Effect of amplicon size on PCR detection of bacteria exposed to chlorine

The effect of amplicon size on the PCR detection of Legionella pneumophila after chlorine inactivation was investigated. Two amplicons specific to the L. pneumophila mip gene were used for the PCR analyses: a 650-bp amplicon and smaller 168-bp amplicon within the 650-bp amplicon; a 108-bp amplicon specific to species rRNA coding sequence also was used. After exposure to chlorine, viable agar grown cells were not detected by plate counts or direct counts with p-iodonitrotetrazolium (INT) after 1 min for treatment at 10 mg/l, after 2 min for treatment at 5 mg/l, and after 4 min for treatment at 2.5 mg/l; viable water grown cells were present at least 4 min after biocide addition even with a chlorine dose of 5 mg/l. At the 10-mg/l dosage, PCR products from the 168-bp amplicon were detected on agarose gels up to 16 min after chlorination; even after 24 hr of PCR the 168-bp products were detectable using a capture probe hybridization assay. However, the 650-bp target was not detected after 4 min chlorine contact time at the same biocide dosage using agarose gels, and PCR products could not be detected by hybridization after 32 min. At lower chlorine concentrations, a similar pattern was seen with the 168-bp amplicon detectable longer after biocide addition than the 650-bp mip amplification target. On the basis of these data, larger amplicons appear to correlate better with viability of L. pneumophila in water samples.