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

Abstract

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 viability-polymerase 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.

Keywords: Bacillus spores; Quantitative PCR; Rapid; Ultrafiltration; Viability; Water samples.

Fig. 1.

Schematic of overall experimental workflow.

Fig. 2.

Diagram of the minimate TFF capsule secondary ultrafiltration.

Fig. 3.

Diagram showing the steps of the RV-PCR method. (Ct, cycle threshold)

Fig. 4.

Comparison of percent recoveries for three secondary concentration methods in 7 trials. The chlorine-demand-free (CDF) buffer in trials 1 through 3 was seeded with a concentration of B. atrophaeus spores as follows: 900, 1000, and 540 cfu/sample, respectively. Concentrated tap water samples in trials 4 through 7 were seeded with concentrations of B. atrophaeus spores as follows (from left to right): 690, 230, 2100, 2200, 2100, 2600, and 3100 cfu/sample, respectively.

Fig. 5.

RV-PCR delta Ct values (A) and membrane filtration results (B) versus chlorine concentrations in CDF-buffer samples seeded with B. atrophaeus spores. The line in plot B indicates the detection limit of the membrane filtration method (0.05 spore per milliliter).

Fig. 6.

Ct values for T0 and T9 samples versus membrane filtration results in samples with equal numbers of B. atrophaeus spores. Non-detections by qPCR were assigned Ct values of 45. Non-detections by membrane filtration were assigned concentrations of 0.5.