Development of pooled testing system for porcine epidemic diarrhoea using real-time fluorescent reverse-transcription loop-mediated isothermal amplification assay

Abstract

BackgroundPorcine epidemic diarrhoea (PED) is an emerging disease in pigs that causes massive economic losses in the swine industry, with high mortality in suckling piglets. Early identification of PED virus (PEDV)-infected herd through surveillance or monitoring strategies is necessary for mass control of PED. However, a common working diagnosis system involves identifying PEDV-infected animals individually, which is a costly and time-consuming approach. Given the above information, the thrusts of this study were to develop a real-time fluorescent reverse transcription loop-mediated isothermal amplification (RtF-RT-LAMP) assay and establish a pooled testing system using faecal sample to identify PEDV-infected herd.ResultsIn this study, we developed an accurate, rapid, cost-effective, and simple RtF- RT-LAMP assay for detecting the PEDV genome targeting M gene. The pooled testing system using the RtF-RT-LAMP assay was optimized such that a pool of at least 15 individual faecal samples could be analysed.ConclusionsThe developed RtF-RT-LAMP assay in our study could support the design and implementation of large-scaled epidemiological surveys as well as active surveillance and monitoring programs for effective control of PED.Electronic supplementary materialThe online version of this article (10.1186/s12917-018-1498-9) contains supplementary material, which is available to authorized users.

Evaluating the Risk Factors for Porcine Epidemic Diarrhea Virus Infection in an Endemic Area of Vietnam

“…Then, 20 individual fecal samples from each farm were combined into two pooled samples (with a pooled size of 10 samples) for the test. Pooled samples were tested with reverse transcription (RT)–loop-mediated isothermal amplification (LAMP) ( 27 ). A “PEDV-positive farm” was defined as any swine herd with at least one positive result in two pooled samples from 20 samples collected from all-aged pigs.…”

A descriptive survey of porcine epidemic diarrhea in pig populations in northern Vietnam

“…The sensitivity of the pooled testing system indicated that a pool of up to 15 individual fecal samples could be analyzed (Mai et al 2018 ). In addition, based on preliminary data, we confirmed the efficiency of the pooled testing system for identification of PEDV-infected herds (Supplementary Data 2 ).…”

“…All samples were collected in ice boxes, transported on dry ice, and stored at − 20 °C until use. Briefly, fecal samples were diluted in phosphate-buffered saline and centrifuged at 2300× g at 4 °C for 10 min as previously described (Mai et al 2018 ). After centrifugation, 100 μL of supernatant from each pooled sample (10 animals each) were transferred to a new tube.…”

Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of porcine pegivirus

“…LAMP is a nucleic acid amplification-based method that generally requires a group of four specific external and internal primers (Notomi et al, 2000). Besides, additional loop primers can be used to further accelerate the reaction (Nagamine et al, 2002). In this study, a reverse transcription LAMP (RT-LAMP) assay was established and evaluated for surveying the prevalence of PPgV in porcine serum samples.…”

“…RT-LAMP is considered to be an alternative diagnostic tool in terms of its ability to rapidly amplify target nucleotide sequence(s) under isothermal conditions. LAMP has been widely used to detect a variety of important swine pathogens, including PDCoV (Zhang et al, 2017) and PEDV (Mai et al, 2018). In the present study, the RT-LAMP reaction conditions were optimized in terms of reaction time and temperature.…”