High throughput screening for spores and vegetative forms of pathogenic B. anthracis by an internally controlled real-time PCR assay with automated DNA preparation

Abstract

Human infections with Bacillus anthracis have become rare but in cases of intentional release, masses of samples would have to be expected. Current PCR assays for anthrax are appropriate for use in single cases, but they have not been formulated for high throughput screening. This article describes a high throughput real-time PCR for anthrax, including automated sample preparation without the need for pre-culturing of samples. The assay detects single copies of target gene. An internal control monitors the whole assay including sample preparation. The limit of detection in blood was 1,066 (95%CI, 741-1,739) copies/ml, corresponding to 4.4-32.3 organisms/ml. Using spore preparations, 20 colony-forming units (CFU) per sample could be detected reliably (0.8 CFU per PCR). The extraction procedures depleted viable spores from solution by factors of 10,000 (automated procedure) and >100,000 (conventional column procedure). One hundred and ten clinical and environmental specimens were retested, 50 of them sampled during a period of heightened anthrax awareness in 2001. A widely used assay yielded two false positive results (cross-reaction with B. cereus), while the new assay tested all samples negative. The internal control operated stable in all clinical samples. The assay is capable of testing for anthrax in the high throughput mode.

Quantification of Periodontopathic Bacteria in Saliva Using the Invader Assay

“…However, the use of real-time PCR in clinical practice is impractical for the quantification of periodontopathic bacteria in spite of its high sensitivity and specificity (11), because real-time PCR involves gene amplification and requires expensive equipments such as thermal cyclers. Furthermore, because the oral cavity's complex aerobic and anaerobic flora contain more than 300 different bacterial species (12), bacterial counts may be underestimated due to false positive results (13,14). When bacteria that have sequences similar to those of the periodontopathic bacteria are contaminated, they could be amplified and detected even at low concentrations (10 2 -10 4 /tube).…”

Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification

“…Several reports have described real-time PCR assays for the detection of B. anthracis [7-10], Y. pestis [6,11,12] and F. tularensis [13-15]. Some assays were designed in multiplex format, but only few of these included internal controls for DNA amplification [10,16] and none included an internal control for successful DNA extraction.…”

Identification and application of AFLP-derived genetic markers for quantitative PCR-based tracking of Bacillus and Paenibacillus spp. released in soil

“…However, it is anticipated that the screening process can become more efficient as, for example, next-generation high-throughput sequencing technologies become more widespread and affordable and automated systems are applied to routine tasks like samplehandling and bioinformatic analysis. Similar considerations are applicable to the environmental persistence studies in that certain aspects of the microcosm analysis are amenable to automation (for example, see Panning et al 2007). …”