Objective: To establish a rapid GeXP-based multiplex reverse transcription-PCR assay (GeXP assay) for simultaneous detection of 5 subtypes of diarrheogenic Escherichia coli. Methods: Specific primers were designed according to reserved sequences of 12 virulence genes in enterotoxigenic E. coli (ETEC), enterinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (ETEC), and PCR amplification was performed with a single pair of primers to validate the specificity of PCR assay with a single template and a single pair of primers. The specificity of the GeXP assay was evaluated with the genomic DNA of 5 subtypes of diarrheogenic E. coli as the template in a mixture of 12 pairs of primers, and the sensitivity of the GeXP assay was evaluated with the mixed suspensions of 5 subtypes of diarrheogenic E. coli at concentrations of 106, 105, 104 and 103 CFU/mL as the template. Foods purchased from supermarkets and agricultural retail markets were prepared into 34 spiked samples, and 5 subtypes of diarrheogenic E. coli were detected using the GeXP assay and compared with the fluorescent real-time multiple PCR assay. Results: The sizes of sth, pic, bfpB, astA, lt, escV, aggR, stx1, uidA, invE, stx2 and stp genes amplification products were consistent with expected sizes using a single template and a single pair of primers, with a fluorescent signal intensity of more than 25 000 A.U. The sizes of the GeXP assay amplification products of 12 virulence genes in 5 subtypes of diarrheogenic E. coli were consistent with expected sizes, with a high specificity. If the concentration of the mixed suspensions of 5 subtypes of diarrheogenic E. coli was 103 CFU/mL, the GeXP assay was effective for simultaneous detection of 12 virulence genes, with a high fluorescent signal intensity, consistent repeated detection results and a less than 10% coefficient of variation. The GeXP assay detected 3 ETEC isolates, 12 EAEC isolates, one EIEC isolate, one EPEC isolate and one EHEC isolate among the 34 spiked samples, which was in agreement with the detection of 5 subtypes of diarrheogenic E. coli with commercial fluorescent real-time multiple PCR assay kits. Conclusions A GeXP assay has been successfully established for simultaneous detection of 12 virulence genes in diarrheogenic E. coli, which is effective for clinical differential diagnosis and epidemiological surveys of diarrheogenic E. coli.

Objective: To investigate the contamination status and assess the potential consumption risk of Listeria monocytogenes ( L. monocytogenes ) in cooked meat products in bulk in Zhejiang Province, so as to provide strategy for food safety supervision and management. Methods: A total of 2 320 cooked meat products were sampled from eleven cities in Zhejiang Province during 2018-2020. The detection of L. monocytogenes was carried out in accordance with the national standard GB/T 4789.30-2016. Risk Ranger software was used for the semi-quantitative risk assessment on the whole population and pregnant women. Results: The total detection rate of L. monocytogenes in cooked meat products in bulk in Zhejiang Province was 2.97% ( 69/2 320 ). The detection rates in stewed, smoked/roasted, fried, dried products and others were 3.85%, 1.81%, 0.59%, 0% and 0.94%, which were significantly different ( P<0.05 ). There were 28 positive samples in 1 069 samples collected in 2020, with the concentration ranging from 5 to 590 CFU/g and averaging 6.8 CFU/g. The estimated number of listeriosis cases each year caused by consumption of cooked meat products in bulk was 131 in the whole population with a risk score of 42, and 1.44 in pregnant women with a risk score of 54. The risk coefficient could reduce to approximate zero after sufficient heating before intake. Conclusion The prevalence of L. monocytogenes in cooked meat products in bulk in Zhejiang Province during 2018-2020 poses a potential risk in food safety. Pregnant women should avoid eating.

Objective: To establish real-time recombinase polymerase amplification（RPA）for the rapid detection of Vibrio parahaemolyticus（VP）. Methods: An exo probe and primers were designed according to the conserved sequence of thermolabile hemolysin（tlh）gene of VP and then RPA for detection of VP was established. The sensitivity of the assay was evaluated by detecting different concentration of VP;the specificity was evaluated by detecting different bacteria;the stability was evaluated by repeat trials;the application effect was evaluated by detecting food samples which were simultaneously tested with traditional culture method according to GB 4798.7-2013 Detection of VP. Results: A real-time RPA was established to complete VP amplification within 20 min at a constant temperature of 39 ℃. The analytical sensitivity of the assay was five pg per reaction and no cross-reactivity with other pathogenic bacteria observed. The RPA detection results with different concentration of VP and E. coli DNA templates at three time points were consistent. The detection results of 51 food samples by RPA were the same as those by traditional culture method. Conclusion The established real-time RPA can qualitatively detect VP,with simple operation and interpretation of results,which is suitable for rapid detection of VP in public health emergencies and food safety supervision.

Objective:In this study, we compared the funding rates of applicants who participated in the expert preliminary counseling with those who did not, to explore the role of the expert preliminary counseling in improving the funding rate of the National Natural Science Fund, so as to provide further reference for the management of scientific research.Methods:Statistical methods was used to analyze relevant data from 2012 to 2019 to compare the approval and funding rate of the applicants that participated in and did not participate in the expert preliminary counseling using.Results:From 2012 to 2019, the proportion of funded applicants in the expert preliminary counselling group was 30.45%, and the overall funding rate was 18.51%. Under the same research basis, the funding rate of the group was 29.91%, which was significantly higher than the group did not participate in the expert preliminary counselling (funding rare: 13.59%, P<0.001). Conclusions:We should pay more attention to and participate actively in the expert preliminary counselling, which can increase the quality of the projects and improve their funding rate significantly.

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.