A method for the detection and quantitation of PCR template in environmental samples by high performance liquid chromatography

Abstract This study describes methodology for the detection and quantitation of PCR amplified DNA. Specifically we report the estimation of the prevalence 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 concentrations to amplified product DNA concentrations within a template range of 520 to 5.2×10 7 cells. The standard curve was used to determine initial template concentrations of the lamB gene sequence present within 11 different environmental samples. Quantified PCR analyses were most useful when samples contained only low numbers of target organisms, and when environmental samples contained few PCR inhibitory substances, as for example in marine water samples. Quantitation of amplified DNA and comparison with culture data also suggested the presence of viable but nonculturable organisms in some environmental samples. Overall these data are unique in that they indicate the successful use of HPLC to quantitate PCR amplifications with concomitant estimation of PCR template within environmental samples.