The Development Of Cycling Probe Real-time PCR Assay For Brucella Identification And Discrimination To The Species Level And Its Visual Detection By Loop-mediated Isothermal Amplification With Hydroxynaphthol Blue

Brucellosis is an important anthropozoonosis caused by a number of species of the genus Brucella, which has global implications both economically and socially. The genus Brucella consists of many species and various biotypes that can infect a variety of animal species. Differentiation to species level within the genus is based on both different phenotypic characteristics and the host from which it was initially isolated. These divisions are also supported by molecular biology, which can use suitably validated genetic markers for objective and unambiguous identification of groups. This paper established a cycling probe real-time PCR to quickly identify seven major Brucella based on the single nucleotide polymorphism (SNP) sites in different kinds of target genes, which cloud be used for Brucella species high-throughput identification. At the same time, a visualization loop-mediated isothermal amplification (LAMP) assay was also developed for diagnosis of Brucella in our country and provide an effective technique for purification and control the brucellosis.1. The establishment and application of a cycling probe real-time PCR assay for detection of Brucella spp. A cycling real-time PCR assay was developed for detection of all species and bivors of Brucella. According to the sequences of BCSP31gene, a set of probe and primers was designed. Meanwhile, a recombinant plasmid was constructed with a pair of outer primers based on BCSP31gene. The sensitivity and standard curve of the cycling real-time PCR assay were tested with ten fold serial dilution of the recombinant plasmid. The results showed that the detection limit of the assay was about10copies and the standard curve was favorable with0.999linear correlation coefficient. The specificity was perfect with typical "S" curve amplification of Brucella and negative results for other bacterias including Escherichia coli K99. Pasteurella multocida C48-1, Streptococcus ST171and Pseudomonas aeruginosa within40cycles. Furthermore, the cycling probe real-time PCR assay was evaluated by40clinical samples (blood, milk and serum)from brucellosis epidemic areas, and the results showed35positive samples that include all27B4/B5-PCR positive samples. The coincidence rate of two methods was80.0%. All results suggested that the cycling probe real-time PCR assay was specific, sensitive and steady, which could be used to diagnosis of clinical samples and investigation of epidemic outbreak.2. Development of seven cycling probe real-time PCR assays for identification and discrimination Brucella at the species level.The aim of this work was to use the SNP site in different target genes of Brucella provided by existing MLST studies to develop a rapid, unambiguous assay for the real-time PCR platform capable of identifying Brucella isolates to species level. According to the SNP sites in different kinds of target genes of B.menlitensis、 B.abortus、B.suis、B.canis、B.ovis、B.neotomae and B.maris, seven species specific cycling probes were designed. The genomic DNA of five different Brucella reference strains(Menlitensis、Abortus、Suis、Canis and Ovis) and the two recombinant plasmids contain PO gene of B.neomae and trpE gene of B.maris were tested and verified respectively. All results showed that each specific probe detected only a particular species of Brucella (except B. suis), and the detection limit of these assays was about10～100fg(2.8～28copies) Brucella genomic DNA. The specificity and the sensitivity of cycling probe real-time PCR assays for identification Brucella at the species level were perfect, and it can be used for unknown Brucella strains rapid identification and pathogen outbreak investigation.3. Visual Detection of Brucella by Loop-Mediated Isothermal Amplification with Hydroxynaphthol Blue Dye.In this study, a loop-mediated isothermal amplification (LAMP) assay targeting of the omp25gene for rapid visual detection of Brucella was developed. After a set of LAMP primers were selected, the reation system(ex. Mg2+, Betaine, dNTP and Bst DNA polymerase) were optimized using Brucella DNA as templates. The results showed the optimal reaction components in the LAMP were0.2μM F3/B3,1.2μM FIP/BIP,0.8μM LF/LB,10μM MgSO4,0.8M Betaine,1.5mM dNTPs and8U Bst DNA polymerase, and finished in60min under isothermal condition at63℃. The reaction system was preadded HNB with the final concentration of120μM. A positive reaction is indicated by a color change from violet to sky blue. The detection limit of the visual LAMP assay was17fg Brucella genomic DNA, which was similar to that of electrophoresis results, and the specificity of the LAMP assay showed there was no cross reactivity with other related bacterias. Furthermore, the LAMP assay was evaluated using40clinical samples from chapter one, and the results indicated the positive samples were31, with the coincidence rate of85.0%to PCR and90.0%to cycling probe real-time PCR.