| Brucellosis is a zoonotic disease caused by Brucella, which has been seriously harmful to both animal husbandry and human health. Brucella is widely distributed around the world and divided into seven main "species", including B. abortus, B. suis, B. melitensis, B.neotomae, B. ovis, B. canis and B.maris. Live attenuated vaccines immunization is one of most important measurment to prevent and control Brucellosis. Nowadays, foreign countries are widely using live attenuated vaccines strains S19, RB51and Rev.l. In China, strains BA19, S2, M5,104M are in use. Though the pathogenicity of the live vaccine strains has been highly attenuated, some strains still have virulence and infectivity to human and other animals.Conventional serological methods are unable to strictly discriminate immunity of the live attenuated vaccines and infection of field strains. Sometimes, this disadvantage makes confusion on accurate epidemiological surveillance and clinical diagnosis of brucellosis. In consideration of accurate surveillance and diagnosis of brucellosis, and also safety of public health, it has practical importance to establish methods to discriminate the live vaccine strains and field strains. According to different genetic characteristics of Brucella vaccine strains, this study established dual-PCR to B.suis vaccine strain S2, Real-time PCR to B.abortus vaccine strain BA19and B.melitensis vaccine strain M5.1. Establishment of dual-PCR for identification of Bsuis vaccine strain S2According to genome of B.suis vaccine strain S2in NCBI, a25bp deletion in IclR gene was blasted and found, which was specific to strain S2. Based on this deletion, a pair of specific primers was designed. Usage of above primers and B.suis biovar1specific primers from AMOS, a dual-PCR was established for detection strain S2. The standards of this dual-PCR were as following:when500bp and285bp bands were appearance at the same time, it was strain S2positive; otherwise, it was negatinve result meaned non-S2strain; when only285bp band was appearance, it was B.suis biovar1positive. The specificity experiments showed that only strain S2was positive result. Eighteen Brucella reference strains and four other live attenuated vaccine strains including BA19, S19, M5and104M were all negative results. Sensitivity of the dual-PCR was about30fg gemonic DNA equating to9cells.58domestic isolates of Brucella were tested. The results showed all these isolates were negative meaned non-S2strain, and12isolate were B.suis biovar1positive. Another panel including145serum samples and20milk samples were also identified. The results showed all these samples were negative meaned non-S2strain, and11samples were B.suis biovar1positive. We inferred that the dual-PCR for strain S2was sensitive, specific, rapid and accurate, which was suitable for discrimination of the strain S2and field strains.2. The establishment of cycling probes Real-time PCR for identification of B.abortus vaccine strain BA19and S19In China, B.abortus vaccine strain BA19homologied to S19is being used in Brucellosis imumunization. Based on SNP of S19CIpX gene verified by Whatmore, specific primers and probes were designed and a Real-time PCR was founded for identification of BA19. The standards of this Real-time were as following:when amplication curves were appearance before35cycles and RFU values of curves were greater than0.10at40cycles, it was typical amplification curve meaned Brucella positive; as detection with CIpX-P1probe marked with FAM, the typical amplification curve meaned strain BA19(or S19) positive; as detection with ClpX-P2probe marked with HEX, the typical amplification curve meaned the strain was Brucella but not BA19(or S19); if no amplification curve was found, it would be negative result meaned non-brucella strain. The specificity experiments showed that only strain BA19and S19got positive result. Eighteen Brucella reference strains and three other live attenuated vaccine strains including S2, M5and104M were identified as Brucella strains but not BA19(or S19), and the CT values ranged form11.21to25.62;4bactieral strains other from Brucella had no amplification cuvres.Sensitivity of the Real-time PCR was about60fg genome DNA equating to18cells. Repeatability of the Real-time PCR were tested by10-3～10-5dilution samples of BA19genomic DNA, and the result showed the variation coefficient was less than1%. Then, the Real-time PCR was examined by three larger panels.58domestic isolates of Brucella were tested. The results showed all these isolates were identified as Brucella strains but not BA19(or S19). With another panel,125serum samples collected from Brucellosis infected areas were tested.39samples were BA19(or S19) positive, and81samples were Brucella strains but not BA19(or S19), and5samples were negative meaned non-brucella strains.20serum samples and20milk samples from five non-vaccination cattle farms were identified as Brucella strains but not BA19(or S19). All the above results inferred that the Real-time PCR method for BA19and S19was sensitive, specific, rapid and repeatable, which was suitable for discrimination of BA19(or S19) immunization and field strains infection.3. The establishment of MGB probes Real-time PCR for identification of B.melitensis live vaccine strain M5and maternal strain M28According to genome of B. melitensis vaccine strain M5(maternal strain M28) in NCBI, a SNP site in glycosyltransferase gene was found. Based on this SNP, specific primers and probes were designed and a Real-time PCR was founded for identification of M5(or M28). The standards of this Real-time PCR were as following:when ampliation curves were appearance before35cycles and RFU values of curves were greater than0.03at40cycles, it was typical amplification curve meaned Brucella positive; as detection with M5G probe marked with VIC, the typical amplification curve meaned strain M5(or M28) positive; as detection with M5A probe marked with FAM, the typical amplification curve meaned the strain was Brucella but not M5(or M28); if no amplification curve was found, it would be negative result meaned non-brucella strain. The specificity experiments showed that only strain M5got positive result. Eighteen Brucella reference strains and four other live attenuated vaccine strains including S2, BA19, S19and104M were identified as Brucella strains but not M5(or M28), and the CT values ranged from13.03to28.06;4bactieral strains other from Brucella had no amplification cuvres.Sensitivity of the Real-time PCR was about300fg genomic DNA equating to90cells. Repeatability of the Real-time PCR were tested by10-1～10-3dilution samples of M5genomic DNA, and the result showed the variation coefficient was less than1%. Then, the Real-time PCR was examined by three larger panels.58domestic isolates of Brucella were tested. The results showed4isolates were M5(or M28) positive and others were Brucella strains but not M5(or M28). With another panel,125serum samples collected from Brucellosis infected areas were tested.15samples were M5(or M28) positive, and105samples were Brucella strains but not M5(or M28), and5samples were negative meaned non-brucella strains.20serum samples and20milk samples from five non-vaccination cattle farms were identified as Brucella strains but not M5(or M28). All the above results inferred that the Real-time PCR method for M5and M28was sensitive, specific, rapid and repeatable, which was suitable for discrimination of M5(or M28) and field strains. |
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