Development of a Rapid Reverse Transcription-Recombinase Polymerase Amplification Couple Nucleic Acid Lateral Flow Method for Detecting Porcine Epidemic Diarrhoea Virus

Abstract

Porcine epidemic diarrhea virus (PEDV) infection is an important acute diarrheal disease of swine that results in economic and industrial losses worldwide. The clinical manifestations in infected piglets are severe diarrhea, dehydration with milk curd indigestion, leading to death. The diagnosis of PEDV is essential for monitoring and managing the disease. PEDV can be detected and identified by serology and the nucleic acid of the virus in clinical samples. Therefore, a novel isothermal amplification and detection technique, reverse transcription-recombinase polymerase amplification couple nucleic acid lateral flow (RT-RPA-NALF) was developed for the rapid detection of PEDV. Qualitative reverse transcription-polymerase chain reaction (RT-qPCR) was established as the gold standard assay to compare results. Specific primer pairs and probes were designed, and RT-RPA conditions were optimized to amplify the M gene of PEDV. The established RT-RPA-NALF assay could finish in 25 min at a temperature of 42 °C and the amplicon interpreted by visual detection. The developed RT-RPA-NALF assay was specific to the M gene of PEDV, did not detect other common swine diarrhea pathogens, and showed minimal detection at 102 TCID50/mL PEDV. The RT-RPA-NALF assay can detect PEDV in 5 simulated fecal samples. Furthermore, in 60 clinical fecal samples, the results of RT-RPA-NALF correlated with RT-qPCR assay, which provides sensitivity of 95.65% and specificity of 100%, with a coincident rate of 98.33%. The rapid RT-RPA-NALF is simple and rapid, increases high sensitivity, and can be used in the field.

Development of DNA-Based Lateral Flow Assay for Detection of LDLR Gene Mutation for Familial Hypercholesterolemia

“…The reverse transcriptase polymerase chain reaction method was used to validate the results. The study results showed that the RT-RPA-NALF can detect the amplicon virus within 25 min at a temperature of 42 °C, with a sP and sN of 100% and 95.65%, respectively, with a 98.33% coincidence rate ( 42 ).…”

Pyrococcus furiosus Argonaute-mediated porcine epidemic diarrhea virus detection

“…Currently, numerous techniques for detecting PEDV have been reported, including conventional virus isolation (Valkó et al 2019 ), enzyme-linked immune sorbent (ELISA) (Gerber et al 2014 ), and molecular biological techniques (Kim et al 2007 ). Virus isolation and identification is a laborious process that takes a lot of time and necessitates specialized equipment and personnel; ELISA has been widely used to detect the level of antibodies in serum, but it was not a good choice for early diagnosis of viral infection as antibody generation required a few days, it was also laborious and time-consuming (Pewlaoo et al 2022 ; Wang et al 2020 ). Several molecular biological techniques commonly employed for PEDV diagnostics are quantitative polymerase chain reaction (qPCR) (Pan et al 2020 ), recombinase polymerase amplification (RPA) (Li et al 2021b ), and loop-mediated isothermal amplification (LAMP) (Yu et al 2015 ).…”