Tantawiwat et al. also developed a method of multiplex
PCR amplification of lacZ, uidA and plc genes for
the simultaneous detection of total coliform bacteria,
E. coli and Clostridium perfringens, in drinking water.
Detection by agarose gel electrophoresis yielded a band
of 876 bp for the lacZ gene of all coliform bacteria; a
band of 147 bp for the uidA gene and a band of 876 bp
for the lacZ gene of all strains of E. coli; a band of
280 bp for the plc gene for all strains of C. perfringens
and a negative result for all three genes when tested with
other bacteria. The detection limit was 100 pg for E. coli
and C. perfringens, and 1 ng for coliform bacteria when
measured with purified DNA. Spiked water samples with
0–1000 cfu/ml of coliform bacteria and/or E. coli and/or
C. perfringens were detected by this multiplex PCR after
a pre-enrichment step to increase the sensitivity and to
ensure that the detection was based on the presence of
cultivable bacteria. The result of bacterial detection from
the multiplex PCR was comparable with that of a standard
plate count on selective medium (P = 0.62). On
using standard plate counts as a gold standard, the sensitivity
for this test was 99.1% (95% CI 95.33, 99.98) and
the specificity was 90.9% (95% CI 75.67, 98.08).
Tantawiwat et al. also developed a method of multiplexPCR amplification of lacZ, uidA and plc genes forthe simultaneous detection of total coliform bacteria,E. coli and Clostridium perfringens, in drinking water.Detection by agarose gel electrophoresis yielded a bandof 876 bp for the lacZ gene of all coliform bacteria; aband of 147 bp for the uidA gene and a band of 876 bpfor the lacZ gene of all strains of E. coli; a band of 280 bp for the plc gene for all strains of C. perfringensand a negative result for all three genes when tested withother bacteria. The detection limit was 100 pg for E. coliand C. perfringens, and 1 ng for coliform bacteria whenmeasured with purified DNA. Spiked water samples with0–1000 cfu/ml of coliform bacteria and/or E. coli and/orC. perfringens were detected by this multiplex PCR aftera pre-enrichment step to increase the sensitivity and toensure that the detection was based on the presence ofcultivable bacteria. The result of bacterial detection fromthe multiplex PCR was comparable with that of a standardplate count on selective medium (P = 0.62). Onusing standard plate counts as a gold standard, the sensitivityfor this test was 99.1% (95% CI 95.33, 99.98) andthe specificity was 90.9% (95% CI 75.67, 98.08).
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