Propidium Monoazide Combined With RT-qPCR Detects Infectivity of Porcine Epidemic Diarrhea Virus

Abstract

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) allows sensitive detection of viral particles and viruses in epidemic samples but it cannot discriminate noninfectious viruses from infectious ones. Propidium monoazide (PMA) coupled with quantitative polymerase chain reaction (qPCR) was assessed to detect infectious viruses. Currently, there is no established test method to detect the infection of the porcine epidemic diarrhea virus (PEDV). In this study, propidium monoazide coupled with qPCR detects infectivity of PEDV. We optimized the method from the selection of primers, the working concentration of PMA, and the inactivation method using heat or ultraviolet (UV). The viruses which were treated with PMA before qPCR were inactivated using heat or UV. However, the addition of PMA alone did not affect the detection of live viruses, which indicates that a viral capsid break may be essential for PMA to bind to the genome. A repetition of the method on naked PEDV RNA suggests that it can be used to detect potentially infectious PEDV. The results indicated that an optimal plan of PMA could be extremely useful for evaluating infectious and noninfectious viruses.

Propidium monoazide PCR, a method to determine OsHV-1 undamaged capsids and to estimate virus Lethal Dose 50

“…Inside the cells, the dye can intercalate into nucleic acids and inhibit PCR amplification. The PMA binding mechanism has previously been used to differentiate between live and dead microorganisms including bacteria, fungi and parasites ( Brescia et al., 2009 ; Nocker et al., 2009 ; Pan and Breidt, 2007 ; Rousseau et al., 2019 ; Vesper et al., 2008 ), or between infectious and non-infectious viruses ( Canh et al., 2021 , 2022 ; Coudray-Meunier et al., 2013 ; Fongaro et al., 2016 ; Fittipaldi et al., 2010 ; Kim and Ko, 2012 ; Lee et al., 2015 ; Leifels et al., 2015 , 2021 ; Liang et al., 2021 ; Moreno et al., 2015 ; Parshionikar et al., 2010 ; Prevost et al., 2016 ; Sanchez et al., 2012 ). Thus, PMA-qPCR could potentially be used to measure the integrity of OsHV-1 particles.…”

Selective Elucidation of Living Microbial Communities in Fermented Grains of Chinese Baijiu: Development of a Technique Integrating Propidium Monoazide Probe Pretreatment and Amplicon Sequencing

“…PMA is a compound extensively employed in the surveillance of viable microorganisms [ 42 ]. At present, PMA combined with amplicon sequencing technology has been successfully applied to the analysis of the structure of living microbial communities in soil, sewage and human gut [ 20 , 43 , 44 ].…”

Triplex-Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Immunoassay for the Simultaneous Detection of Three Pathogens of Porcine Viral Diarrhea Syndrome in Swine

“…Laboratory diagnostic methods, which are commonly used for porcine viral diarrhea detection, include the virus isolation method [ 33 ], enzyme-linked immunoadsorption assays (ELISAs) [ 34 ], indirect immunofluorescence assays (IFAs) [ 35 ], neutralization assays (NTs) [ 36 ], and real-time fluorescence quantitative PCR (rt-qPCR) [ 37 ]. The virus isolation method is time-consuming and complicated [ 38 ].…”