The method of detection of fusobacterium necrophorum in nested polymerase chain reaction

 

The invention relates to biotechnology, namely the genetic engineering of animals, and can be used in veterinary Microbiology to identify the causative agent of microbacteria ruminants Fusobacterium necrophorum and differentiating it from atypical forms of Fusobacterium pseudonecrophorum and other microflora. The method involves the synthesis of external 1 5'- AGT ATC TGA TTT TST ACG CC-3', 2 5' - CAG AAT HUNDRED ATA TTG TAC AC-3' and internal 01 5' - CAT GTA GAT GTC ATT GCG-3', 02 5' - ATT TCA AGA GTC CTT CCG-3' oligonucleotide primers, the synthesis of complementary DNA on the matrix chromosomal DNA, then the synthesis of external and then internal fragments. The amplification of the synthesized DNA is carried out by polymerase chain reaction (PCR) and analyze the size of the PCR products. The proposed method has high specificity and sensitivity and allows to differentiate Fusobacterium necrophorum isolated from the lesions, atypical forms and associated microflora. table 1.

The invention relates to biotechnology, namely the genetic engineering of animals, and can be used in veterinary Microbiology and BioIndustry to identify diseases of farm animals. To nastiashurigina examination (microscopy, culture medium, the bioassays in laboratory animals) [guidelines for the laboratory diagnosis of nitrobacteria. - M., 1987].

This diagnostic method has its drawbacks. So, from the lesions, along with Fusobacterium necrophorum with virulence, distinguish atypical forms of Fusobacterium pseudonecrophorum that never cause disease, and morphological and biochemical characteristics are very similar and associated flora, such as Staphylococcus, Streptococcus, micrococci, potato, Escherichia coli and other microorganisms, therefore, be isolated pure cultures is difficult. Laboratory animals (rabbits, white mice), along with the causative agent of microbacteria also sensitive and the accompanying microflora. In General, the diagnosis takes 12-16 days, and in case of significant contamination of biological samples vulgar microflora time of diagnosis increased by 6-10 days and this reduces the reliability of the diagnosis. The closest solution adopted for the prototype, is a method of detection of Fusobacterium necrophorum, based on polymerase chain reaction (PCR), including the selection of chromosomal DNA from infected tissues, amplificatoare with the help of the of Genta DNA using agarose gel electrophoresis (Electrophoretic mobility anomalies associated with PCR amplification of the intergenic spacer region between 16S and 23S ribosomal RNA genes of Fusobacterium necrophorum. SK.Narayanan, TG.Nagaraja, MM.Chengappa, GC.Stewart//J. Environ.Methods.-2001 (aug).-v.46.-.N2.-p.l65-169; Unexpected Cross-Reaction with Fusobacterium necrophorum in a PCR for Detection of Mycoplasmas: J. S. Jenson, B. Bruun, B. Gahrn-Hansen//J. Clin.Micob.-1999 (rt).-V. 37.- 3.-R. 828-829).

The disadvantages of this method include the fact that it is not possible to detect a significant part of virulent strains of the pathogen Fusobacterium necrophorum circulating among animals, because only one pair of primers. The technical purpose of our invention is to improve the specificity, sensitivity and the ability to differentiate from F. pseudonecrophorum and other gram-negative microflora present in the lesions. The invention consists in that at the first stage are selected and synthesized oligonucleotide primers, complementary to the region of greatest similarity between the genomes of different strains of F. necrophorum with virulence and that are different from the nucleotide sequences of DNA other related microorganisms. Then there is the amplification of chromosomal DNA by polymerase chain reaction using the aforementioned primers. To increase the sensitivity synthesized internal (nested, nested) primers and with them and the product of the first amplification Provo is desirous two oligonucleotide pairs of primers, for example: exterior 1 5'- AGT ATC TGA TSH TST ACG CC-3' 2 - 5' CAG AAT HUNDRED ATA TTG TAC AC-3' and internal 01 5'- CAT GTA GAT GTC ATT GCG-3' 02 5'- ATT TCA AGA GTC CTT CCG-3.

The primers are chosen in a region of chromosomal DNA, characteristic of virulent strains of F. necrophorum and absent in F. pseudonecrophorum and associated flora, such as Staphylococcus, Streptococcus, micrococci, potato, Escherichia coli. Primer 1 is used for the synthesis of the first chain complementary chromosomal DNA of F. necrophorum, primer 2 for synthesis of the second chain cDNA using the enzyme Tag polymerase. Then the two primers used for amplification of the selected area of the chromosomal DNA in polymerase chain reaction. Further to increase the sensitivity spend reamplification where primer 01 is used for the synthesis of the first chain complementary DNA fragment synthesized using primers 1 and 2, and primer 02 - for the synthesis of the second circuit internal fragment of the enzyme Tag polymerase. Then the two primers used for amplification of an internal fragment.

A significant difference of the proposed method from the known prototype is that as primers used nucleotide sequence that is the same for most virulent strains of F. nec is Teya and other related microorganisms. Thus, the claimed method meets the criterion of "novelty". New signs of technical decisions on the totality of symptoms achieve the goal of the invention: detected a larger number of strains of F. necrophorum and differentiation. Therefore, we can assume the claimed technical solution meets the criterion "inventive level".

An object of the invention is to increase the specificity and the ability to differentiate F. necrophorom from F. pseudonecrophorum and other microorganisms without reducing the sensitivity of the method of detection.

This goal is achieved by the selection and synthesis of two paragonroulette primers: exterior 1 5'- AGT ATC TGA TTTTCT ACG CC-3' and 2 5'- CAG AAT HUNDRED ATA TTG TAC AC-3', synthesizing the fragment 546 NP. and internal 01 5'- CAT GTA GAT GTC ATT GCG-3' 02 5'- ATT TCA AGA GTC CTT CCG-3', synthesizing the fragment within the first 187 NP. Primers were selected in the General area for the genomes of virulent strains of F. necrophorum, but differs from the nucleotide sequences of the chromosomal DNA of F. pseudonecrophorum and associated microflora. The first pair of primers used for synthesis and amplification of a larger region of the genome of the bacteria in the polymerase chain reaction, after 29 cycles when the following is 73oC for 1 min and one cycle (docentes) at 73oC for 4 minutes, the Second pair for the synthesis of smaller fragments, structurally included in the greater, and its amplification in polymerase chain reaction mode: denaturation at 95oWith over 0,8 min, hybridization - with 48oWith over 0,8 min, synthesis - by 73oC for 1 min, 29 cycles + 1 cycle docentes at 73oC for 4 minutes About the positive result of the analysis to be judged by the size of the synthesized cDNA fragment migrating 0.8% agarose gel.

A significant difference of the proposed method from the known prototype is that as primers used nucleotide sequence that is the same for most virulent strains of F. necrophorum and with the greatest differences from DNA sequences F. pseudonecrophorum and other microorganisms.

The essence of the technical solutions disclosed in the specific examples of the method.

Example. Operation 1. Isolation of the pathogen from pathological material. A sample of pathological material 500-600 mcg pound in a mortar with glass powder (100-200 µg) and add 1000 ál of 0.9% NaCl, stirred and centrifuged 8000 rpm for 8 min in a centrifuge T23D in the angular rotor. NEMA 2 ml.

Step 2. Isolation of the pathogen from pathological material. Make the suspension 1: 10 sample of pathological material from the BCH or 0.9% NaCl, carry out sowing in the BCH by adding 10% of aminacrine, cultivated in an incubator at 37oC for 1 day. 10 ml daily culture centrifuged in a centrifuge T23D in the angular rotor at 8000 rpm for 8 minutes. The supernatant discarded and the sediment resuspended in 500 ál of TE buffer and transferred to another test tube with a volume of 2 ml.

Step 3. In the test tube after the operation 1 or 2, add 150 ál of lysozyme (20 mg/ml), mixed and placed on ice for 25 minutes Then add 65 ál of 10% SDS and 100 μl of pronase (20 mg/ml), mixed and incubated at +55oWith 15-18 hours. Then add an equal volume mixture of phenol/chloroform/isoamyl alcohol (25: 25:0.5), and gently pipeinput and centrifuged 10 minutes at 14 thousand Rev/min the Aqueous phase is transferred into another test tube and poured it 1/2 volume mixture of chloroform: isoamyl alcohol (25:0,5), centrifuged and the aqueous phase is transferred into another tube. To the aqueous phase add 1/10 volume of 3 M sodium acetate pH 4.8 and 2 volumes of ethanol and placed in 23 hours and 20oC. Centrifuged 15 min at 14 thousand rpm in a centrifuge MPW 310, the precipitate is washed with 70% this is ilen bidistilled water or TE buffer pH 8.0.

Step 4. Polymerase chain reaction. 1 μl of the solution containing the chromosomal DNA of F. necrophorum, added to 24 μl of a solution containing 67 mm Tris-HCl, pH 8.8; 15 mm (NH)SO, 3 mm MgCL, in 0.01% TWeen-20; 0.2 mm each of the four detoxicification, 2 units of Taq polymerase and 200 ng of primers 1 and 2. Over reaction mixture layer 30 ál of mineral oil. PCR consists of 29 cycles: 95oWith over 0,8 min, 56oWith over 0,8 min, 73oC for 1 min and a final synthesis at 73oC for 4 minutes

Operation 5. Determine the size of PCR products. a 12.5 μl of a solution of PCR products mixed with 3 µl of a solution containing 50% glycerol and bromophenol blue, and put in the "pocket" 0.8% agarose gel. The molecular weight marker consists of products of DNA cleavage of plasmid pUC18 DNA restriction endonuclease Alul. The analysis considered positive if the PCR product corresponding to the expected size of the fragment in 546 base pairs. In the absence of the calculated fragment surgery is performed 6.

Step 6. In the absence of the calculated fragment 546 NP. take 1-2 ál of the reaction mixture and have reamplification with the second pair of primers 01 - 02. The reaction mixture composition and mode amplifica. The analysis considered positive if the PCR product corresponding to the expected size of the fragment in 187 nucleotide pairs.

The experimental results show that the positive analysis of PCR products receive only when as a matrix using the DNA of the pathogen microbacteria. The tests were negative when used DNA F. pseudonecrophorum and other bacteria (Staphylococcus, Streptococcus, E., hay Bacillus, Proteus). The sensitivity of the proposed method of detecting chromosomal DNA of F. necrophorum is quite high and is 1 PG (see table).

Claims

The method of detection of Fusobacterium necrophorum in nested polymerase chain reaction (PCR), comprising the synthesis of oligonucleotide primers, the synthesis of complementary DNA on the matrix chromosomal DNA and amplification of the synthesized complementary DNA by PCR using oligonucleotide primers followed by size analysis of PCR products, characterized in that synthesize two pairs of oligonucleotide primers - exterior 1 and 2 for the synthesis of a DNA fragment in 546 nucleotide pairs and internal (female) 01 and 02 for the synthesis of fragment in 187 nucleotide pairs with the following GTC CTT CCG-3'; and identify Fusobacterium necrophorum, if the PCR product corresponds to the fragment size in 546 or 187 nucleotide pairs.

 

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