Rapid and sensitive detection of porcine epidemic diarrhea virus by reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip

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Abstract

Porcine epidemic diarrhea virus (PEDV) is an important pathogen that causes vomiting, diarrhea, and dehydration, leading to serious damage to the swine industry worldwide. The establishment of effective diagnostic methods is imperative. However, traditional methods are often unsuitable. In this study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) was combined with a vertical flow (VF) nucleic acid detection strip to detect PEDV. Parameters that affect the RT-LAMP reaction were optimized. The RT-LAMP-VF assay that we established was performed at 62 °C for 40 min, and then directly evaluated on the VF visualization strip cassette. The method demonstrated high specificity for PEDV. The detection limit was 10 pg of ribonucleic acid, consistent with RT-PCR, RT-LAMP detected products on agarose gels and by direct calcein fluorescence. Application of this method to clinical samples yielded a positivity rate that was comparable to that obtained for 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.

Introduction

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 confused with transmissible gastroenteritis virus (TGEV), as both of them can cause watery diarrhea [2]. PEDV is an enveloped virus, whose genome contains ∼28 kb of positive-sense, single-stranded ribonucleic acid (RNA) with a 5′ cap and a 3′ polyadenylated tail. The RNA comprises a 5′ untranslated region, a 3′ untranslated region, and at least seven open reading frames (ORFs). They are arranged in the genome in the order 5′-replicase (1a/1b)-S-ORF3-E-M-N-3′. The spike (S), envelope (E), membrane (M), and nucleocapsid (N) genes encode four separate structural proteins, while replicase 1a and 1b as well as the ORF3 gene encode 3 separate non-structural proteins [3], [4].

The similar clinical signs associated with PEDV and TGEV infections are problematic for the differential clinical diagnosis of PED, necessitating the establishment of effective laboratory diagnostic methods [5]. Virus isolation is considered the standard method, but PEDV is not easily adapted to cell lines. Although the addition of trypsin offers a possible solution, the viral titer of PEDV in cell lines remains low [6]. Other methods, such as immuno-electron microscopy, immunofluorescence and immunohistochemical techniques, have also been applied to detect PEDV [7], [8]. In addition, serum neutralization assays and enzyme-linked immunosorbent assay (ELISA)-based techniques have been proven effective for the detection of antibodies against PEDV [9], [10], [11]. In situ hybridization and polymerase chain reaction (PCR)-based methods possess the advantages of high specificity and sensitivity [12], [13], [14], [15]. However, both of them require either high-precision instruments or well-trained personnel to operate. Therefore, they are unsuitable for use in practice and in poorly equipped laboratories.

Loop-mediated isothermal amplification (LAMP) is a deoxyribonucleic acid (DNA) amplification method, with high specificity and sensitivity reported [16]. The method has many advantages, including accomplishing amplification in an ordinary water bath and yielding results that can be visually examined after the addition of a fluorescent dye. RT-LAMP has been used in the diagnosis of PEDV [17]. However, the use of dyes, such as calcein or picogreen, incubated with the reagent may cause false positive results, and the addition of SYBR Green I after the reaction may lead to subsequent contamination once the tubes are opened. LAMP combined with the use of a lateral flow dipstick has recently been developed for the detection of several viruses [18], [19], [20]. Despite remarkable specificity, this method had the shortcoming of a leakage of amplification products when the labeled probe was added to the LAMP reaction tube for hybridization, and hybridized product was added to the assay buffer in a new tube for lateral flow dipstick detection.

Recently, a vertical flow nucleic acid detection strip housed in a sealed plastic device called vertical-flow (VF) visualization strip cassette was designed to control the contamination of amplicons [21]. In this study, we combined RT-LAMP with the VF visualization strip to develop a convenient method for the diagnosis of PEDV. This method showed good sensitivity and specificity in the experimental and clinical tests.

Section snippets

Viruses

PEDV (CV777 strain), TGEV (attenuated H strain), porcine reproductive and respiratory syndrome virus (PRRSV; isolate 08-2) and Escherichia coli were obtained from the repository in the College of Veterinary Medicine, South China Agriculture University. Porcine circovirus (PCV; LG strain) and pseudorabies virus (PRV; Bartha-K61 strain) are commercial vaccines that were purchased from Shanghai Hile Bio-Pharmaceutical in China.

Clinical samples collection, and testing

A total of 20 clinical intestinal samples were collected from different

Initial RT-LAMP-VF assay

In the initial RT-LAMP-VF assay, the tubes were incubated at 61 °C for 60 min and then directly determined using the VF visualization strip cassette. The procedure is shown in Fig. 2A. Once the handle of the detection chamber is pressed, with the cartridge reaching the bottom of the detection chamber, the razor blade cuts the PCR tube and the plastic pin pricks a bulb contained the running buffer. The amplicon products mix with the running buffer and flow to the DNA detection strip lodged in

Discussion

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 [24]. Traditional techniques, such as virus isolation and immunofluorescence, have been proved high specificity and low sensitivity [5], [7], [14]. Although PCR- and ELISA-based techniques demonstrate good specificity and sensitivity, high-precision instruments and complicated

Authors' contributions

HG carried out the design of primers, participated in the collection of clinical samples and drafted the manuscript. JD carried out the optimization of this method. JW participated in the analysis of specificity and sensitivity. JP performed the statistical analysis. WL helped to draft the manuscript. MZ participated in the design of the study. JC conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.

Acknowledgments

This research was supported by grants from the Special Fund for Agro-Scientific Research in the Public Interest (No. 201203056), the National Natural Science Foundation of China (Nos. U1405216, 31172321 and 31472200), and the Special Project for Scientific and Technological Innovation in Higher Education of Guangdong, China (No. 2012CXZD0013). All authors have seen the final version of this manuscript and approve it for publication. We ensure that this original manuscript has not been published

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    These authors contributed equally to this work.

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