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Enteroaggregative E. coli(EAggEC) is a common cause of infantile diarrhoea. In vitro study, shows that EAggEC strains established characteristic "stacked-brick" on the surface of tissue culture cells and such adhrence pattern was termed as aggregative adherence(AA). Recently, Natatro et al. and Savarino et al. found that EAggEC expressed an inducible aggregative adherence fimbriae I(AAF/I) associated with the presence of AA on HEp-2 or HeLa cells. The cloned regulator gene aggR and structural gene aggA encoding AAF/I were found to be specific for EAggEC and not in other enteropathogens. The purpose of this study is to design PCR primers for the specific detection of the aggR and aggA genes of 7 EAggEC strains which were screened by the pCVD432 primers which were designed by Schmidt et al.. The results obtained were compared with the in vitro HeLa cells adherence test, bacteria clump formation test, hemagglutination test and DNA sequencing of suspected agg genes so that the accuracy of primers pCVD432, AggR-1/R-2, AggRI/RII, AggR5/R3, and AggA1/A2 were checked. In addition, the multiplex PCR systems for the simutaneous detection of EAggEC and enteropathogenic E. coli( EPEC) cells were also developed. On the other aspect, combined LTI and STII gene specific PCR primers were combined into a multiplex PCR system and used for the simultaneous detection of LTI and STII ETEC cells contaminated in tap water and environmental waters. By comparison of the sequences of aggR and aggA genes from GenBank/EMBL, primers AggRI/RII and AggA1/A2 were designed for the specific detection of aggR and aggA genes of EAggEC. Only four strains of seven EAggEC-like strains showed the 610 bp and 352 bp PCR products positive results with AggRI/ RII and AggA1/A2 as PCR primers. On the other hand, all of the seven EAggEC-like strains produced expected 355 bp PCR products with AggR5/R3 primer sets. Similar positive PCR results were obtained when reporeted pCVD432 and AggR-1/R-2 primers were used. Based on the above facts, in vitro HeLa cells adherence test, bacteria clump formation test and hemagglutination test were thus used to check the seven EAggEC-like strains. Results showed that AggRI/RII and AggA1/A2 primer pairs developed by us are more specific than pCVD432, AggA1/A2 and AggR-1/R-2 primer pairs. Under the PCR conditions as described, bacteria strains including enterohemorrhagic E. coli(EHEC), enterotoxigenic E. coli(ETEC), enteroinvasive E. coli(EIEC), EPEC, non-pathogenic E. coli and non-E. coli strains including strains of the family of enterobacteriaceae, would not produce the specific PCR product. The detection sensitivity of AggA1/A2 primers(50 pmole each) was 100-101 CFU. In detection of EAggEC cells in milk samples, tap water, ground water and infantile stool samples, the detection sensitivity would be 100 CFU if a 8~9 hr pre- enrichment step using MacConkey broth was performed prior to the PCR. The specific PCR system for EAggEC detection could be combined with BFP1/2 primers a multiplex PCR system. The detection sensitivity for EAggEC and EPEC cells with the multiplex PCR system using AggA1/A2 and BFP1/2 primers was 103/ 103 for each target cells per assay. Finally, LTI1/I2 and STII1/ II2 primers were combined into a multiplex PCR system and used for the inspection of tap water and environmental waters near Taichung City. On detection of ETEC T02 and T10 cells in tap water and environmental waters, if a 8 hr pre-enrichment step was performedprior to PCR, the detection sensitivity for target cells in water would be reach to 100 CFU per assay.
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