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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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