Abstract Porcine epidemic diarrhea， a highly contagious enteric infectious disease caused by the porcine epidemic diarrhea virus （PEDV） with symptoms of vomit， diarrhea， loss of appetite of suckling pig， has led to serious economic loss to the global swine industry. In this study， a real-time fluorescence reverse transcription loop-mediated isothermal amplification （RT-LAMP） assay was developed to detect PEDV RNA. The real-time fluorescence RT-LAMP assay was performed at 62 ℃ for 60 min， using a simple and portable device， the ESE-Quant Tube Scanner. The detection limit of RNA was 2.9×106 copies/μl， 10 times as sensitive as RT-PCR， and the detection was specific only to PEDV. Application of this method to clinical samples yielded a positivity rate of 93%， which was higher than that of RT-PCR. This technique saves time and is efficient， and is thus expected to be useful for the diagnosis of PEDV infection in the field.
　　Key words Porcine epidemic diarrhea virus； Real-time fluorescence RT-LAMP； Detection
　　Porcine epidemic diarrhea （PED）， which is characterized clinically by vomiting， diarrhea， and dehydration， causes serious damage to the swine industry worldwide[1]. Its causative agent， porcine epidemic diarrhea virus （PEDV）， is easily confound with transmissible gastroenteritis virus （TGEV）， as both of them can cause watery diarrhea[2]. PEDV is an enveloped， single-strand， and positive-sense RNA virus， which belongs to the family Coronaviridae[3]. The PEDV genome is 28 kb in length and comprised of a 5′untranslated region （UTR）， a 3′ UTR， and at least seven open reading frames （ORFs） that encode four structural proteins[spike（S）， envelope（E）， membrane（M）， and nucleocapsid（N）][4] and three non-structural proteins （replicase 1a and 1b， and ORF3）[5]. In China， the first outbreak of PEDV was noticed in Shanghai in 1973. Until now， PEDV infection has been observed on most swine breeding farms in most provinces since late 2010[6]， and has caused serious economic losses caused to swine industry in China[7]. The rapid detection of PEDV would provide important information for the control of PEDV circulation. The currently available diagnostic methods for PEDV include virus isolation， haemagglutination test， fluorescence assay， enzyme linked immunosorbent assay （ELISA） and molecular biological characterization[8]， such as reverse transcription-polymerase chain reaction （RT-PCR） and real-time RT-PCR assays[9]. However， these methods have some disadvantages such as time-consuming and the requirement for special instruments. With this in mind， a rapid， simple， sensitive， and more cost-effective assay for diagnosis of PEDV in clinical samples is required. 　　Loop-mediated isothermal amplification （LAMP） is a novel method that can be used to rapidly amplify a specific nucleic acid with high specificity under isothermal conditions， with the use of four to six specifically designed primers[10]. This technique can amplify target nucleic acids from a few to 109 copies within 1 h based on the auto-cycling strand displacement DNA synthesis activity of Bst DNA polymerase large fragment[10]. RT-LAMP has been developed for detection of several RNA viruses[11]. The results are visible to naked eyes when a nucleic acid stain （SYBR Green） is added. However， such method is of limited sensitivity compared with the fluorescence-based LAMP. In this study， we used the simple portable ESE-Quant Tube Scanner device that contains both the amplification platform and the fluorescent detection unit. This conveniently portable detection system for simple and rapid real-time fluorescence RT-LAMP assay provides promising applications in both clinical diagnosis and field surveillance of PEDV.
　　Materials and Methods
　　Virus
　　PEDV LX strain used for the study was isolated and identified in the laboratory of Hebei Engineering and Technology Research Center of Veterinary Biotechnology. It was treated and then inoculated into Vero cells containing a final concentration of 50 mg/L trypsin for virus isolation at 37 ℃ under 5% CO2.
　　Design of primers
　　The real-time fluorescence RT-LAMP primers were designed to the conserved sequences of the N gene （GenBank accession No. JX406145.1） using the Primer Explorer version 4 software （http：//primerexplorer. jp/ elamp 4.0/index. html）， and synthesized by Shanghai Sangon Co.， Ltd. The primers included two outer primers （forward outer primer F3 and reverse outer primer B3）， two inner primers （forward inner primer FIP and reverse inner primer BIP）， and two loop primers （loop primer LF and loop primer LB） （Table 1）.
　　RNA extraction
　　Viral RNA was extracted from 200 μl of supernatant from virus-infected Vero cells or tissue samples using the RNA extraction kit （Qiagen Inc.， USA） following the manufacturer’s instruction. The extracts were resuspended in 25 μl of distilled water， aliquoted and stored at -80 ℃ before the real-time fluorescence RT-LAMP amplification was carried out.
　　The real-time fluorescence RT-LAMP assay was performed in a 25 μl reaction mixture that contained 40 pmol of the inner primers （FIP and BIP）， 5 pmol of the outer primers （F3 and B3）， 20 pmol of the loop primers （LF and LB）， 0.5 μl AMV reverse transcriptase （Transgen， Beijing， China）， 2 μl extracted RNA template， 1 μl calcein fluorescence dye （Invitrogen， Shanghai， China）， 8 units of Bst DNA polymerase （New England Biolabs， Beijing， China）， 5 mmol/L MgSO4， 2.5 μl 10×Bst DNA Buffer. Amplification and detection were performed with an ESE Quant tube scanner （ESE Gmbh， Stockach， Germany） at 62 ℃ for 60 min. The tests were performed in duplicate more than three times. 　　Specificity of the real-time fluorescence RT-LAMP
　　To evaluate the specificity of the real-time fluorescence RT-LAMP assay， amplification was performed using the optimized reaction parameters （Table 2） on a PEDV strain LX. In addition， porcine rotavirus （PRV）， Lawsonia intracellularis （LI） and transmissible gastroenteritis virus （TGEV） were included. The specificity was confirmed by ESE Quant tube scanner.
　　Sensitivity of the real-time fluorescence RT-LAMP
　　To examine the sensitivity of the real-time fluorescence RT-LAMP for PEDV amplification， real-time fluorescence RT-LAMP， visual RT-LAMP and RT-PCR reactions were conducted using various concentrations of PEDV RNA as template. PEDV RNA was extracted from the culture supernatants of infected cells by using EasyPureR Viral DNA/RNA Kit. The RNA was quantified by NanoDrop 2000e， diluted serially 10-fold from 2.9×1011 to 2.9×105 copies/μl and used as the template for the three methods. Real-time fluorescence RT-LAMP and visual RT-LAMP was performed using the optimized reaction parameters. The sensitivity was confirmed by ESE Quant tube scanner. RT-PCR was performed using PEDV-specific primers （N-F 5′-ACG GGT GCC ATT ATC CCT CTA T-3′；N-R5′-GAC TGG TTG TTG CCT CTG TTG T-3′）. Briefly， the conventional RT-PCR assay for the detection of PEDV was performed in a 25 μl reaction mixture containing 2 μl cDNA， 12.5 μl conventional PCR Mixture， 0.5 μl Taq DNA polymerase， and 10 μmol/L each primer. The thermocycling program consisted of an initial step at 94 ℃ for 3 min， followed by 30 cycles denaturation at 94 ℃ for 30 s， annealing at 55 ℃ for 30 s， and extension at 72 ℃ for 30 s. Five microliters of RT-PCR products were analyzed by agarose gel electrophoresis.
　　Clinical samples
　　Sixty clinical samples （including feces and intestinal samples） were collected from different pig farms in Hebei Province. The samples were homogenized in 0.9% normal saline （NS） and centrifuged for 5 min at 9 000 r/min and 4 ℃ to obtain a cell-free supernatant. The sample RNAs were extracted as described above. The sixty clinical samples were tested by both the real-time fluorescence RT-LAMP and RT-PCR assays.
　　Results and Analysis
　　Optimization of the real-time fluorescence RT-LAMP assay
　　In this study， a real-time fluorescence RT-LAMP assay using the ESE Quant tube scanner system was developed and optimized for the proper detection of PEDV. During real-time fluorescence RT-LAMP optimization， 2 μl RNA was used as the template， and only one variable was changed at a time. More amplified products were obtained when the temperature varied between 61 and 64 ℃， and 62 ℃ was optimal （data not shown）. In addition， the yield of products changed significantly with different ratios of outer and inner primers. The optimal ratio was 1∶4， for which 0.5 μl of each outer primer was sufficient for the activation of the reaction. The optimized reaction parameters are given in Table 2. The real-time fluorescence RT-LAMP amplification was finished within 1 h by using ESE-Quant Tube Scanner. 　　Specificity of real-time fluorescence RT-LAMP
　　The specificity of the real-time fluorescence RT-LAMP assay was evaluated using other viruses， and water in the negative control. Amplification curves were observed only in the detection of PEDV. No amplification was found with all other viruses tested and the water control after 60 min of incubation （Fig.1）.
　　Sensitivity of real-time fluorescence RT-LAMP
　　The sensitivity of the real-time fluorescence RT-LAMP assay was evaluated by testing 10-fold serial dilutions of RNA templates （2.9×1011- 2.9×105 copies/μl）. The detection limit of real-time fluorescence RT-LAMP and visual RT-LAMP assay was 2.9×106 copies/μl （Fig. 2 and Fig. 3）， whereas that of RT-PCR was 2.9×107 copies/μl （Fig. 4）. Comparisons between the RT-PCR amplification indicated that real-time fluorescence RT-LAMP and visual RT-LAMP assay are 10-fold more sensitive than RT-PCR.
　　Detection of PEDV in clinical samples
　　Real-time fluorescence RT-LAMP and conventional RT-PCR were preformed simultaneously on 60 clinical samples. The results are shown in Table 3. Fifty of sixty samples （83%） were positive by RT-PCR analysis， whereas fifty-six of sixty samples （93%） were positive by real-time fluorescence RT-LAMP （Table 3）. Fifty samples （83%） were positive by both methods. Six samples （10%） were positive by real-time fluorescence RT-LAMP， but negative by RT-PCR analysis. No sample （0%） was positive by RT-PCR and negative by real-time fluorescence RT-LAMP. The results showed that real-time fluorescence RT-LAMP was more sensitive than conventional RT-PCR assay.
　　Discussion and Conclusions
　　Porcine epidemic diarrhea virus （PEDV） was initially recognized as a pathogen in Europe in 1971， followed by its introduction into Asia and， in 2013， North America[12]. Clinical signs include acute vomiting， anorexia and watery diarrhea in pigs of all ages， with up to 90%-95% mortality in suckling pigs[12]. PEDV belongs to the family Coronaviridae， which includes transmissible gastroenteritis virus （TGEV） and porcine respiratory coronavirus （PRCV）. At present， the control of PEDV infection primarily depends on the early identification to prevent the further spread of the virus. Therefore， accurate diagnostic methods are useful to assess and monitor the transmission of pathogens， which is particularly important for PEDV， considering that its clinical signs are similar to those caused by other viruses[1]. Traditional techniques， such as virus isolation and immunofluorescence， have been proven to have high specificity but low sensitivity[13]. Although PCR and ELISA-based techniques demonstrate good specificity and sensitivity， high-precision instruments and complicated procedures restrain their application for clinical diagnosis[14]. In this study， we combined RT-LAMP with ESE Quant tube scanner to detect PEDV. The ESE Quant tube scanner is comparable to the real-time PCR machine in the sense that both are capable of detecting samples in real-time as well as analysis of the melting curves using a computer with the corresponding software. To reach maximum accuracy of detection， the primers were designed from the sequence of the highly conserved N gene （GenBank accession No. JX406145.1）. In order to reach high specificity and sensitivity in the reaction， the real-time fluorescence RT-LAMP assay parameters were optimized. The inner and outer primers， working in combination to start the reaction， had a positive effect. It has been reported that the inclusion of loop primers improves the efficiency of LAMP[15]， which was confirmed in the present assay. It was also reported that， while optimizing the concentrations of Mg2+ can accelerate the reaction to some extent， excessive concentrations could have a negative effect on the specificity of the real-time fluorescence RT-LAMP. Interestingly， once dNTPs and Bst DNA polymerase were abundant， the yield of products decreased， indicating that excessive Bst DNA polymerase might reduce the specificity of the reaction. No false positive test results were observed in the specific assay due to the use of six primers targeting amplified regions[10]. In contrast to the use of gel electrophoresis to detect LAMP products， ESE Quant tube scanner does not require that reaction tubes are opened， thus minimizing the potential risks for exposing the laboratory and subsequent experiments to potential cross-contamination. Moreover， the real-time fluorescence RT-LAMP method also avoids the use of the carcinogen ethidium bromide. The results from RT-LAMP visualized by calcein can be readily confirmed by the real-time fluorescence RT-LAMP. Comparisons between the real-time fluorescence RT-LAMP and conventional RT-PCR amplification indicated that real-time fluorescence RT-LAMP is 10-fold more sensitive than conventional RT-PCR. However， the use of the lateral flow dipstick has the shortcoming of a leakage of amplification products when the labeled probe was added to the LAMP reaction tube for hybridization， it may cause false positive results. Using extracted RNA from a clinical sample， the RT-PCR assay took about 4 h to yield a result， but the real-time fluorescence RT-LAMP assay was faster， which needed only 15-60 min for amplification of PEDV specific sequences. 　　In summary， RT-LAMP assay， owing to its high sensitivity， specificity and rapidity， can be considered as a highly promising and effective tool for large-scale screening of PEDV in clinical， which is an increasingly important issue worldwide. In addition， it serves as a model platform that could be adapted easily to detect other pathogens using the ESE Quant tube scanner.
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