Quantification of the carbazole 1,9a-dioxygenase gene by real-time competitive PCR combined with co-extraction of internal standards

The fluorogenic probe assay, competitive polymerase chain reaction (PCR) an d co-extraction with internal standard cells were combined to develop a rap id, sensitive, and accurate quantification method for the copy number of a target carbazole 1,9a-dioxygenase gene (carAa) and the cell number of Pseud omonas sp. strain CA10. The internal standard DNA was modified by replaceme nt of a 20-bp long region with one for binding a specific probe in fluoroge nic PCR (TaqMan). The resultant DNA fragment was similar to the correspondi ng region of the intact carAa gene in terms of G+C content. When used as a competitor in the PCR reaction, the internal standard DNA was distinguishab le from the target carAa gene by two specific fluorogenic probes with diffe rent fluorescence labels, and was automatically detected in a single tube u sing the ABI7700 sequence detection system. To minimize variations in the e fficiency of cell lysis and DNA extraction between the samples, the co-extr action method was combined. A mini-transposon was used to introduce competi tor DNA into the genome of other pseudomonads, and the resultant construct was used as the standard cell. After the addition of a fixed amount of the internal standard cells to soil samples, total DNA was extracted (co-extrac tion). Using this method, the copy number of the carAa gene and the cell nu mber of strain CA10 in soil samples could be quantified rapidly. (C) 2001 P ublished by Elsevier Science B.V. on behalf of the Federation of European M icrobiological Societies.