Sensitive and Rapid Quantitative Detection of Anthrax Spores Isolated from Soil Samples by Real‐Time PCR

Abstract

Quantitative analysis of anthrax spores from environmental samples is essential for accurate detection and risk assessment since Bacillus anthracis spores have been shown to be one of the most effective biological weapons. Using TaqMan real-time PCR, specific primers and probes were designed for the identification of pathogenic B. anthracis strains from pag gene and cap gene on two plasmids, pXO1 and pXO2, as well as a sap gene encoded on the S-layer. To select the appropriate lysis method of anthrax spore from environmental samples, several heat treatments and germination methods were evaluated with multiplex-PCR. Among them, heat treatment of samples suspended with sucrose plus non-ionic detergent was considered an effective spore disruption method because it detected up to 10 5 spores/g soil by multiplex-PCR. Serial dilutions of B. anthracis DNA and spore were detected up to a level of 0.1 ng/l and 10 spores/ml, respectively, at the correlation coefficient of 0.99 by real-time PCR. Quantitative analysis of anthrax spore could be obtained from the comparison between CT value and serial dilutions of soil sample at the correlation coefficient of 0.99. Additionally, spores added to soil samples were detected up to 10 4 spores/g soil within 3 hr by real-time PCR. As a consequence, we established a rapid and accurate detection system for environmental anthrax spores using real-time PCR, avoiding time and labor-consuming preparation steps such as enrichment culturing and DNA preparation.

In silico and in vitro evaluation of PCR-based assays for the detection ofBacillus anthracischromosomal signature sequences

“…30 These genetic markers provide limited specificity and require additional timeconsuming and labor-intensive post-PCR analysis steps. Other areas of the chromosome have also been investigated as potential DNA-targets for identification purposes, including the so-called BA813 [31][32][33][34][35][36][37][38] and BA5510 sequences, 19 genes bclB, 39 sap, 40,41 saspB, 5,42 and sspE, 22,43 the B-type small acid-soluble spore protein gene (SASP), 44 a glycosyltransferase group 1 family protein, 45 a protein showing similarities with an abhydrolase, 18 and several DNA loci located on prophage regions, 17 i.e., BA5345, 21 BA5357, 46 and PL3. 47 Although most of these regions have been claimed to be anthrax-specific, B. cereus strains sometimes yield false positive results.…”

Application of Genetic Marker and Real-Time Polymerase Chain Reaction for Discrimination between Forsythia viridissima and Forsythia suspensa

“…[23][24][25][26][27][28][29][30][31] TaqMan probes have a fluorescent reporter dye at one end and a quencher dye that inhibits fluorescence at the other end. During the extension stage, the prove is broken apart by the DNA-polymerase and begins to fluorescence with more intensity.…”

Comparison of nucleic acid extraction platforms for detection of select biothreat agents for use in clinical resource limited settings

“…This is especially true for the potential of an intentional release of a biological agent. In this context, real-time PCR is currently implemented for the highly specific identification of pathogens in both clinical and environmental settings (Belgrader et al, 1998;Christensen et al, 2006;Higgins et al, 1998;Makino et al, 2001;Ryu et al, 2003;Shchelkunov et al, 2011;Skottman et al, 2007;Tomaso et al, 2008).…”