We designed, optimized, and extensively tested several sensitive and specific real-time PCR assays for rapid detection of both smallpox and pan-orthopox virus DNAs. The assays are based on TaqMan 3-minor groove binder chemistry and were performed on both the rapid-cycling Roche LightCycler and the Cepheid Smart Cycler platforms. The hemagglutinin (HA) J7R, B9R, and B10R genes were used as targets for the variola virus-specific assays, and the HA and DNA polymerase-E9L genes were used as targets for the pan-orthopox

the unique RET mutations was not possible when the derivative curves overlapped. Although not all pathogenic RET mutations were available for analysis, a recent systematic study of high-resolution melting detection of heterozygous point mutations within a PCR amplicon found a sensitivity and specificity of 100% for amplicons Ͻ400 bp in size (15 ). High-resolution melting analysis for mutation scanning is a rapid (1-2 min after PCR), costeffective assay that requires no processing or separation steps. As applied to RET mutation scanning, accuracy of heterozygote detection appears to be 100%, and some (but not all) sequence variations can be distinguished from each other. Because samples are immediately available for further processing after high-resolution melting analysis, the detected variant samples can be sequenced for confirmation of genotype.

No abstract

Aims:  The Cepheid GeneXpert® is a four‐site, automated sample preparation and real‐time PCR detection system. In this study, the capability of the GeneXpert® to isolate and detect nucleic acid from Bacillus anthracis Ames spores was assessed. Methods and Results:  A four‐plex, dried‐down bead cartridge containing PCR reagents specific for the pXO1 and pXO2 plasmids as well as sample processing and inhibition controls was evaluated. For B. anthracis Ames spores harbouring pXO1 and pXO2, samples containing 68 CFU per ml (148 spores per ml) were positive in all four replicates. A limited cross‐reactivity panel, which included closely related Bacillus species, was also tested to determine the specificity of the pXO1 and pXO2 assays. No cross‐reactivity occurred. Further, B. anthracis Sterne spore samples were analysed to compare results when processed using the GeneXpert® to those run directly on the Cepheid SmartCycler® without sample processing. The GeneXpert® detection capability was three logs lower than the SmartCycler® indicating the benefit of incorporating a nucleic acid extraction procedure. Conclusions:  This study demonstrates that the GeneXpert® is a rapid and reliable system for simultaneously detecting the B. anthracis virulence plasmids pXO1 and pXO2. Significance and Impact of the Study:  The GeneXpert® is the only platform currently available that is capable of both nucleic acid purification and real‐time PCR detection enclosed within a single system. Further, all sample manipulations are automated, thus reducing errors associated with manual processing.

Polymerase chain reaction (PCR) is commonly used for pathogen detection in clinical and environmental samples. These sample matrices often contain inhibitors of PCR, which is a primary reason for sample processing; however, the purification process is highly inefficient, becoming unacceptable at lower signature concentrations. One potential solution is direct PCR assessment without sample processing. Here, we evaluated nine inhibitor-resistant PCR reagents for direct detection of Francisella tularensis in seven different clinical and environmental samples using an established real-time PCR assay to assess ability to overcome PCR inhibition. While several of these reagents were designed for standard PCR, the described inhibitor resistant properties (ex. Omni Klentaq can amplify target DNA samples of up to 20% whole blood or soil) led to our evaluation with real-time PCR. A preliminary limit of detection (LOD) was determined for each chemistry in whole blood and buffer, and LODs (20 replicates) were determined for the top five chemistries in each matrix (buffer, whole blood, sputum, stool, swab, soil, and sand). Not surprisingly, no single chemistry performed the best across all of the different matrices evaluated. For instance, Phusion Blood Direct PCR Kit, Phire Hot Start DNA polymerase, and Phire Hot Start DNA polymerase with STR Boost performed best for direct detection in whole blood while Phire Hot Start DNA polymerase with STR Boost were the only reagents to yield an LOD in the femtogram range for soil. Although not the best performer across all matrices, KAPA Blood PCR kit produced the most consistent results among the various conditions assessed. Overall, while these inhibitor resistant reagents show promise for direct amplification of complex samples by real-time PCR, the amount of template required for detection would not be in a clinically relevant range for most matrices.

No abstract

Simultaneous analysis of three targets in three colors on any real-time polymerase chain reaction (PCR) instrument would increase the flexibility of real-time PCR. For the detection of Bacillus strains that can cause inhalation anthrax-related illness, this ability would be valuable because two plasmids confer virulence, and internal positive controls are needed to monitor the testing in cases lacking target-specific signals. Using a real-time PCR platform called MultiCode-RTx, multiple assays were developed that specifically monitor the presence of Bacillus anthracis-specific virulence plasmid-associated genes. In particular for use on LightCycler-1, two triplex RTx systems demonstrated high sensitivity with limits of detection nearing single-copy levels for both plasmids. Specificity was established using a combination of Ct values and correct amplicon melting temperatures. All reactions were further verified by detection of an internal positive control. For these two triplex RTx assays, the analytical detection limit was one to nine plasmid copy equivalents, 100% analytical specificity with a 95% confidence interval (CI) of 9%, and 100% analytical sensitivity with a CI of 2%. Although further testing using clinical or environmental samples will be required to assess diagnostic sensitivity and specificity, the RTx platform achieves similar results to those of probe-based real-time systems.

No abstract