| Background:Diarrhea caused by viral and bacterial infections is a major health problem in developing countries and the clinical presentation of the patients with diarrhea symptoms is not generally indicative of a specific virus or bacteria. Conventional diagnostic methods for routine detection of enteric pathogens within the clinical microbiology setting rely on microscopy, culture, and enzyme immunoassays. However, these procedures are either laborious or have limited sensitivity and specificity. Few study reported the simultaneous detection of viral and bacterial pathogens within the same sample, which is essential to elucidate the potential synergy between enteric virus and bacteria and reveal the clinical relevance between viral and bacterial infections. Although several multiplex molecular assays such as Lumiex GPP and Seegene Diarrhea ACE are commercialy available, the extrmely high price made them unaffordable methods for routine use in provincial Centers for Diseases Control and Prevention, China. Therefore, a rapid, sensitive, specific and cost-effective diagnostic method which detects virus and bacteria simultaneously would be highly preferred for routine laboratory testing.Objective:Here we describe the development of a one-step multiplex PCR assays (two-tube assay) for the simultaneous detection of13most commonly found diarrhea-causative viruses or bacteria. And a new Resequencing Pathogen Microarray (RPM)-based multiple gastrointestinal pathogens detection assay was developed to simultaneously detect14Rotavirus,7Calicivirus,8human Astro virus,28Enteroviruses and16rare gastrointestinal viruses. Method:The two tube assay was designed to detect rotavirus A, norovirus GI and Gâ…¡, human astrovirus, enteric adenoviruses and human bocavirus (tube V), and Salmonella, Vibrio parahaemolyticus, diarrheogenic Escherichia coli, Campylobacter jejuni, Shigella, Yersinia and Vibrio cholera (tube B). The RPM assay divided all the primer in to6groups. The analytical specificity of the two tube assay and the RPM assay was examined with positive controls for each pathogen. The analytical sensitivity was evaluated by performing the assay on serial ten-fold dilutions of in vitro-transcribed RNA, recombinant plasmids or bacterial culture. The positive decision threshold of RPM was determined by testing the quantified control RNA, and the verification steps were optimized to type the Enterovirus. A total of122stool samples were tested by this two-tube assay and the results were compared with those obtained from reference methods, and10negative samples were tested by RPM assay.Result:The two-tube assay and the RPM assay both achieved a sensitivity of20-200copies for a single virus and102-103CFU/mL for bacteria. The clinical performance demonstrated that the two-tube assay had comparable sensitivity and specificity to those of reference methods, and the RPM assay demonstrated a better sensitivity. Nucleic acids of10stool samples with unexplained gastrointestinal infections were detected and6of them showed viral positive result.Conclusion:In conclusion, the two-tube assay is a rapid, cost-effective, sensitive, specific and high throughput method for the simultaneous detection of enteric bacteria and virus. And the high-throughput RPM assay with high specificity and sensitivity, which demonstrated to have great potential for the identification of unexplained gastrointestinal samples and to improve the capacity for emergency management. |
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