Multiplex PCR: Optimization and Application in Diagnostic Virology

Abstract

SUMMARY PCR has revolutionized the field of infectious disease diagnosis. To overcome the inherent disadvantage of cost and to improve the diagnostic capacity of the test, multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than one pair of primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction tube have been described. Early studies highlighted the obstacles that can jeopardize the production of sensitive and specific multiplex assays, but more recent studies have provided systematic protocols and technical improvements for simple test design. The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids. This article reviews the principles, optimization, and application of multiplex PCR for the detection of viruses of clinical and epidemiological importance.

Comparison of fast-track diagnostics respiratory pathogens multiplex real-time RT-PCR assay with in-house singleplex assays for comprehensive detection of human respiratory viruses

“…of respiratory virus infections (Beck and Henrickson, 2010;Kehl and Kumar, 2009), but the large and increasing number of viruses makes laboratory testing with individual (singleplex) virus assays challenging. Conversely, multiplex PCR assays that combine multiple individual assays in a single reaction facilitate more rapid, high throughput and cost-effective testing and are generally preferred in the clinical setting (Elnifro et al, 2000;Jansen et al, 2011).…”

Diagnostic et surveillance épidémiologique des infections grippales et à virus respiratoire syncytial: intérêt de la PCR multiplex

“…Depuis quelques années, il a été montré que l'utilisation de la PCR (poymerase chain reaction) pouvait améliorer le diagnostic des viroses respiratoires en alliant sensibilité, spécificité et rapidité [5]. La PCR multiplex présente l'avantage de permettre, à partir du même échantillon, l'amplification de plusieurs séquences génomiques virales différentes [6].…”

Integrated Capillary Electrophoresis Microsystem for Multiplex Analysis of Human Respiratory Viruses

“…A lower sensitivity obtained from the RNA samples compared to their DNA counterparts resulting from the known limited efficiency of reverse transcription has been previously observed [1]. In particular, for a multiplex one-step RT-PCR where more than one virus-specific RT primer is present, the optimal choice of RT conditions can become more difficult than in hexamer primed reactions [6]. Nevertheless, the results obtained here demonstrate the ability of the microdevice to process RNA samples, which opens the door to other RNA-based applications such as detection and genotyping of other viruses, HIV viral load testing, and gene expression studies.…”