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.
At present, there is no effective experimental method for detecting whether the suid herpesvirus 1 (SHV-1) detected in pigs is infectious. Although the technique of quantitative polymerase chain reaction (qPCR) has significantly improved the detection rate and accuracy of the disease, it does not differentiate between infective and non-infective status of the virus. Propidium monoazide (PMA) is a dye that can be combined with DNA molecules. The decomposition of PMA produces an azene compound covalently crosslinked with DNA molecules, thereby inhibiting PCR amplification of DNA. In this study, the combination of PMA and qPCR was used to determine the infectivity of SHV-1. We optimized the method from the selection of primers, the working concentration of PMA, and the method of inactivation using UV or heat inactivation. We found that when specific primer 1 was used and a PMA working concentration was 50–100 μM, heat inactivation was able to distinguish whether SHV-1 was infectious or not. We also showed that UV prevented the virus from replicating, it did not destroy the capsid of the virus, and therefore, PMA cannot enter the virus and bind to the nucleic acid of the virus. Consequently, there is no way to identify the infectivity of the virus using UV inactivation. The study showed that the method was stable and the detection rate reached 96%. In conclusion, this method exhibited strong specificity and high sensitivity and can identify the infectivity of SHV-1. This method has practical significance for clinical virus isolation and the effects of disinfection of farms.
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