A METHOD FOR THE DETECTION AND QUANTITATION OF PCR TEMPLATE IN ENVIRONMENTAL-SAMPLES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

This study describes methodology for the detection and quantitation of PCR amplified DNA. Specifically we report the estimation of the preva lence of E. coli in marine waters and other environmental samples from Mamala Bay, Hawaii. High performance liquid chromatography (HPLC) was used to quantitate PCR products containing between 1 and 250 ng DNA. PCR was used to amplify E. coli DNA through the use of lamB primers. A standard curve was generated that related initial cell template conce ntrations to amplified product DNA concentrations within a template ra nge of 520 to 5.2 X 10(7) cells. The standard curve was used to determ ine initial template concentrations of the iamB gene sequence present within 11 different environmental samples. Quantified PCR analyses wer e most useful when samples contained only low numbers of target organi sms, and when environmental samples contained few PCR inhibitory subst ances, as for example in marine water samples. Quantitation of amplifi ed DNA and comparison with culture data also nonculturable organisms i n some environmental samples. Overall these data are unique in that th ey indicate the successful use of HPLC to quantitate PCR amplification s with concomitant estimation of PCR template within environmental sam ples. (C) 1997 Elsevier Science B.V. All rights reserved.