Design, optimization, and application of multiplex rRT-PCR in the detection of respiratory viruses

Viral respiratory infections are common and serious diseases. Because there is no effective treatment method or vaccine for respiratory tract infection, early diagnosis is vital to identify the pathogen so as to determine the infectivity of the patient and to quickly take measures to curb the spread of the virus, if warranted, to avoid serious public health problems. Real-time reverse transcriptase PCR (rRT-PCR), which has high sensitivity and specificity, is the best approach for early diagnosis. Among rRT-PCR methods, multiplex rRT-PCR can resolve issues arising from various types of viruses, high mutation frequency, coinfection, and low concentrations of virus. However, the design, optimization, and validation of multiplex rRT-PCR are more complicated than singleplex rRT-PCR, and comprehensive research on multiplex rRT-PCR methodology is lacking. This review summarizes recent progress in multiplex rRT-PCR methodology, outlines the principles of design, optimization and validation, and describes a scheme to help diagnostic companies to design and optimize their multiplex rRT-PCR detection panel and to assist laboratory staff to solve problems in their daily work. In addition, the analytical validity, clinical validity and clinical utility of multiplex rRT-PCR in viral respiratory tract infection diagnosis are assessed to provide theoretical guidance and useful information for physicians to understand the test results.