Rapid-viability PCR method for detection of live, virulent Bacillus anthracis in environmental samples

Abstract

In the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulent Bacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for the B. anthracis chromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulent B. anthracis in environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples.

Fig. 1.

Summary of RV-PCR sample processing steps, with corresponding sample volumes.

Fig. 2.

Manual and automated RV-PCR results obtained for clean wipe, air filter, and water samples spiked with Bacillus anthracis Ames spores (samples processed with the manual protocol were spiked with 31 ± 2 CFU/sample and samples processed with the automated protocol were spiked with 26 ± 1 CFU/sample, as measured by plating). Each C_T value is the average of 4 replicate samples. Error bars represent 1 standard deviation above and below the average C_T value.

Fig. 3.

Manual and automated RV-PCR on dirty wipe, air filter, and water samples spiked with Bacillus anthracis Ames spores (samples processed with the manual protocol were spiked with 49 ± 3 CFU/sample and samples processed with the automated protocol were spiked with 40 ± 2 CFU/sample, as measured by plating). Each C_T value is the average of 4 replicate samples. Error bars represent 1 standard deviation above and below the average C_T value.

Fig. 4.

Manual and automated RV-PCR results obtained for clean wipe, air filter, and water samples spiked with Bacillus anthracis Ames spores (samples processed with the manual protocol were spiked with 14 ± 1 CFU/sample and samples processed with the automated protocol were spiked with 38 ± 2 CFU/sample, as measured by plating) in a background of 10⁶ autoclaved Bacillus anthracis Ames spores/sample. Each C_T value is the average of 4 replicate samples. Error bars represent 1 standard deviation above and below the average C_T value.

Fig. 5.

Manual and automated RV-PCR results obtained for clean wipe, air filter, and water samples spiked with Bacillus anthracis Ames spores (samples processed with the manual protocol were spiked with 33 ± 2 CFU/sample and samples processed with the automated protocol were spiked with 21 ± 1 CFU/sample, as measured by plating) in a combined background of 10³ live Bacillus atrophaeus and 10⁶ Pseudomonas aeruginosa CFU/sample. Each C_T value is the average of 4 replicate samples. Error bars represent 1 standard deviation above and below the average C_T value.

Fig. 6.

Manual RV-PCR results obtained for clean wipe samples spiked with 3 levels of Bacillus anthracis Ames (54 ± 9 CFU/sample, 813 ± 43 CFU/sample, and 8,130 ± 430 CFU/sample). The incubation time was varied from 8 to 7 and then 6 h (noted as T₈, T₇, and T₆, respectively). Each C_T value is the average of 7 replicate samples. Error bars represent 1 standard deviation above and below the average C_T value.