at a given position in the genome. In highly conserved genes, such as 16S rRNA in bacteria, the SNP are highly specific in relation to the type and strain, which contributes to obtaining accurate information about genotyping. Other highly conserved region of microorganisms include bacterial gene 32S rRNA, yeast genes 16S and 18S rRNA genes and the viral polymerase. However, the competence of a specialist to apply the methods of bioinformatics to identify SNP in other alternative conservative regions of the genome of this microorganism. Polymorphisms, such as SNPs, described above, can be associated with specific phenotypic characteristics in the tested model body. For example, antibiotic resistance due to mutation and, thus, polymorphism. However, the method according to the present invention is not limited to the identifier is a function only of polymorphic positions in the genomes of the studied microorganisms. If this conservative target sequence are competitive control sequence, the term "polymorphic allele" is used approximately to denote a variant on this site sequence between the sequence of wild-type (which is checked) and artificial competitive sequence. In this case, it is clear that the so-called polymorphism simply contributes to the differentiation of the products of the extension reaction of a primer on competitive matrix compared with a matrix of wild-type, since the corresponding reaction products have different relative molecular mass.
The present invention is based in part on the way to reliable and high performance testing of one or more biological samples for the presence of microorganisms in the sample. Performance analysis favors the use of a diagnostic multiplex panel (DMP), which is designed for genotyping of many microorganisms, theoretically present in the biological sample. DMP includes a combination of primers, each of which undergoes specific hybridization of highly conserved sequences in DNA isolated from microorganisms that may be present in the biological sample. DMP makes possible the YMI simultaneous primer extension reaction with the purpose of definition, whether or not one or more microorganisms are present in the isolated sample. DMP according to the present invention can be appropriately designed for a specific treatment or diagnosis, when tested in a broad sense, according microorganisms area or type of disease. In the example used in the invention, described in more detail below, collect DMP designed to diagnose the presence of infection, sexually transmitted diseases in biological samples taken from people. This form of DMP is suitable for checking the presence of bacterial pathogens, species such as Mycoplasma (Mycoplasma spp.); chlamydia (Chlamydia spp.); Ureaplasma (Ureaplasma spp); Neisseria (Neisseria spp.); Gardnerella (Gardnerella spp.); Trichomonas (Trichomonas spp.); Treponema (Treponema spp); or yeast Candida albicans (Candida albicans); or viral pathogens, such as cytomegalovirus (CMV, CMV), hepatitis viruses (e.g., HAV, HBV, and HCV, etc.); human immunodeficiency virus (HIV, HIV); human papilloma virus (HPV, HPV), herpes simplex virus (HSV, HSV); molluscum contagiosum virus (Molluscum contagiosum, MCV); influenza virus; Epstein-Barr (EBV, EBV) and the virus stays zoster (VZV, VZV). Other diseases, to diagnose which is well suited DMP include: food poisoning, tuberculosis, viral cancer; encephalitis; malaria; hepatitis; meningitis; leishmaniasis; sleeping more is ery; pneumonia; plague; SARS SARS; methicillin-resistant Staphylococcus aureus (MRSA); rabies; anthrax; rift valley fever; tularemia; shigellosis; botulism; yellow fever; Q fever; fever, Ebola, dengue fever, West Nile fever (West Nile virus); dysentery; influenza; measles and typhoid.
In addition, the invention makes possible the detection of sequences that give bacterial pathogens and antibiotic sensitivity, due to the inclusion of such sequences in the design DNP-test. This allows you to accelerate the onset the treatment of patients who are diagnosed with pathogens such as falls an additional separate stage microbiological determination of sensitivity to antibiotics. In addition, the invention can provide data about the progression of some diseases by determining the concentration of the detected pathogens, which in many cases reflects the progression of the disease. Concentration may include, for example, assessment of viral load. Quantitative data can be obtained in the reaction phase primer extension, for example, including a competitive sequence in the target sequence, and this competitive sequence contains typed polymorphism in position compared to Clevo the sequence. Competitive sequence may include alternative nucleotide at position known SNP, but in everything else is identical. If a competitive sequence in a known concentration (or a known number of copies) is added at a stage of amplification of the nucleotide, then it can serve as a starting point for the quantitative determination of the concentration of containing a polymorphism in a target sequence of interest microorganism. In a specific embodiment of the invention can include additional competitive sequences in various concentrations (e.g., low, medium or high concentration), all of them with the option of a sequence, aimed at a specific site in the target sequence that facilitates a more accurate quantitative determination of the concentration of microorganisms in the original biological sample. In addition to quantification, the introduction of competition sequences also provides an internal control for all stages enzyme diagnostic method according to the invention.
DMP according to the invention are provided in the form of a set of corresponding plexus (plexed) primers. However, the DMP may also contain the primers are immobilized on a solid surface, for example, in the form of microci the and. The solid surface may be in the form of a silicon substrate or glass substrate.
Separation (resolution) of the reaction products on the DMP after primer extension can be done by various methods, including mass spectrometry (e.g., MALDI-TOF), electrophoresis (e.g., capillary electrophoresis), DNA microarrays (e.g., GeneChip™ company Affymetrix or DNA chips on the printed matrix), the incorporation of nucleotides labeled with a fluorescent label (e.g., SNPstream®from Beckman Coulter or Applied Biosystems SNPlex®), or other labels (e.g., antigenic tag, biotina or RFID tags). The preferred method of separation of the products of the primer extension reaction includes the determination of relative molecular mass, for this purpose, preferred as mass spectrometry and capillary electrophoresis.
In one specific embodiment, the present invention changes the total length of the nucleotide sequence of each primer, available in the DMP, so that no two primers with the same relative molecular mass either before or after the primer extension reaction. The reaction products cleanse for optimization of mass spectrometric analysis. After cleansing products in the form of patches applied to the corresponding element, preferably, a silicon chip, including high-density, Svetosti is a new set of sites for mass spectrometric analysis (e.g SpectroCHIP ®) and analyzed by time-of-flight mass spectrometer with laser desorption/ionization sample (MALDI-TOF) (e.g., mass spectrometer Mass Array®the company Sequenom, as described in U.S. patents№ 6500621, 6300076, 6258538, 5869242, 6238871, 6440705 and 6994969). The results of mass spectrometric analysis process using the appropriate software package to obtain data on the presence or absence of products of primer extension, which correlate with the presence or absence of specified microorganisms in a biological sample.
Further, the invention is illustrated using the following non-limiting example.
The authors present invention has created a cost-effective, reliable and high-precision test, which allows you to define any number of infections in a separate sample. The test consists of two parts: a simple set of sampling at home (using paper Whatman FTA Elute®as substrate (substrate), which caused the sample) and a new multiplex panel of sexually transmitted infections (STIMP), as a Diagnostic multiplex panel (DMP), which allows you to define methods on the basis of DNA, whether or not any given individual with one or more sexually transmitted bacterial, viral, protozoal (easiest is) and/or fungal pathogens.
This example shows that:
paper Whatman FTA Elute®is an adequate medium for bacterial, fungal, and protozoal (protozoa) pathogens from a sample of human urine.
- compounds present in paper Whatman FTA Elute®or urine, do not interfere with subsequent enzymatic processes testing.
- new STIMP functions as DMP and is able to detect individual pathogens from a mixture of pathogens.
MATERIALS AND METHODS
- CLINICAL SAMPLES (TESTS)
Only 44 of the sample taken from patients of a private clinic GUM (genito-urinary diseases in Ukraine.
Patients in the experimental group were asked to provide a sample (a sample) of the first portion of urine (about 30-50 ml) in a sterile container after consultation. All samples were obtained with informed consent of patients, and all ethical requirements and technical standards (including the sampling) was observed and was consistent with the requirements of the Ministry of health of Ukraine.
Each urine sample is transferred to the card (charge) for the samples of Whatman FTA Elute®(Whatman plc, Brentford, UK), once plunging sterile sponge applicators (Puritan®, Maine, USA) in a vessel with a sample of urine, and then four times cause spots in four different places card (the card).
After transfer of samples each separate map Whatman FTA Elute®dried in to the room temperature until completely dry. To prevent cross-contamination dry card with samples separately placed in samonapisavshaiasia plastic bags and then stored at room temperature.
Then samples (samples from batches 1 and 2 (31 and 13 individual samples, respectively) are sent in the form of two parcels (courier service fedex®in place of testing (analysis, testing) to the laboratory in Germany. Sampling in party 1 is performed in the period from 12 December to 26 December 2006. Sampling in the party 2 is carried out in the period from 5 January to 12 January 2007 inclusive.
All samples analyzed in parallel independent local laboratory (Kyiv, Ukraine) experts in the field of analysis of sexually transmitted infections, conventional detection methods based on DNA, recommended by the Ministry of health of Ukraine.
All samples tested for the presence of the following microorganisms:
Candida albicans (Candida albicans)
Chlamydia trachomatis (chlamydia specific antibody in the sample)
Gardnerella vaginalis (Gardnerella vaginalis)
Mycoplasma genitalium (Mycoplasma of genitalium)
Mycoplasma hominis (Mycoplasma, hominis)
Neisseria gonorrhoeae (Neisseria gonorrhea)
Trichomonas vaginalis (Trichomonas vaginalis)
Treponema pallidum (Pale Treponema)
Ureaplasma urealyticum (Ureaplasma urealyticum)
The results obtained in local analytic La the oratorios (marked as 'clinical', 'clinic') then compare with the results obtained by the method STIMP/DMP (designated as 'lab', 'lab').
EXTRACTION of DNA
To account for the difference of concentrations of DNA from each card for samples Whatman FTA Elute®manual punch cut out six discs, 6 mm samples in the following sequence - 1 x circle for test tube 2 ml, 2 X circle for test tube 2 ml, 3 x circle for tubes in 2 ml After cutting out each card for samples punch cleaned three times Prodanova through a clean filter paper, and then three more times Prodanova through filter paper, moistened with 70% tO. To prevent cross-contamination, then push the net card FTA Elute®and extracted DNA, as indicated below (tube 4).
DNA is extracted according to the extraction Protocol DNA with cards Whatman FTA Elute®in our modification, given that the perforated disk with a sample size of 6 mm, and the recommended disk size sample 3 mm One, two and three CDs with sample 6 mm, or cut pieces of paper (Excised Paper Fragments, EPF) is placed in a separate round-bottom tubes Eppendorf 2 ml and add, respectively, 0.7 ml, 1.4 ml, and 2.1 ml of N2O.
Each sample three times mix on vortex for 5 seconds and drives the sample is transferred into a clean Eppendorf tubes n is 0.5 ml.
To each tube containing one, two and three disk with the sample, add to, respectively, 50 μl, 80 μl and 100 μl ddH2Oh, after which the tubes incubated at 95°C for 30 minutes. After incubation, the samples centrifuged at 12000 g for 2 minutes and stored at +4°C. For PCR amplification, 1 µl of each sample are used directly or in the form of a dilution 1:1 with ddH2O.
As positive controls use the following cell line was obtained from National collection of type culture (NCTC).
Cell line No.
Cells are diluted with 500 μl of fresh urine, and then take 50 ál of each sample to prepare a positive control by ledouxnebria.
The positive control No. 1
The positive control No. 2
The positive control No. 3
Positive controls and a sample of fresh urine, used for cultivation of cells, put the eyedropper on a separate card (card) Whatman FTA Elute®in accordance with the manufacturer's recommendations Whatman (50 μl, approximately 1 cm2). After applying the sample card (card), dried at 60°C for one hour and extracted DNA as described above.
In addition, an aliquot of each of the positive control and sample of fresh urine (for cultivation of cells) are used, both directly and in a dilution of 1:5 in ddH2For PCR amplification.
During the experiment, we were unable to obtain positive controls for C. trachomatis, T. vaginalis or T. pallidum.
Design DMP analysis based on DNA sequences, highly conserved in different strains of each interest type. Given the high variability of three different plot in conservative areas used for planning (design) of the three tests for each pathogen. For all of the pathogens selected conservative region is the 16S rRNA gene, except .albicans, where selected conservative region is gene 18S rRNA (see Figure 1).
Conduct analyses of the two types. The first type includes a control competitive sequences for each target, the second does not include any competitors. The role of a competitor is to serve as an internal positive control for PCR, RE and other enzymatic reactions, as well as when applying spots on the chip. It is expected that in the sample with a competitor will be visible, at least the signal of a competitor, even if the sample does not contain the target DNA of the pathogen. Competitors create so that they were identical to the target DNA sequence with a known only different nucleotide at the SNP site of interest, the sequence of the competitors shown in Table 1. Competitive sequence type in the amount of about 30 copies on the target for PCR amplification.
In the samples analyzed without competitors, the expected signal only when the sample is present in the target DNA of the pathogen. The target of the same multiplex serve as an internal positive control for each other, however, if no signal is present for all targets amplified together, nevoso is but to conclude, whether or not the sample contains no target DNA of a pathogen or several enzymatic reactions has not passed.
PCR amplification is carried out in vitro in a volume of 5 μl containing h HotStar®buffer for PCR, 1.625 mm MgCl2, 0.04 mm of each dNTP, 0.1 μm of each primer and 0.1 Units. polymerase HotStarTaq®. The characteristics of the cycles of the following:
95°C 15 min
94°C 10 sec
56°C 30 sec
72°C 1 min
72°C 3 min
4°C 5 min
Amplification is performed on thermocycler MJR Tetrad Thermo Cycler.
TREATMENT of ALKALINE PHOSPHATASE SHRIMP (SAP)
After PCR amplification in each tube add: 1.53 μl of N2Oh, 0.17 μl buffer TS and 0.30 ál of SAP. The reaction is carried out using the following characteristics:
37°C for 20 min
85°C 5 min
The reaction is carried out at thermocycler MJR Tetrad Thermo Cycler.
The REACTION PRIMER EXTENSION
The elongation of the primers is carried out in accordance with the Protocol iPLEX™ (Sequenom, Inc., San Diego, CA, USA). Incubated with 2 ál of the mixture to primer extension iPLEX containing 1x iPLEX buffer, 1x iPLEX termination mix, then in each tube add 0.625 μm of each primer for elongation and 1x iPLEX enzyme. The iPLEX reaction carried out using the following characteristics:
94°C 30 sec
94°C 5 sec
52°C 5 sec
80°C 5 sec
The transition to stage 3×5 times.
The transition to stage 2×40 times>
72°C 3 min
The reaction is carried out at thermocycler MJR Tetrad Thermo Cycler.
To the above mixture add 16 ál of N2Oh and 6 mg of SpectroCLEAN resin®and rotate on a circular shaker for 30 minutes and then centrifuged 5 min at 3000 g to precipitate the resin.
APPLYING STAIN SAMPLES
9 nanolitres each sample is applied in the form of spots on a 384 SpectroCHIP®with the help of a robot MassARRAY®Nanospotter in accordance with the manufacturer's instructions. Then, each chip is read at mass spectrometer MALDI-TOF, compact MassARRAY analyzer®Compact Analyzer, and data are obtained using Typer v3.3 (or above) and stored in the database.
ASSESSMENT of GENOTYPE AND DETERMINATION of infection STATUS SAMPLE
For each analysis (target DNA) create some competitive sequence that differs from the target DNA artificially introduced by single nucleotide polymorphism (SNP). The last design as MUT (Mutant) genotype using genotyping. Pathogenic SNP create as C (Control) genotype. When competitor and target DNA present in the reaction mixture, the heterozygous genotype, denoted either C.MUT or MUT.C, estimated with application programs. The order of the alleles in the heterozygous genotype reflects the relative proportion of the corresponding DNA in the reaction mixture.>
Suppose that the sample is infected when at least two of the three analyses show the presence of pathogenic DNA in an individual DNA sample. Because of card samples each patient will receive three different DNA sample, we should expect that all three samples should give identical results (NB: since each sample is theoretically contains a different number of copies of a pathogen, believe that the DNA samples obtained from two and three drives pattern, gives more reliable results due to the increased amount of pathogen DNA compared to DNA sample extracted from a single disk with the sample). Sometimes only one or two analyses show the presence of pathogenic DNA in the sample taken from the patient, while the majority (main part) of the tests for this pathogen on all DNA samples are negative. I believe that these results are artifacts (most likely the result of cross-contamination), however, samples are evaluated as positive and are indicated with an 'x' after the result in the summary table of results (table 2).
RESULTS AND DISCUSSION
Paper Whatman FTA Elute®
In order to examine whether any of the compounds contained in the paper Whatman FTA Elute®or urine, to interfere with subsequent enzymatic processes, a number of control samples, starasiahleeque known high purity ("benign") DNA diluted 1:1 eluent, obtained after extraction of one, two or three disks, or fresh urine. Then the samples used for PCR and other subsequent enzymatic reactions. For all samples obtained pure PCR products and mass spectra (data presented). This shows that the chosen method of extraction and washing Protocol is applicable for MALDI-TOF analysis and that substances in human urine would not significantly affect the reliability DMP analysis.
In order to check whether pathogenic DNA from urine samples to immobilizates and stored on paper Whatman FTA Elute®several DNA samples (extracted from the card (cards)) patients, the analysis in the local laboratory (clinical)identified as positive (positive), amplified in a reaction volume of 50 µl using primers from STIMT/DMP and the products separated on a 2% agarose gel in 1× TBE buffer (see Figure 1). In all cases there is a clear signal of the amplicons of the desired size, which indicates the presence of pathogenic DNA on paper Whatman FTA Elute®in quantities sufficient for reliable PCR amplification.
The same experiment is carried out using urine samples with positive control, and get similar results (data not shown).
These results confirm that products based on cellulose, such as paper Whatman FTA Elute®can be used as a solid substrate (substrate), suitable for the equipment and transportation of patients ' samples, and do not display a visible degradation in time. However, it seems possible that for testing real STIMT/DMP method can also be applied unprocessed urine samples and other biological samples immobilized on substrates of other types.
All samples tested in reactions in the presence or in the absence of competitive control sequences. In the experiment, which involves competitive sequence, signals appear competitive DNA, in most cases, including positive controls.
In this section we discuss the results obtained in experiments in which the reaction does not participate in competitive sequences.
The results of the experiments are presented in Table 2.
In order to confirm whether the method STIMT/DMP can be used to detect specific pathogens in a mixture of pathogens, three positive control obtained by the mixing of pathogens from NCTC, sexually transmitted diseases, create, using the environmental quality of the urine (see above). Samples positive controls analyze both samples in the urine, and samples on paper Whatman FTA Elute®(Table 3). In all cases, the presence of each of the CSOs of the pathogen in the sample will be detected regardless of its nature. Positive controls are distinguished by the presence of strains of N. gonorrhoeae. In all analyses for this method STIMT/DMP allows you to detect the presence of N. gonorrhoeae regardless of the type strain. This shows that DNA, which was chosen to develop analyses of N. gonorrhoeae, are dependent strain specificity and can be used to detect any known species N. gonorrhoeae in the sample.
In the analysis of clinical samples for laboratory STIMT/DMP results in all cases confirm the results obtained and analyzed independently in the clinic (table 2). Some samples, not identified in the analytical laboratory, also identified as positive for the infection. However, since the pathogen was not detected in all analyses, and because only one (rarely two) DNA sample have a positive signal, these results should be viewed as artifacts of this pilot study. Most of this occurs when the patient sample in the previously punched by a punch card for samples gives a positive response to a particular infection (for example, patients 2 and 3 - U. urealyticum in patients 11 and 12 M. genitalium and so on). The most likely explanation for these artifacts is the transfer of DNA from the previous sample. This is confirmed by the fact discovered by the fact that the samples for which ntrolle impurities (net card (fee) Whatman FTA Elute ®used between samples taken from patients are also positive for the same infections. This clearly demonstrates the high sensitivity of the DMP according to the invention, and subsequent experimental methods will be optimized to avoid cross-contamination.
For each card patient samples are taken three different DNA sample. The samples contain a different number of copies of pathogenic DNA (in case of its presence). In most cases, it is possible to identify the infection in the DNA samples of patients with a positive reaction, if the extraction is carried out two or more disk drives.
In most DNA samples of patients with a positive reaction from all three of the tests to identify the infection. In a very small number of cases, one of the three tests does not give a clear signal and it is celebrated with the program genotyping.
You can also define the number of copies of target DNA present in the reaction mixture, tetroe number of competitor or including three different competitor in a known initial concentration in the same reaction mixture. The latter approach is recommended for pathogens, infectious load which is important from the point of view of clinical perspectives, for example, for patients infected with HIV or G. vaginalis.
Although the specific variants of the invention disclosed in detail herein, they are given only as examples and illustrations. It is not intended that the above options limit the scope of the attached claims. Applicants believe that various substitutions, changes and modifications can be made in this invention without departing from the essence and scope of the invention defined by the claims.
1. The way to determine whether two or more of certain microorganisms in the biological sample, and the microorganisms are bacteria, fungi, viruses and protozoa, including:
(a) immobilization of the biological sample and/or hard substrate in one, the first location;
(b) the transfer of the immobilized biological sample, at least in a different location and implementation phase extraction on a solid substrate so that was extracted DNA of any microorganism immobilized on and/or in a solid substrate;
(C) amplification of nucleic acids in the DNA of microorganisms extracted at the stage (b), and stage of amplification is amplification, less than the least one highly conserved sequence of two or more of certain microorganisms, and amplificatoare sequence referred to as the target sequence;
(g) a mixture of target sequences with sequences together primers contained on diagnostic multiplex panel (DMP), and the sequence of each primer helps genotyping target sequence;
(d) carrying out a primer extension reaction using a combination, a mixture of target sequences and DMP presented on stage (g)obtaining the reaction product with DMP; and
(e) analysis of the reaction product in order to determine the genotype of any present target sequence, and correlation of the genotype of the target sequences present in the reaction product, with the identification of specific microorganisms in a biological sample.
2. The method according to claim 1, characterized in that the biological sample is at least one sample from the group consisting of urine, saliva, blood, sputum, semen, faeces, nasal smears, tears, vaginal swab, rectal swab, cervical secretions, tissue biopsies and urethral smear.
3. The method according to claim 1 or 2, characterized in that the solid substrate is an absorbent fibrous material, p is opetany one or two reagents, which contribute to immobilization and inactivation of all microorganisms in a biological sample, and an absorbent fibrous material selected from cellulose paper, membranes microfiber, glass fibers, the material of the polymeric fibers, woven fabric and non-woven material, and a solid substrate contains a reagent Whatman FTA® or Whatman FTA Elute® and paper Whatman FTA Elute®.
4. The method according to claim 1, characterized in that two or more infectious pathogens include human pathogens, causing one or more diseases selected from the group consisting of infections, sexually transmitted diseases; food poisoning; tuberculosis; viral cancer; encephalitis; malaria; hepatitis; meningitis; leishmaniasis; sleeping sickness; pneumonia; plague; SARS SARS; methicillin-resistant Staphylococcus aureus (MRSA); rabies; anthrax; rift valley fever; tularemia; shigellosis; botulism; yellow fever; Q fever; Ebola; fever dengue; West Nile fever (West Nile virus); dysentery; influenza; measles and typhoid, and the bacteria are selected from the group consisting of Mycoplasma (Mycoplasma spp.); Chlamydia (Chlamydia spp.); Ureaplasma (Ureaplasma spp); Neisseria (Neisseria spp.); Gardnerelly (Gardnerella spp.); Trichomonas (Trichomonas spp.) and Treponema (Treponema spp), yeasts are Candida albicans (Candida albicans), viruses wybir the Ute from the group consisting of a cytomegalovirus (CMV, CMV), hepatitis A virus (HAV); hepatitis b virus (HBV); hepatitis C virus (HCV), hepatitis E virus (HEV), hepatitis G and GB (GBV-C); the human immunodeficiency viruses (HIV, HIV), human papilloma virus (HPV, HPV), herpes simplex viruses (HSV, HSV); virus molluscum contagiosum (Molluscum contagiosum, MCV); influenza virus; Epstein-Barr (EBV, EBV) and the virus stays-zoster (VZV, VZV).
5. The method according to claim 1, characterized in that the DMP is designed to identify alleles from a combination of microorganisms, presumably present in the biological sample, and this combination includes bacteria, viruses, and fungi, all of the microorganisms are preferably pathogens.
6. The method according to claim 5, characterized in that the DMP is used for identification of microorganisms associated with a specific type of disease is selected from the group consisting of infections, sexually transmitted diseases; food poisoning; tuberculosis; viral cancer; encephalitis; malaria; hepatitis; meningitis; pneumonia; plague and influenza, preferably is a sexually transmitted infection, and DMP contains primernye sequences that hybridize with one or more target sequences, derived from a microorganism selected from the group consisting of Mycoplasma of genitalium (Mycoplasma genitaliu); Mycoplasma, hominis (Mycoplasma hominis); Chlamydia specific antibody in the sample (Chlamydia trachomatis); Ureaplasma urealyticum (Ureaplasma urealyticum); Neisseria gonorrhoea (Neisseria gonorrhoea); Gardnerelly vaginalis (Gardnerella vaginalis); Trichomonas vaginalis (Trichomonas vaginalis); Pale Treponema (Treponema pallidum); CMV; HAV; HBV; HCV; HEV, GBV-C, HIV-1; HIV-2; HPV; HSV-1; HSV-2; MCV; VZV; VEB and Candida albicans (Candida albicans).
7. The method according to claim 1, characterized in that the highly conserved polymorphic allele contains a gene or part of a gene of the microorganism, and a gene selected from bacterial 16S rRNA gene; bacterial gene 32S rRNA; 16S rRNA gene of yeast; gene 18S rRNA yeast and viral polymerase, and a highly conserved sequence is a polymorphic allele selected from the group consisting of single nucleotide polymorphism (SNP); insertions; deletions; inversion and replacement.
8. The method according to claim 1, characterized in that, when present in a biological sample of two or more of certain microorganisms in the reaction primer extension is formed DMP reaction product containing at least two elongated primernye sequence is known for a given molecular weight different from one another, and DMP reaction products analyzed by the method that is used to separate long primernye sequence according to their molecular weight, preferably MALDI-TOF mass spectrum is scopy and/or capillary electrophoresis.
9. The method according to claim 1, characterized in that the primer extension reaction involved the labeling reagent primer, so if two or more of certain microorganisms present in the biological sample, during the primer extension reaction, the labeling reagent is included in the primer extended product thus formed DMP reaction product containing labeling reagent, and DMP reaction products analyzed by a method that allows to determine the presence of included labeling reagent product containing elongated primer, preferably the labeling reagent primer contains a label, a radioactive label selected from; fluorescent labels and antigen, and a method of analysis choose from SNPstream® and/or SNPlex®.
10. The method according to claim 1, characterized in that the set of primernih sequences contained in the DMP, immobilized on a solid substrate, preferably selected from glass and silicon.
11. The method according to claim 1, characterized in that stage of amplification of the nucleotide sequence includes a set of amplification primers that are designed to amplify the totality of highly conserved sequences of two or more of certain microorganisms, preferably a set of primers represents one or more primer pairs selected from the group, with the standing of SEQ ID NO: 1/2; 3/4; 5/6; 7/8; 9/10; 11/12; 13/14; 15/16; 17/18; 19/20; 21/22; 23/24; 25/26; 27/28 and 29/30, and/or one or more primer pairs selected from the group consisting of SEQ ID NO: 46/47; 48/49; 50/51; 52/53; 54/55; 56/57; 58/59; 60/61; 62/63; 64/65; 66/67; 68/69; 70/71; 72/73 and 74/75.
12. The method according to claim 1, characterized in that the DMP contains one or more primernih sequences selected from SEQ ID NO: 31-45, and/or one or more primernih sequences selected from SEQ ID NO: 76-90.
13. The method according to claim 1, wherein the one or more control competitive sequences combine with the target sequences before stage nucleic acid amplification, each competitive sequence is identical to the corresponding target sequence except that, compared with the corresponding target sequence of a competitive sequence is a variant sequence at a particular position, preferably the variant sequence contains artificially introduced SNP, and/or one or more control sequences combine with the target sequences before stage nucleic acid amplification, and one or more control sequences contain a DNA sequence selected from the DNA sequences that are not related sequence in the biological sample; DNA p is coherence, not related to the test (test) microorganisms (microorganisms); and synthetic DNA sequences, and stage nucleic acid amplification and DMP contain the appropriate primernye sequences that specifically hybridize with one or more control sequences.
14. The method according to claim 1, wherein the biological sample is collected from human, non-human animal, plant, food, preferably in humans.
15. Diagnostic multiplex panel (DMP), suitable for use in the genotyping of pathogenic microorganisms causing at least one infectious disease that may be present in a biological sample that contains a set primernih sequences that are designed to identify at least two or more highly conserved sequences of at least two microorganisms that cause infectious disease, using the primer extension reaction, and infectious disease selected from one or more diseases in the group consisting of infections, sexually transmitted diseases; food poisoning; tuberculosis; viral cancer; encephalitis; malaria; hepatitis; meningitis; pneumonia; plague and influenza, and DMP contains primernye consistently the tee, suitable for genotyping of microorganisms selected from the group consisting of Mycoplasma of genitalium (Mycoplasma genitalium); Mycoplasma, hominis (Mycoplasma hominis); Chlamydia specific antibody in the sample (Chlamydia trachomatis); Ureaplasma urealyticum (Ureaplasma urealyticum); Neisseria gonorrhoea (Neisseria gonorrhoea); Gardnerelly vaginalis (Gardnerella vaginalis); Trichomonas vaginalis (Trichomonas vaginalis); Pale Treponema (Treponema pallidum); CMV; HAV; HBV; HCV; HEV, GBV-C, HIV-1; HIV-2; HPV; HSV-1; HSV-2; MCV; VZV; VEB and Candida albicans (Candida albicans), while the DMP contains one or more primernih sequences selected from SEQ ID NO: 31-45, or one or more primernih sequences selected from SEQ ID NO; 76-90.
16. Diagnostic multiplex panel (DMP) of clause 15, wherein the highly conserved sequence contains a gene or part of a gene of the microorganism, and a gene selected from bacterial 16S rRNA gene; bacterial gene 32S rRNA; 16S rRNA gene of yeast; gene 18S rRNA yeast and viral polymerase, preferably a highly conserved sequence is a polymorphic allele selected from the group consisting of single nucleotide polymorphism (SNP); insertions; deletions; inversion and replacement.
17. Diagnostic multiplex panel (DMP) of clause 15, wherein the set of primernih sequences contained in the DMP, immobilized on a solid substrate, predpochtitelno selected from glass and silicon.
18. The test microorganisms suitable for personal use by the user, containing a surface testing, and containing a solid substrate capable of mobilitat biological sample or in or on its surface, and a chamber for accommodating the surface with deposited sample, sealing the space around the surface of the test.
19. The test set for p, wherein the solid substrate is an absorbent fibrous material is impregnated with one or more reagents that facilitate immobilization and inactivation of all microorganisms in a biological sample, such as Whatman FTA® or Whatman FTA Elute®, preferably solid substrate is a material selected from cellulose paper, membranes microfiber, glass fibers, the material of the polymeric fibers, woven fabric and non-woven material, such as paper Whatman FTA Elute®.
20. The test set for p or 19, characterized in that the biological sample is at least one sample from the group consisting of urine, saliva, blood, sputum, semen, faeces, nasal smears, tears, vaginal swab, rectal swab, cervical secretions, tissue biopsies, and urethral smear.
21. The test set for p different those who, what biological sample taken from the man.
22. The test set for p, characterized in that the microorganism causing the infection, sexually transmitted diseases.
SUBSTANCE: at least one bacterium or a portion of a bacterium of oral bacteria species contacts with a gingival cell, and the presence of an indicator material is detected. The indicator material may be selected from a group consisting of molecules generated by modulation of a toll-like receptor or a NFk-B path. The absence of the indicator material shall mean that the given bacteria species is not malignant.
EFFECT: method simplification.
10 cl, 2 dwg, 1 tbl
SUBSTANCE: diagnostic technique for pseudotuberculosis involving infectious agent antibody determination in patient's blood serum by enzyme-linked immunosorbent assay with using the diagnostic antigen - a species-specific thermostable lethal toxin Y. pseudotuberculosis of certain molecular weight, estimated analysis result shown by optical density.
EFFECT: method enables reducing the analysis stages and providing more rapid diagnostics of pseudotuberculosis.
4 dwg, 1 tbl
SUBSTANCE: invention refers to methods of in vitro detection of infection by Mycobacterium tuberculosis in mammals and to methods of in vitro differentiation of a Mycobacterium tuberculosis infected mammal suffering a latent form of tuberculosis, and a mammal suffering an active form of tuberculosis, and to a method of differentiation of a mammal suffering the active form of tuberculosis and a mammal non-infected by Mycobacterium tuberculosis, or a mammal who has the latent form of tuberculosis. The present invention also refers to kits for detection and differentiation of the infected mammal presenting tuberculosis symptoms, and the infected mammal with no disease development, and to a kit for distinction of a mammal suffering the active form of tuberculosis, and the mammal non-infected by Mycobacterium tuberculosis, or the mammal suffering the latent form of tuberculosis.
EFFECT: use of the methods and the kits under the present invention allows detecting in vitro the infected mammals with the active and latent forms of tuberculosis.
29 cl, 4 tbl, 15 dwg, 7 ex
SUBSTANCE: cultivated cell strain of Mus. musculus L animals that is a producer of monoclonal antibodies (MCA) specific to LPS of cholera vibrios 0139 serogroup is produced. Hybridoma is deposited in Specialised Collection of continuous somatic cells of the Vertebrata Institute of Cytology (St.-Petersburg), No. RKKK (P) 674D. The specific activity of the MCA produced by hybridoma under the invention as a diagnostic reagent is proved in a slide agglutination reaction. The produced hybridoma can be applied in creating diagnostic test systems of the monoclonal antibodies for accelerated identification of cholera vibrios O139 serogroup.
EFFECT: invention use allows producing the diagnostically significant monoclonal antibodies to LPS epitopes of cholera vibrios 0139 serogroup that allows higher effectiveness of laboratory cholera researches.
3 tbl, 4 ex
SUBSTANCE: method of producing diagnostic immunoglobulins for HBsAg detection is presented. The method involves the following stages: HBsAg introduction to 20-25-days-old female quails directly in a thymus region, batch of eggs after the injected bird reaches the age of 50 days and production of the appropriate immunoglobulins from egg yolks.
EFFECT: method allows producing a large amount of specific immunoglobulins.
SUBSTANCE: faeces sample is examined in patients with acute enteric infections by bacteriological and microscopical methods. One faeces sample of the patient is concentrated by acetate sedimentation, and the produced sediment is used to prepare simultaneously 3 smears: the first one is stained with Ziehl-Nielsen carbolic fuchsin for acid-fast intestinal protozoa indication, the second one is stained with 1% Lugol's solution for lamblia detection, the third one is stained with polyvalent adsorbed serums in an indirect immunofluorescence reaction for salmonella indication, and is observing specific luminescence, the salmonellosis-protozoal acute enteric infection is diagnosed. 1-4 cells in a field of view enable to diagnose a mild form, and 5-12 cells - a severe infection.
EFFECT: use of the declared method allows producing results already in 1-1,5 h after sampling the material and providing higher diagnostic accuracy ensured by quantitative analysis of adhesive activity of simultaneously examined faeces samples, both for salmonellas, and for intestinal protozoa.
2 ex, 1 dwg
SUBSTANCE: analysed human blood serums are brought in wells of 96-well polystyrene plates with an immobilised complex of pertussis microbe antigens, added with a human IgG3 antibody peroxidase conjugate, added with a substrate mixture that is followed with recording the optical density of the mixture to derive a titre of immunosorbent attached human IgG3 antibodies. The substrate mixture is a ready form of tetramethylbenzidine and hydrogen peroxide. The human antibody titre 1:40 is accepted as pertussis related.
EFFECT: higher sensitivity and specificity of pertussis diagnostics.
1 ex, 2 tbl
SUBSTANCE: on the 2nd-7th postoperative day, microbial flora is sampled from at least five skin areas of a patient's body, specifically in the zones of a palmar surface, a bend of elbow, a postoperative suture and a therapeutic and/or diagnostic system and life sustenance junction, and also on surfaces of said systems and aids which directly contacts with the patient's body. The samples are analysed for the microbial flora composition, and if observing at least in three samples at least one microorganism of: Pseudomonas spp; Acinetobacter spp; Klebsiella spp; Enterobacter spp; Serratia spp, Citrobacter spp, Escherichia spp and/or microorganisms S. aureus, developing infectious complications within the next 7-14 days are predicted.
EFFECT: use of the declared method allows higher prognostic reliability with respect to developing infectious complications in cardiosurgical patients at a stage preceding said pathology.
SUBSTANCE: analysed sample is studied simultaneously by two methods: the first one is a indirect immunofluorescence (IIMF) reaction, while the second one involves a polymerase chain reaction (PCR); the IIMF provides using monoclonal antibodies "BCKK" (P-384D and 434D). The antibodies interact with the capsular antigen F1 specific for Y.pestis species, or the plasmid-temperature-independent surface protein PFV which is found in all Y.pestis strains and rare R-form Y.pseudotuberculosis strains. The bacteria luminescence shows the presence of native or fraction-less Y.pestis bacteria, or typical and PFV-atypical Y.pseudotuberculosis strains in the sample. The PCR is conducted by two pairs of primers vlm33for/ISrevl754 - specific for Y.pestis species, and JS - specific for Y. pseudotuberculosis species. The values derived with the first pair of the primers are estimated as positive if observing the amplicons in 400 bps, and with the second pair if observing the amplicons in 223 bps; the analysed sample is identified by the matching the IIMF and PCR values with the reference strains.
EFFECT: method provides quick identification and high-reliability differentiation of all strains.
7 tbl, 6 ex
FIELD: veterinary science.
SUBSTANCE: method is intended to detect antibodies to agents of infectious rhinotracheitis, parainfluenza-3, virus diarrhea, mucous membranes disease, respiratory syncytial and adenoviral infection of cattle. The proposed method makes it possible to detect specific antibodies per saltum in a single dilution procedure. For this purpose optimal working dilutions of serums is determined, as well as a positive-negative threshold of reaction, permissible values of optical densities of negative and positive serums, and also diagnostic sensitivity and specificity of the method. Then immune-ferment analysis is carried out. Analysis results are considered to be valid, if values of optical density of negative serums do not exceed 0.150 p.u., values of the positive control are at least 0.439 p.u., the upper and lower limits of the positive-negative threshold of reaction make 0.261 and 0.216 p.u. accordingly.
EFFECT: method is effective, cost-efficient, convenient in use and makes it possible to make preliminary forecasts about herd infection.
3 tbl, 4 ex
SUBSTANCE: invention relates to devices and methods with increased sensitivity in carrying out diagnostics, for instance, optic biopsy. Method of determining area of interest includes providing one or more ionic probes suitable for demonstration of avalanche effect with shift of emitted irradiation to higher frequency, impact on ionic probe(s) by activation energy and determination of one or more areas with higher concentration of ionic probe(s) on the basis of emission, connected, at least, partially with avalanche effect. System ensures determination of area of interest in accordance with the method.
EFFECT: application of invention makes it possible to considerably reduce or eliminate intensity of background signal from ionic probes, distributed in lower concentration throughout volume of measurements, also in application of invention, in fact, there is no background autofluorescence of surrounding healthy tissue.
16 cl, 5 dwg
SUBSTANCE: immunoglobulin-A is sorbed in microplate wells, then after incubation and treatment, a solution containing a proteolytic enzyme is introduced in the microtablet wells. It is followed with incubation; the contents of the wells are poured out, and then an enzyme conjugate, e.g. peroxidase, with immunoglobulin-A Fc-fragment antibodies and a substratum of this enzyme are introduced. After incubation of a reaction mixture, reaction results are accounted by a spectrophotometre. IgA-protease activity is calculated by the amount of hydrolysed substratum of the enzymatic reaction.
EFFECT: use of the invention allows facilitating determination of the IgA-protease activity level, the method exhibits good result reproducibility, high sensitivity, low substratum consumption and the absence of necessity to use of immunoglobulins of certain specificity.
1 dwg, 1 tbl, 4 ex
SUBSTANCE: method of blood plasma proximate analysis for cardiomyoglobin by means of an electrochemical immunosensor consists in the fact that a blood plasma sample is applied on a surface of a main electrode pre-modified by colloidal gold and monoclonal cardiomyoglobin antibodies; the prepared sample immunosensor is kept, preincubated in a buffer solution; cardiomyoglobin is electrochemically recorded in the sample by the prepared calibration diagram under certain conditions.
EFFECT: more sensitive analysis procedure, and reduced analysis time.
2 dwg, 2 tbl, 4 ex
SUBSTANCE: method of detecting anthrax pathogen in environment and biological fluids is based on functional detection of activity of one of anthrax toxin components, weak protease of anthrax lethal factor (LF). Applied principle of highly sensitive detection of LF proteolytic activity is based on the system of amplification of sugnal, originating from the act of substrate cutting, by means of present in substrate composition auxiliary enzyme of alkaline phosphatase of Escherichia coli (AP). In composition of recombinant LF substrate in addition to alkaline phosphotase and specific for LF substrate sequence RRKKVYPYPME, there is a peptide which is biotinilated in vivo and in vitro by means of biotin-ligase of E.coli and ensuring substrate immobilisation on solid phase (plaque surface) due to interaction with avidin and its derivatives, and sequence of six histidines for purification of recombinant substrate from periplasm of E.coli by means of metal-chelate resin.
EFFECT: method in accordance with the invention possesses high sensitivity, with application of fluorescein diphosphate makes it possible to detect up to 1 pM of lethal factor, rapidity of execution, high specificity, low risk of false-positive signals.
1 dwg, 3 ex
SUBSTANCE: set of reagents for quantitative determination of avermectins contains an ivermectin conjugate with bovine serum albumin which is immobilised on a polystyrene dish, a peroxidase conjugate of highly specific mouse monoclonal antibodies to ivermectin, and ivermectin calibration samples (with concentration between 0 and 100 ng/ml). The monoclonal antibodies in the set are produced by a strain of hybrid cultured cells of Mus. Museums L., which are deposited in the Collection of transferred vertebrate somatic cells of the D. I. Ivanovsky Research Institute of Virology under No. 05/09. From non-specific components, the set contains a buffer for culturing the monoclonal antibody conjugate with horseradish peroxidase, a buffer for culturing the analysed samples and washing the trays, reagents for detecting peroxidase activity, a substrate solution, hydrogen peroxide and tetramethylbenzidine and a stop solution (1M sulphuric acid). The disclosed set is designed to detect residual quantities of avermectins in tissue and biological fluids for monitoring chemical contamination of animal products.
EFFECT: use of the set enables to determine main components of an avermectin complex in animal products, while providing high sensitivity and specificity of detection.
1 dwg, 2 tbl, 4 ex
SUBSTANCE: proposed device comprises sensitive surface consisting of binding sections that allow specific attachment to biological objects with magnetic labels, magnetic pickup element, appliance to differentiate between magnetic labels connected to binding sections and magnetic labels, not bound, to act on binding sections. System to determine concentration of target objects consists of measuring device and electronic circuit to detect changes in magneto resistive resonance of magnetic pickup element. Note here that electronic circuit stays in or out of substrate. To determine concentration of target objects in fluid, fluid medium containing magnetic labels is subjected to magnetic field to differentiate between bound and unbound magnetic labels for period when binding process occurs on sensitive surface.
EFFECT: higher accuracy and rate of determining concentration of target molecules in fluid.
24 cl, 9 dwg
SUBSTANCE: method of obtaining erythrocyte diagnosticum for reaction of indirect hemagglutination (RIHG) in case of cattle coccielosis consists of fractional formalinisation of sheep erythrocytes and their sensibilisation with coccielosis antigen at 70°C for 30 minutes. For sensibilisation used are erythrocytes, loaded with sensitin obtained from vaccine strain C.burnetii M-44, heated on water bath at 70°C for 30 minutes, with further triple washing of erythrocytic diagnosticum with buffer solution with pH -7.2.
EFFECT: invention ensures increase of specificity and activity of diagnosticum in RIHG in case of cattle coccielosis.
SUBSTANCE: claimed is method of obtaining reactive sugar which includes the following stages: i) providing sample, containing reducing sugar; ii) providing solid substrate covalently bound to linker, which includes catch group containing -NH2 group, in which said linker is optionally bound to said solid substrate via spacer; iii) reaction of said reducing sugar with said -NH2 group resulting in obtaining immobilised sugar; iv) reaction of free -NH2 groups with camping-agent, with camping-agent containing reactive group able to react with -NH2 group and v) reduction of C=N bonds with reducer resulting in obtaining reactive sugar of SugarCHn-NH- structure, bound with solid substrate via linker and, optionally, spacer, where n is 1 or 2 manipulation with immobilised carbohydrates by derivation.
EFFECT: improvement of method
45 cl, 2 tbl, 7 ex, 25 dwg
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention concerns development of a diagnostic test system in immunochip format and a method of simultaneous and differential detection of reaginic antibodies and antibody spectrum to diagnostically significant mmunologically relevant proteins Treponema pallidum of G (IgG) and M (IgM) classes. The diagnostic test system in immunochip format for differential serum diagnostics of syphilis consists of an immunosorbent with separately immobilised antigens Treponema pallidum Tp15, Tp17, TmpA, Tp47, conjugate and reactants required to detect an antigen-antibody complex. Cardiolipin is additionally immobilised on the immunosorbent; antigens Treponema pallidum and cardiolipin are immobilised at least, in two repetitions, and conjugate consists of mixed antispecies human IgG antibodies and human IgM antibodies modified by two phosphors with different spectral characteristics. On the immunosorbent, there can be additionally immobilised at least, one antigen and/or peptide Treponema pallidum specified e.g. of Tp39, Tp41, Tp42, Tp44.5, Tp92, Tp0453 at least in two repetitions. Cardiolipin can represent an oxidised cardiolipin derivative bounded with protein. The differential serum syphilis diagnostic technique with using the diagnostic test system in immunochip format involves application of human IgM modified by two phosphors with different spectral characteristics. On the immunosorbent, there can be additionally immobilised at least one antigen and/or peptide Treponema pallidum, specified e.g. of Tp39, Tp41, Tp42, Tp44.5, Tp92, Tp0453 at least in two repetitions. Cardiolipin can represent an oxidised cardiolipin derivative bounded with protein. The differential serum syphilis diagnostic technique with using the diagnostic test system in immunochip format implying that a cultivation solution for check and test samples is introduced on the immunosorbent with separately immobilised antigens Treponema pallidum and cardiolipin; the check and test samples are introduced; the prepared mixture is incubated at temperature 20-42°C for 15-60 min to prepare the antigen-antibody complex; the immunosorbent is rinsed; then a conjugate solution of mixed human IgG antibodies and human IgM antibodies modified by two phosphors with different spectral characteristics is introduced on the immunosorbent; it is followed with incubation at temperature 20-42°C for 15-60 min; the immunosorbent is rinsed, dried to detect the prepared antigen-antibody complex.
EFFECT: improved diagnostic accuracy.
20 cl, 5 dwg, 2 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: method for combined immunobiological analysis of cells using a biochip involves incubation of the biochip which contains immobilised antibodies, with suspension of cells, washing the biochip from non-bonded cells, determination of coexpression of antigens on the bonded cells. The obtained result is assessed by determining presence of bonded cells in the region of the stain of the biochip and bonding density of cells and interpretation of the obtained result. Coexpression of antigens on cells bonded to the biochip is determined by carrying out one or more immunocytochemical reactions. When reading out the result, morphological analysis of cells bonded to the biochip is also carried out and presence and character of colouring of cells and their components with the reaction product are determined.
EFFECT: use of the disclosed method provides high reliability and information content of analysis.
9 cl, 6 dwg, 2 ex
SUBSTANCE: method provides contact of said cell with a peptide nucleic acid (PNA) molecule and a photosensitising agent and cell exposure to light at wave length effective to activate the photosensitising agent where said PNA molecule is conjugated with positive peptide. Also, there are described compositions containing such conjugated PNA molecules, the cells produced with the use of the method, and application of the method.
EFFECT: effective cell capture of peptide nucleic acid molecules conjugated with positive peptide.
37 cl, 13 dwg, 9 ex