Objective To establish a rapid,sensitive and specific assay based on real-time PCR combined with reverse transcription for detecting and identifying viable Listeria monocytogenes.Methods The hlyA gene of Listeria monocytogenes was chosen as target,and then the primers and TaqMan probe were designed.Both ends of probe were modified with two different fluorescence groups.The PCR reaction was optimized systematically.The mRNA of Listeria monocytogenes was extracted,and then reverse transcription was performed through random primer.The cDNA Was detected by real-time PCR.Then the specificity,sensitivity and reproducibility of real-time PCR were estimated.In final,real-time PCR was applied to detect 20 mocked double-blind samplea.Results Viable Listeria monocytogenes were detected by real-time PCR accurately and quickly,and meanwhile,none of other bacteria and non-viable Listeria monocytogenes could be identified.The sensitivity was 10 CFU/ml in pure culture and 103CFU/ml for mocked samples respectively.The coefficient of variation of intra-assay and inter-assay Was less than 5%.When this assay was applied directly to identify 20 mocked double-blind samples,10 of these were positive to viable Listeria monocytogenes,5 were negative to non-viable Listeria monocytogenes,and 5 were negative to other pathogens.Conclusion It is demonstrated that real-time PCR is a reliable,accurate and feasible assay for viable Listeria monocytogenes.The establishment of this assay provided complete data for analysis and diagnosis in the field of food safety and epidemiologic survey.

Objective To develop a loop-mediated isothermal amplification method for rapid diag-nosing Vibrio cholerae O139 in inspection department and small-scale laboratory. Methods Four primers (2 inner primer and 2 outer primer) for the LAMP test were designed by targeting the wbfR gene of Vibrio chol-erae O139, and the reaction condition and reaction system of LAMP were optimized. Thirty Vibrio cholerae O139, 13 Vibrio cholerae reference strains, 10 O1 biotype Vibrio cholera* and 32 other enterobacterias were analyzed and the LAMP results were determined by visual inspection or electrophoretie analysis . Results All of the Vibrio cholerae O139's amplification products were observed as green by visual inspection and had a ladder-like pattern on the gel, but O1 biotype Vibrio cholera* and other enterobacteria's products were dis-played as orange by visual examination and had no ladder-like pattern on the gel. In addition, the reaction time of the LAMP method was only 1.5 h and the detection limit of this assay was 63 CFU/reaction. Con-clnsion LAMP assay targeting the wbfR gene of Vibrio cholera* O139 is rapid, specific, and sensitive for the detection of Vibrio cholerae O139. This method not only reduced the dependence of complicated equip-ment but also had a potential for wider use in inspection department, small-scale laboratory, emergency mo-tor vehicles and field survey.

Objective:To determine the molecular characteristics of Streptococcus suis type 2 (SS2) in Zhejiang Province. Methods:Twenty-nine SS2 sporadic human isolates in Zhejiang Province from Januery 2005 to July 2021 were genotyped by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and minimum core genome (MCG) sequence typing.Results:Among 29 strains, 10 PFGE patterns and three main clusters were obtained by PFGE. Twenty-one (72.41%) of the strains were divided into two main branch groups and the remaining eight (27.59%) showed genetic diversity with the similarity ranging from 49.7% to 94.7%. Three sequence types were obtained from 29 strains by MLST, including ST7 (86.21%(25/29)), ST1 (10.34%(3/29)) and ST25 (3.45%(1/29)). In addition, three genotypes were obtained from 29 strains by MCG, including genotype E (41.38%(12/29)), genotype group 1 (55.17%(16/29)) and genotype group 4 (3.45%(1/29)).Conclusions:Two large clonal groups of highly pathogenic strains of SS2 have been prevalent in Zhejiang Province. A few strains display genetic diversity, indicating genetic variation may exist during transmission.

Objective:To understand the in vitro antimicrobial resistance and resistance phenotypes profile of Streptococcus suis strains isolated from human in Zhejiang Province. Methods:The strains of sporadic Streptococcus suis infections were isolated during 2005 to 2021 in Zhejiang Province, and were subjected to antimicrobial resistance analysis using agar dilution method. Polymerase chain reaction (PCR) technology was also used to detect 70 resistance genes including tetracyclines, macrolides and aminoglycosides. Results:The results of antimicrobial resistance analysis showed that these strains were sensitive to eleven kinds of antimicrobial agents with a sensitivity rate ≥96.8%, including cefepime, cefotaxime, ceftriaxone, chloramphenicol, daptomycin, ertapenem, levofloxacin, linezolid, meropenem, penicillin and vancomycin. These strains were mainly resistant to tetracycline, clindamycin, azithromycin and erythromycin, especially resistant to tetracycline with a rate of 93.5%(29/31). Fourteen strains (45.2%) exhibited multidrug resistant patterns. The PCR analysis of 70 drug resistance genes showed that 14(20.0%) different resistance genes were detected. The highest detection rate of resistant genes came from tetracycline, including tet ( O) gene (58.1%, 18/31), tet ( M) gene (48.4%, 15/31), tet ( 40) gene (35.5%, 11/31), followed by ermB gene (41.9%, 13/31) in the class of macrolide. Fourteen strains (45.2%) with more than three drug resistance genes were detected, of which eight strains (25.8%) detected 10 drug resistance genes. The analysis of antibiotic resistance and resistance phenotypes profile showed that tet ( M)+ ST7 accounted for 35.5%(11/31), tet( O)+ tet( 40)+ ermB+ mef( A)+ mef( A/ E)+ msrD+ Ant( 6)- Ⅰb+ aph( 3′)- Ⅲa+ aadB+ sat4+ ST7 accounted for 25.8%(8/31). Conclusions:The antimicrobial resistance and resistance phenotypes profile of sporadic Streptococcus suis strains isolated from human in Zhejiang Province are endemic, with mainly two types of characteristic genetic cloning of drug resistance genes.