Evaluation of a modified rapid viability-polymerase chain reaction method for Bacillus atrophaeus spores in water matrices

A rapid method that provides information on the viability of organisms is needed to protect public health and ensure that remediation efforts following a release of a biological agent are effective. The rapid viabilitypolymerase chain reaction (RV-PCR) method combines broth culture and molecular methods to provide results on whether viable organisms are present in less than 15 h. In this study, a modified RV-PCR (mRV-PCR) method was compared to a membrane-filtration culture method for the detection of viable Bacillus spores in water matrices. Samples included small and large volumes of chlorine and non-chlorine treated tap water. Large volume water samples (up to 100 L), were processed by ultrafiltration using a semi-automated waterborne pathogen concentrator, followed by centrifugation as a secondary concentration technique. The concentrated samples were analyzed by mRV-PCR and culture methods. The overall agreement between the mRV-PCR and culture methods when seed concentrations were greater than 10 spores per sample volume analyzed was 96%. The total time from the start of sample processing to the final sample result for the mRV-PCR method was decreased by approximately 2 h, in comparison to the previously published RV-PCR method because of the incorporation of shorter, more efficient primary and secondary concentration steps and a shorter DNA extraction technique. Overall, this study confirmed that RV-PCR is a promising approach for identifying viable Bacillus spores in small- and large-volume water samples and for producing results in less time than traditional culture methods.

Automated summary

The study compared a modified RV-PCR method to a membrane-filtration culture method for detecting viable Bacillus spores in water samples. Results showed that there was 96% overall agreement between the two methods when spore concentrations were greater than 10 per sample volume analyzed. The modified RV-PCR method demonstrated shorter processing time and faster results compared to the previously published method.