Detection of Biological Threat Agents by Real-Time PCR: Comparison of Assay Performance on the R.A.P.I.D., the LightCycler, and the Smart Cycler Platforms

Abstract

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.

Antimicrobial peptides as new recognition molecules for screening challenging species

“…The key advantages of ELISA are its overall sensitivity and the lack of requirement for significant sample preparation. PCR, on the other hand, is generally more sensitive and specific and, once nucleic acids are extracted, rapid; a number of excellent, high sensitivity PCR-based assays have been described [4][5][6][7][8][9][10][11]. However, both antibody-and nucleic acid-based detection methods require prior development of target-specific reagents whose efficacy is highly dependent on assumed genetic sequences and/or antigenic motifs.…”

Comprehensive Panel of Real-Time TaqMan™ Polymerase Chain Reaction Assays for Detection and Absolute Quantification of Filoviruses, Arenaviruses, and New World Hantaviruses

“…Recent data indicated an easy transfer of both probe chemistries from one platform to another through similar assay performance, sensitivity, and specificity on three of these instruments. 20 In a pilot evaluation of the TaqMan™-MGB assay designed to detect Lassa Josiah genomic RNA, 39 blood samples from patients infected with Lassa virus in Sierra Leone 21,22 were tested as part of the Lassa surveillance and treatment program based at Kenema Government Hospital. 23,24 Five of these samples were serially collected from one patient (#G-104) on days 1, 2, 3, 8, and 15 after admission to the Lassa Fever Ward.…”

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

“…Five μls of extracted nucleic acids were used for each PCR reaction in a 20 μl reaction volume. For all of the DNA-extracted organisms, the cycling conditions were 45 cycles of 95°C for 0 s and 60°C for 20 s. DNA extracted from B. anthracis Ames was detected using a pX01-specific assay as previously described (Christensen et al, 2006). Y. pestis was detected using a PIMspecific assay as previously described (Christensen et al, 2006).…”

“…For all of the DNA-extracted organisms, the cycling conditions were 45 cycles of 95°C for 0 s and 60°C for 20 s. DNA extracted from B. anthracis Ames was detected using a pX01-specific assay as previously described (Christensen et al, 2006). Y. pestis was detected using a PIMspecific assay as previously described (Christensen et al, 2006). VACV primers targeting the polymerase were used at a final concentration of 0.5 mM for VACA25L-F151 (5′-CACCTCCACCAAAACCTAAAACTC) and VACA25L-R231 (5′-AAAATgggCgTggATTgTTAAC).…”

“…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).…”