Application of polymerase chain reaction to detect RNA coliphage Qβ in environmental water samples

Abstract

An RT-PCR technique for direct detection of enteric viruses in environmental water samples was developed by using RNA coliphage Qβ as a model virus. RNA coliphage Qβ was prepared from the lysate broth of single-plaque-isolated stock and extracted by direct heating. The sensitivity of the PCR amplification was improved by optimizing the parameters to be evaluated for efficient PCR including KCl in buffer components, Taq polymerase, annealing temperature, and number of cycles. The higher KCl concentration (up to 90 mM) in reaction incorporating with lower Taq polymerase concentration (0.5 unit/40 μl total reaction) caused quite low sensitivity and inconsistency of RT-PCR. The KCl concentration (30 to 90 mM) implied no significant effect to the yields of PCR products when higher Taq polymerase (1.0 to 2.5 units/40 μl total reaction) was included. Increasing the number of cycle from 25 to 35 cycles was able to increase the sensitivity. The increasing of annealing temperature reduced amplifying background products seen on gel electrophoreses. The maximum sensitivity is 0.3 PFU/reaction using condition of 35-cycle amplification with Taq polymerase 2.5 units and 55°C annealing temperature. This RT-PCR technique will be used as a protocol for further study for enteroviruses. The success of detection of indigenous RNA coliphages (group III) in night soil sample using the developed RT-PCR methods means that the developed method has great potential as a direct detection method of viruses in environmental sample. The inconsistency of the detection limit between plaque assay and RT-PCR assay may be caused by the effects of inhibitors in night soil sample. Further study on the relationship between plaque assay and PCR assay is required.