Quantitative Polymerase Chain Reaction (PCR) for Detection of Aquatic Animal Pathogens in a Diagnostic Laboratory Setting

Abstract

Real-time, or quantitative, polymerase chain reaction (qPCR) is quickly supplanting other molecular methods for detecting the nucleic acids of human and other animal pathogens owing to the speed and robustness of the technology. As the aquatic animal health community moves toward implementing national diagnostic testing schemes, it will need to evaluate how qPCR technology should be employed. This review outlines the basic principles of qPCR technology, considerations for assay development, standards and controls, assay performance, diagnostic validation, implementation in the diagnostic laboratory, and quality assurance and control measures. These factors are fundamental for ensuring the validity of qPCR assay results obtained in the diagnostic laboratory setting.

Evaluation of Non-destructive Molecular Diagnostics for the Detection of Neoparamoeba perurans

“…Two of the protocols were not assessed for DNA copy numbers due to availability of information on the primer sequences for protocol 3 and for protocol 5 which was based on the Young et al (2008) primers which were used to produce the plasmid DNA. From the analysis of the three protocols assessed it was found that protocol 1 was able to detect the lowest concentration of copies of DNA at 2.64 copies µl −1 , which approaches the theoretical limit of detection (Purcell et al, 2011). Analysis of protocol 2 gave a concentration of 14.3 copies µl −1 which is similar to levels reported by Fringuelli et al (2012).…”

“…Additionally, TaqMan R chemistry is generally thought to offer a number of advantages over SYBR R green, in particular, the incorporation of minorgroove-binders (MGB) which allow for the raising of melting temperatures of the probes (enabling the use of shorter probes) and integration of the internal hydrolysis probe providing greater specificity in comparison to the intercalating dye assays, which have reduced specificity because any amplification product incorporates the dye. (Gunson et al, 2006;Purcell et al, 2011). Each assay was designed to amplify specific regions of the 18S rRNA gene, which is generally chosen due to its high copy number, thus potentially increasing sensitivity.…”

Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

“…The high virus copy number in samples may increase the likelihood of cross-contamination of samples during DNA extraction and assay preparation, and negative controls should be included throughout the sample processing. 19 It is advisable that samples representing different populations be processed separately to avoid cross-contamination. One approach commonly used to reduce potential false-positive test results is to select a quantification cycle cutoff value (e.g.…”

PCR-based Methodologies Used to Detect and Differentiate theBurkholderia pseudomalleicomplex:B. pseudomallei,B. mallei, andB. thailandensis

“…However, such assays are difficult to implement due to the high degree of optimization that is required. Overall, qPCR assays are generally considered to have a large dynamic range, low interassay variation, and high reliability (Purcell et al, 2011).…”

“…Melioidosis endemic regions, especially rice-paddy communities and indigenous tribes, may not have access to these resources. However, competition has reduced initial start-up and operating costs of PCR/qPCR systems (Purcell et al, 2011). Although disadvantages of PCRbased methods exist, alternative detection methods have their own disadvantages in that they are usually slower and less accurate.…”