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 identify the avian influenza virus subtype from the avian and environmental samples using the Ion Torrent new-generation semiconductor sequencing technology and to establish a high-throughput sequencing method to identify unclassified influenza A virus. Methods: Virus RNA was extracted from the nine avian swab and environmental samples and real-time RT-PCR was carried out to detect universal fluA, H5N1, H7N9 and H9N2. The whole genome of influenza A virus was amplified by PathAmpFluA kit. Sequencing library was prepared using Next Fast DNA Fragmentation & Library Prep Set for Ion Torrent kit and high-throughput sequencing was done by Ion Torrent Personal Genome Machine(PGM). Data from the PGM was processed and quality evaluated using Ion TorrentSuite v3.0 software. Sequence assembly and influenza database blast were carried out by FluAtyping v4.0 and PathogenAnalyzer bioinformatics software to identify the influenza A virus subtype of these nine samples. Results: The results of real-time RT-PCR for universal fluA of these nine samples were positive but the results for H5N1, H7N9 and H9N2 were negative. Seven subtypes of influenza A virus were identified by high-throughput sequencing and bioinformatics analysis: six samples were H2N3, H5N6, H5N8, H7N1, H7N7, H11N3 subtype respectively and three samples were H6N6 subtype. Conclusions Avian influenza virus has many subtypes in the environment of Zhejiang province. Ion Torrent semiconductor sequencing technology is suitable for fast identification of unclassified influenza virus for avian influenza environment monitoring.

Objective:To understand the pathogens and molecular-epidemiologic characteristics of viral meningo-encephalitis in Zhejiang province during 2002 to 2018.Methods:All the samples were collected from suspected patients admitted to the hospitals under the monitoring program. Of the total samples, 2 173 were cerebrospinal fluids while the other 455 were stool specimens. Cerebrospinal fluid (CSF) samples were subject to real-time qPCR for the detection of Human enterovirus (HEV), Mumps virus (MuV), Herpes simplex virus (HSV), Cytomegalovirus (CMV) and Japanese encephalitis virus (JEV). Stool sample were subject to real-time qPCR for HEV. ELISA was used to detect the IgM antibodies in CSF, in the 5 kinds of virus mentioned above. VP1 genes from all RNA-positive specimen were amplified, sequenced, for typing and for evolution analysis.Results:871 (40.1 %) of the 2 173 samples were detected as HEV nucleic acid positive during 2002 to 2018. 654 (38.1 %) of the 1 718 CSF sample were HEV nucleic acid positive while 217 (47.7 %) of the 455 stool sample were HEV nucleic acid positive. Among the total positive nucleic acid sample, 670 of them were VP1 sequence positive, including 5 HEV-A and 665 HEV-B. There were 23 HEV serotypes, including Coxsackievirus (CV) CVA4, CVA6, CVA9, CVA10, CVB1-5, Echovirus (EchoV; E) E3, E4, E6,E7, E9, E11, E14, E16, E18, E21, E25, E30, E33 and EV-71. The top three serotypes went to E30, E6 and CVB5. These three serotypes presented enhanced viral activity in every several years. 795 CSF samples were detected as virus nucleic acid positive, including 374 HEV, 6 MuV, 5 HSV and 5 CMV, from 2012 to 2015 and in 2018. 5 kinds of IgM antibodies were detected simultaneously in 368 CSF samples, including 2 HEV positive, 6 JEV positive and 1 MuV positive for 5 viruses, respectively. Except for EV-71, there were 517 EchoV and 152 CV viruses presented, with the ratio of 3.4∶1. These two kinds of viruses alternately changed for each predominant epidemic strains in every 3-5 years. Based on VP1, results from the phylogenetic tree showed that HEV from Zhejiang province clustered into HEV-A and HEV-B clades respectively. E30 developed both h and i sub-genotypes. Conclusions:HEV-B seemed the main pathogen for viral meningo-encephalitis in Zhejiang province. Ratio of positive detection on EchoV was significantly higher than that on CV. These two kinds of virus alternately presented changing tendency in every several years. Predominant epidemic strains E30, CVB5 and E6 were presenting enhanced viral activity, also in every several years. High correlation was found in both HEV viral activity from the surveillance sites and in time line of the viral meningo-encephalitis outbreaks.