Development of a real-time reverse transcription loop-mediated isothermal amplification method for the rapid detection of porcine epidemic diarrhea virus

Abstract

BackgroundPorcine epidemic diarrhea (PED) is an acute and highly contagious enteric disease characterized by severe enteritis, vomiting and watery diarrhea in swine. Recently, the outbreak of the epidemic disease has been a serious problem in swine industry. The objective of this study is to develop a rapid, sensitive, and real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of porcine epidemic diarrhea virus (PEDV) in less equipped laboratories.ResultsThe optimal reaction condition of the current real-time RT-LAMP for PEDV was 62 °C for 45 min. It was capable of detecting PEDV from clinical samples and differentiating PEDV from several related porcine viruses, while it did not require additional expensive equipment. The minimum detection limit of the real-time RT-LAMP assay was 0.07PFU per reaction for PEDV RNA, making this assay approximately 100-fold more sensitive than that of one-step RT-PCR. By screening a panel of clinical specimens, the results showed that this method presented a similar sensitivity with real-time RT-PCR and was somewhat sensitive than one-step RT-PCR in detection of clinical samples.ConclusionsIn this study, we have developed a new real-time RT-LAMP method, which is rapid, sensitive and efficient to detect PEDV.This method holds great promises not only in laboratory detection and discrimination of PEDV but also in large scale field and clinical studies.

Development of a real time reverse transcription loop-mediated isothermal amplification method (RT-LAMP) for detection of a novel swine acute diarrhea syndrome coronavirus (SADS-CoV)

“…SADS-CoV N gene was specifically amplified with a detection limit of 1.0 × 10 1 copies/μL and had good reproducibility. The sensitivity of the LAMP assay is similar to real-time RT-PCR (Yu et al, 2015). In this study, the RT-LAMP correctly identified 20 out of 24 samples as positive, showing similar diagnostic sensitivity comparable to the real time RT-PCR.…”

“…LAMP technology is currently used for the nucleic acid detection of a number of animal pathogens (Niessen et al, 2013;Sahoo et al, 2016). The assay offers multiple advantages compared to conventional PCR including a shorter turnaround time (30-60 min) and a sensitivity 1-2 orders of magnitude higher (Huang et al, 2016;Yu et al, 2015). In the present study, the real-time RT-LAMP method was used to detect SADS-CoV and did not cross-react with other porcine pathogens.…”

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

“…The PEDV M protein is a highly conserved trans-membrane protein that is the most abundant envelope component [13,14]. Two reports have shown that the developed RT-LAMP method for the PEDV M gene has a higher sensitivity than the RT-LAMP method developed for the N gene [15,16]. The use of LAMP for detecting PEDV has been reported [15][16][17].…”

Reverse transcriptase loop-mediated isothermal amplification for the detection of feline coronavirus

“…Loop-mediated isothermal amplification (LAMP) is an amplification gene technique developed some years ago (Notomi et al, 2000) which allows to amplify nucleic acids in an hour and under isothermal conditions, and to evaluate the results by observation of the turbidity of the reaction or using different dyes. Reverse transcriptase LAMP (RT-LAMP) has been used to amplify the genome of several coronaviruses of both humans and animals (Amer et al, 2013;Bhadra et al, 2015;Cardoso et al, 2010;Hanaki et al, 2013;Nemoto et al, 2015;Pyrc et al, 2011;Thai et al, 2004;Yu et al, 2015). To our knowledge, RT-LAMP has never been used for the identification of FCoVs.…”