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173
UDC 619:578.832.1
IDENTIFICATION OF BORDETELLA BRONCHISEPTICA BACTERIA WITH THE HELP OF POLYMERASE CHAIN REACTION IN MONO- AND MULTYPLEX FORMAT
Vasylyeva Y.B., Candidate of Veterinary Science Ulyanovsk State Agricultural Academy named after P.A. Stolypin, Ulyanovsk, Russia
E-mail: grant-ugsha@yandex.ru
ABSTRACT
The article gives the results of the original research aimed at working out molecular-genetic methods for the identification of Bordetella bronchiseptica. The parameters for conducting polymerase chain reaction (PCR) with the worked out systems of primers within using monoplex and multiplex format are suggested. The optimal methodology of singling out B. bronchiseptica DNA is selected; the main parameters of the reaction are validated. The research was performed with the financial help of the government represented by the Ministry of Science and Education of the Russian Federation within the implementation of the Federal Target Project “Scientific and Scientific-Pedagogical Staff of the Innovative Russia” for the years 2009-2013 (agreement № 8267 from 10.08.2012).
KEY WORDS
Bordetella bronchiseptica; Bordetellosis; Polymerase chain reaction.
One of the most important points in the process of bordetellosis diagnostics is the impossibility of fast detection of the agent within the bacteriological method and the difficulty of biochemical and serological identification of Bordetella bronchiseptica (B.bronchiseptica). This determines the necessity of working out alternative fast and effective diagnostic ways that can help to accurately detect the agent of bordetellosis.
Laboratory diagnostics based on molecular-genetic research makes it possible to detect specific genetic “targets” in short periods of time and with bigger accuracy in comparison with phenotypic methods of agent identification [1].
One of the key points of working out accelerated molecular-genetic methods of diagnostics is the possibility of detecting agents in the shortest possible time directly from the biomaterial without the stage of getting the “pure” culture [1].
Notwithstanding the fact that molecular-diagnostics method with the PCR for the identification of Bordetella was first described in 1990, there is still much disagreement among scientists in the problem of choosing agent genome fragment as the target, the methods of isolation nucleic acid and the techniques of detection the amplificated fragment and the ways of conducting polymerase chain reaction [3, 4].
Concerning the choice of the targets for the amplification of the genome fragment, the scientists mostly use conservative gene parts, responsible for the expression of bordetella pathogen factors: promotor fragment of pertussis toxin; DNA fragments of porin gene, conservative fragments of adenylate cyclase-hemolysin gene. The test-systems based on these research show rapid response (80-100%), though in some cases the DNA of B.bronchiseptica cannot be differentiated from the DNA of B.parapertussis and B.pertussis that have a high level of homology of the initial targets.
Thus nowadays there are no standardized primer systems that make it possible to conduct accurate intrageneric identification of bordetella.
The analysis of literature showed the urgent necessity of perfecting the methodology of PCR in the monoplex format and constructing multiplex test-systems of B.bronchiseptica identification.
Thus our research is aimed at working out the molecular-genetic methods of B.bronchiseptica identification.
THE MATERIALS AND METHODS OF STUDY
The research was performed on the basis of research-and-development innovative center of microbiology and biotechnology (RDICMB) of FSBEI HPE «Ulyanovsk State Agricultural Academy Named after P.A. Stolypin» and LLC «Medical Center «Akademya» (Ulyanovsk) together with scientific associate A.V.Mastylenko.
The objects of the research consisted in 5 reference strains of B.bmnchiseptica, 25 reference strains of homological genera and species of the bacteria that have common phenotypical signs (biochemical activity, antigen characteristics, etc.), closely related species (B.parapertussis) and possible associates (Pseudomonas spp.), taken from the museum of RDICMB, and 52 strains of B.bronchiseptica isolated from the clinical samples of the biomaterial.
The primers were synthesized in NPF «Lytex» and LLC «Syntol».
The molecular-genetic methods of research included selection of unique primer systems, isolation of nucleic acids (DNA) from cultures, conducting PCR, detection with the help of horizontal electrophoresis [2-3]. The Statistical methods included evaluation of susceptibility and specificity of PCR according to A. Petry and C. Sabin [4].
RESULTS AND DISCUSSION
While working out the system of primers for PCR we performed the study of the genomes of B.bronchiseptica strains for presence of the unique genes - candidates for molecular-genetic identification of the agent. Two unique genes bfrA and bfrZ of the system of TonB-complex of external membrane protein - iron receptors B.bronchiseptica were found. When checked, the genes confirmed their species specificity.
For making faster response of the molecular-genetic diagnostics of the agent within SNP-substitutions in the unique genes bfrA and bfrZ or non-specific anneal of the corresponding primers target-genes were selected for generic typing. They were the gene coding the enzyme cytochrome-c-oxidase (ccox) and the gene 16S of the subunit of bacterial ribosomes (16S rRNA).
After selecting specific gene-candidates for molecular-genetic identification of B.bronchiseptica the most conservative fragments of the selected targets were distinguished by means of their comparison within different strains in the database of GeneBank. The primers meeting the demanded criteria were put on them: the length of the primers must make 20-32 nucleotide pairs, the melting temperature of the primer - 60-70°C, the size of the flanked by primers gene fragment - not less than 100 and not more than 1000 b.p. (table 1).
Table 1 - Characteristics of Primer Systems
Parameters The fragment of B.bronchiseptica gene
bfrA bfrZ ccox 16SrRNA
Direct primer (f) 5'-3' CCTTCCAGCACCTGGC GGTACGAGTTGCTCC GGACGACCAGG AT CACAT CTT CC GCATTGCTCCATC CTGTTGTGCG CTACGGGGGAAAG CGGGGGA
Reverse primer (r) 5'-3' CCCCGTGCCGGGGTGC CTGGACCTGGGCG GCTTTCCTGGTA GTTGGCGTAGG GATGGGTTATCTG AGCGCGC GACCGT ACT CCCC AGGCGGT
Rated melting temperature of direct and reverse primers 62,0C 62,0C 62,0C 62,0C
Theoretical specificity B.bronchiseptica B.bronchiseptica B.bronchiseptica, B. pertussis, B. parapertussis B.bronchiseptica, B. pertussis, B. parapertussis, B. avium, B. hinzii, B. holmseii i/i B. trematum
Length of the specific amplicon (b.p.) 528 298 168 711
The primers were leveled by the program Gene Runner Version 3.05, the dimers were defined at their possible non complementary linking one with itself or in pairs.
One of the important stages in the practice of PCR conducting is preparation of suspension of bacterial mass or clinical biomaterial for the study - isolating the DNA from the bacterial cell. For this purpose were tested several technologies of purifying nucleic acids from enzymes, proteins and ions that can significantly harden the reaction and in some cases even inhibit acting of Taq -DNA-polymerase. Sorbent method was tested with using guanidinium thiocyanate as a chaotropic agent, phenolic-chloromorph extraction of the DNA, thermal lysis and the following deposition of the cellular detritus. The experiments proved that using sorbent gives the best yield of the matrix DNA.
Then optimization of PCR rates was conducted. On the basis of the primers’ melting temperatures the temperature of primer anneal was selected for constructing universal PCR protocol. This saves work resource of the amplifier, shortens the time of the analysis and enables mass research. The most optimal temperature turned out to be 62°C, when the maximal amplification product was observed and the DNA of all the strains in 1000-multiple dilution was found.
Experiments enabled to select optimal in terms of the efficiency of the PCR universal concentrations of the primers. We took into account the fact that paucity of primers can lead to the emergence of longer (more than 1000-2000 b.p.) amplicons. This happens because on each cycle primers get annealed in the matrix and do not take part in the reaction any more, because they make basis of Taq-DNA-polymerase work and enter into the structure of amplicons. When lack of restricting primers takes place and there is enough initial amount of the DNA matrix of B.bronchiseptica, Taq-DNA-polymerase works without restricting the fragment, as much as its activity permits. When the concentration is too big, the primers make dimers annealing one on itself or in pairs and composing no longer than 100 b.p. amplicons. However one should point out that dimers usually begin getting constructed at the final cycles of the reaction.
The optimal universal concentration of each of the primers was 5 pmol per reaction the amount of 25 mcl.
Thus the test system for PCR conducting included the sets of reagents for isolating DNA from bioprobes - "Proba GS”, for amplification of DNA fragment and detection of DNA with the method of gel electrophoresis
For detecting B.bronchiseptica DNA fragment daily cultures obtained through cultivation at 37°C 24-48 h. on beef-peptonic broth and selective diagnostic medium BBR-57 UGSHA were used. The probes of the clinical material (cellular throat swabs and scrapes) were taken from cats and dogs with sterile spatulas and endocervical PCR brushes. Separate culture colonies from the medium BBR-57 UGSHA, wire inoculating loop with broth culture or swabs and scrapes of the clinical material directly from animals were put into 0,5 ml of sterile saline that had been poured beforehand into micro test tubes the type of «Eppendorf» (1,5-1,7 ml) in sterile laminar PCR isolation ward. After that all the probes were delivered to the PCR-laboratory in a thermocontainer.
After DNA isolation the program demonstrated in table 2 was entered on the amplifier.
Table 2 - Program for DNA Amplification
For amplifiers with active regulation (by solution in the test tube) «Tercyk» (NPF «DNK-Technologya», Russia)
№ cycle Step Temperature Duration Number of repetition
1 1 95"C 5 min 1
1 95"C 10 sec
2 2 62C 10 sec 40
3 72"C 20 sec
3 1 72"C 2 min 1
When the reaction was over (on the day of the study or after storing in the morning of the next day) the analysis of PCR products was conducted with the method of horizontal electrophoretic separation of DNA fragments in agarose gel. When the specificity was being detected, all the pairs of the strains were checked in the PCR on field strains of B.bronchiseptica. All the studied agent strains showed positive results.
Since the problem of shortening the price and time of PCR conducting in "line-rate” is
urgent, we revealed the necessity of working out multiplex PCR system for screening animal tests of bordetellosis.
For the multicomponent PCR format it was decided to use 2 systems of primers for bfrA and ccox gene fragments and 3 systems of primers for bfrA, bfrZ and ccox gene fragments. The amount and correlation of primers were selected experimentally: 5 pmol primers of the system for bfrA and bfrZ gene fragments and 15 pmol for ccox gene fragments. We should also state that the universal amplification program stated above was used. The program took 70 minutes on thermal cycler (pict. 1).
Thus, the multiplex PCR format enables to quickly detect the existence of DNA fragment that is specific for the three specimens of Bordetella genus, and isolate B.bronchiseptica DNA fragment separately.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
M
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
m
Picture 1 - Electrophoregram of the multiplex PCR:
1-15 - using the primers for bfrA+bfrZ+ ccox gene fragments; 16-30 - using the primers for bfrA+ ccox gene fragments 1-13, 16-28 - field strains of B.bronchiseptica; 14, 29 - negative check-outs; 15, 30 - molecular weight
marker
PCR-testing sensitivity with the selected primer systems was detected: for bfrA and bfrZ, ccox gene fragments it was 5,5x103 bacterial cells /ml; for 16S rRNA gene fragments -5,5x102 bacterial cells/ml.
CONCLUSION
In the result of the experiments conducted, high efficiency of the worked-out primer systems for B.bronchiseptica identification was proved. For typing the agent of bordetellosis we recommend conducting PCR with the worked-out systems of primers in mono- and multiplex format.
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4. Petri A. Naglyadnaya Statistika v Meditsyne / A. Petry, C. Sabin. - M.: Geoter-med, 2003. -139 p.
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