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.
Highlights
Porcine epidemic diarrhea (PED) is an acute and highly contagious enteric disease characterized by severe enteritis, vomiting and watery diarrhea in swine
PED is caused by porcine epidemic diarrhea virus (PEDV), which was firstly identified in Belgium in 1978 [2]
Primer set screening for real-time reverse transcription loop-mediated isothermal amplification (RT-loop-mediated isothermal amplification (LAMP)) assay To select the optimal primer set for the real-time RTLAMP assay, the primer set screening assay was investigated at 63 °C for 45 min using the LA-320C Loopamp real-time turbidimeter (Teramecs, Japan)
Summary
Porcine epidemic diarrhea (PED) is an acute and highly contagious enteric disease characterized by severe enteritis, vomiting and watery diarrhea in swine. 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. Porcine epidemic diarrhea (PED) is an acute, highly contagious and devastating enteric disease characterized by severe enteritis, vomiting and watery diarrhea in swine [1]. PED is caused by porcine epidemic diarrhea virus (PEDV), which was firstly identified in Belgium in 1978 [2]. PEDV has suddenly emerged in the United States and rapidly spread across the country, resulting in high mortality in infected newborn piglets, which have posed serious economic losses to the swine industry in the USA [7, 8]. Several conventional methods are available for the detection of PEDV, including virus isolation, fluorescence
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