Establishment And Evaluation Of Rapid Detection Process Of Diarrhea Pathogens

Objective:To establish fast detection process of diarrhea pathogens, which was adopted integrated PCR as early screening technology, for the important common pathogens causing diarrhea, such as Salmonella spp, Shigella spp, pathogenic vibrio (mainly include Vibrio cholerae and Vibrio parahaemolyticus), diarrheaenic Escherichia coli (EPEC, ETEC, EHEC, EAEC), Norovirus, Sapovims, Rotavirus, enteric adenovirus and astrovirus, and evaluate the practical application value of the process. Preliminary mastered the pathogenic spectrum characteristics of diarrhea syndrome in monitoring sites, and understood the serotyping, virulence genes and drug resis-tance of isolated pathogens.Methods:1.7sentinel hospitals in Wuxi city were chosen as the monitoring sites. Collect the basic information and stool specimens of diarrhea cases, and identify Salmonella spp, shigella spp, Vibrio cholerae and Vibrio parahaemolyticus and diarrheagenic Escherichia coli with conven-tional culture methods. Serotype, virulence genes and drug sensitive experiments were done for the strains isolated.2. Nucleic acid(contain DNA and RNA), extracted from stool specimens and different links at the bacterial cultivation process(both the enrichment broth and the bacterium lawn on the first area of EMB plate), were detected for different target pathogens. The target gene of diarrheagenic Escherichia coli was identified with multiple conventional PCR, while target genes of Salmonella spp, Shigella spp, Vibrio cholerae and Vibrio parahaemolyticus were tested with multiple real-time PCR. The target vims were detected by single real-time PCR technique.Results:1. A total of334feces samples of diarrhea cases were collected from April to December in2013.With conventional culture method30strains of target bacteria were separated, positive rate was9.0%.There were6strains of diarrheagenic Escherichia coli,18strains of Salmonella spp, and Shigella spp, Vibrio cholerae and Vibrio parahaemolyticus were2strains respectively.2.Results of PCR detection:①diarrheagenic Escherichia coli:18copies were positive in stool specimens,13were confirmed containing the corresponding target sequence by sequenc-ing,5copies of nonspecific,2were consistent with the conventional cultivating results.21copies from bacterial lawn were positive, all confirmed by sequencing, including5consistent with the conventional cultivating results.②Shigella spp:8copies were positive in stool speci-mens, all containing target sequence of Shigella spp confirmed by sequencing, including2posi-tive by traditional cultivating.③Salmonella spp:1from feces was positive, while22from enrichment broth were positive, which were identified by sequencing,18of which consistent with the conventional cultivating method.④Vibrio parahaemolyticus:2copies positive from stools as well as from enrichment broth, were consistent with the conventional cultivating re-sults.⑤Vibrio cholerae:1from feces was positive, while2from enrichment broth were posi-tive, which were consisted with traditional cultivating.3. A total of83nucleic acid were positive for target virus, and the positive rate was24.9%, from high to low as follows:NV(11.7%) and RV(8.4%), EADV (2.1%), ASTV(1.5%), SPAV(1.2%).Virus infection had obvious seasonal characteristic, with peak season occurring in winter and spring.4. Serotype of Salmonella spp presented diversity, with E group as main type, and isolated strains showed drug resistance of different degree to several kinds of antibiotics. Diarrheagenic Escherichia coli were highly resistant to nalidixic acid and trimethoprim/sulfamethoxazole.Conclusions:1. The detection process of diarrhea pathogens, which was based on PCR as early screen-ing technology, was rapid, accurate and efficient. New screening technology solution was as follows:①diaiTheagenic Escherichia coli:Inoculate stool specimens on the plate of intestinal bacteria and detect the first section bacterial lawn with multiple PCR. Single colony picked from the plate was used to identify the corresponding virulence gene, if the sample was positive.②Shigella spp:Samples were detected by real-time PCR directly, and positive samples would be conventional cultivated further.③Salmonella spp, Vibrio parahaemolyticus and Vibrio cho-lerae:Detect enrichment broth after selective enrichment by real-time PCR, and choose selec-tive plates to isolate target bacteria if positive. 2. Salmonella spp, diarrheagenic Escherichia coli, Norovirus and Rotavirus were the main diarrhea pathogen. The bacterial diarrhea was given priority in summer, while viral diarrhea in winter and spring.