[Taq Man probe-based quadruple real-time PCR for detection of Salmonella paratyphi A/B/C and Salmonella typhi].

OBJECTIVE: Establishment and application of Taq Man probe-based quadruple real-time PCR for detection of Salmonella paratyphi A/B/C and Salmonella typhi. Primers specific to Salmonella paratyphi A( SPAP), Salmonella paratyphi B( SPBP), Salmonella paratyphi C( SPCP), and Salmonella typhi( STP) were designed. METHODS: A method of Taq Man probe-based quadruple real-time PCR was established according to the distinction of the 5'end of the probe mark of TET, ROX, FAM and HEX. 5 strains of SPA, 4 strains of SPB, 7 strains of SPC and 11 strains of ST were identified by amplification from SPAP, SPBP, SPCP and STP. RESULTS: While other serotypes of salmonella and17 strains of non-salmonella got negative results of amplification. Amplification rate of SPAP, SPBP, SPCP, and STP were 84. 5%, 101. 8%, 92. 4% and 90. 9%, respectively. The linear correlation coefficient( R~2) were 0. 996, 0. 975, 0. 996 and 0. 984, respectively. CONCLUSION: The PCR system is specific and sensitive for the identification of SPA, SPB, SPC and ST.

[Detection of foodborne Salmonella typhimurium and Salmonella enteritidis by diplex real-time PCR using TaqMan probe].

OBJECTIVE: To establish a method to detect Salmonella typhimurium (ST) and Salmonella enteritidis (SE) simultaneously with a dual real-time PCR assay using double-color fluorescent TaqMan probes. METHODS: The primers and probes were designed based on the conservative domain of STM4599 sequence of ST (GenBank: AERV01000023.1) and the specific sequence of SE ( GenBank: AF370707.1) respectively. The probes were labeled with reporter dye FAM for ST or VIC for SE at the 5' end. The dual real-time fluorescence PCR assay was set up and conditions were modified. RESULTS: The dual real-time fluorescence PCR method for ST and SE was developed successfully. ST and SE specific primers and probes amplified 16 SE and 15 ST strains, while other 28 different Sa serotypes and 17 negative control Proteus strains showed negative results. The amplification efficiency of ST and SE with the dual fluorescent PCR were all 94. 2% and R2 were 0. 998 and 0. 995 respectively, while the minimum detectable concentration reached 300 CFU/ml for ST and 260 CFU/ml for SE. The entire test can be completed within 31 hours. CONCLUSION: The method is highly specific, sensitive, and fast. The present study thus provides a rapid and effective method to detect ST and SE simultaneously from food samples.

[Typing of Listeria monocytogenes in import and export foods with PFGE].

OBJECTIVE: To investigate the molecular type and genetic correlation of Listeria monocytogenes (LMO) isolated from imported and exported foods. METHODS: Pulsed-field gel electrophoresis (PFGE) standard method recommended by american PulseNet for typing LMO was applied. The chromosomal DNA of L. monocytogenes was digested by restriction enzyme Asc I and analyzed by PFGE. The PFGE patterns of L. monocytogenes strains isolated from different areas were compared by using BioNumberics software to analyze the similarity between strains. RESULTS: Twenty-four L. monocytogenes strains were grouped into 20 PFGE subtypes by the similarity of PFGE pattern ranged from 44.45% to 100%. Particularly, it was identified that there were three subtype strain groups, each group sharing with the same PFGE pattern, and the similarity reaching 100%: the first group including LMO16 from Canada, LMO18 from America and LMO13 from Denmark, the second group including LMO7 from Denmark and LMO22 from Shunde, and the third group including LMO21 from Guangzhou and LMO8 from Shanxi. CONCLUSION: Generally, much genetic diversity was shown in Listeria monocytogenes isolated from import and export foods, but the correlations among strains are existed to some extent.

[Research on detection of Vibrio comma and Vibro parahaemolyticus from foodstuffs using MPCR methods].

OBJECTIVE: To establish a set of multi-PCR (MPCR) methods to detect Vibrio comma O1 serogroup (EVC) and O139 serogroup, and Vibrio parahaemolyticus rapidly and sensitively from foodstuffs. METHODS: Using T139 (specific gene of O139 serogroup), ctxB and tcpA genes from V. comma, and tdh gene from V. parahaemolyticus as target sequences, we detected the anticipative amplified bands, whose sizes were relatively 417bp, 564bp, 471bp and 202bp. RESULTS: Excellent specificity of the amplified products could be found from both standard and wild strains of EVC, O139VC and V. parahaemolyticus. It also means that no amplified band was detected from total 35 strains of other bacilli, including salmonella, comma bacillus which do not belong to O1 and O139 serogroups. The detection limits of artificial contaminated samples such as tilapia flesh, oyster and mixture of tilapia intestines and gills were proved to be 22cfu/g in EVC, 50cfu/g in O139 and 65cfu/g in V. parahaemolyticus. Besides, it took only 8-10 hours to finish the whole process. CONCLUSION: Experiment results show that MPCR is a sensitive, convenient and time saving method suitable for detection.