Rapid and sensitive detection of type II porcine reproductive and respiratory syndrome virus by reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip

Highlights

• An easily performed and little equipment required method was established for detecting type II porcine reproductive and respiratory syndrome virus (PRRSV).

• The reverse transcription loop-mediated isothermal amplification (RT-LAMP) was combined with a sealed lateral flow strip.

• Labeled amplicon products were captured by the corresponding antibody coated on the detection strip as the T line and the C line, respectively.

• The whole reaction could be completed in <50 min in a completely enclosed environment.

Abstract

Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was combined with a vertical flow (VF) nucleic acid detection strip to develop a universal assay for the detection of type II porcine reproductive and respiratory syndrome virus (PRRSV). The loop primers were labeled separately with biotin and fluorescein isothiocyanate (FITC) in this assay. Using optimized parameters, the whole reaction could be completed in <50 min in a completely enclosed environment. The detection limit of this assay was found to be 1 pg RNA, 30 tissue culture infective dose 50 (TCID₅₀) virus, or 230 copies of recombinant plasmid DNA, which is relatively higher than that of RT-LAMP analyzed by agarose gel, RT-LAMP visualized by calcein, and the conventional RT-polymerase chain reaction (PCR). No false-positive results were obtained in the specificity assay. The efficiency of the RT-LAMP method was tested by analyzing 43 clinical samples, and the results were compared with those obtained by RT-PCR analysis, with the respective positive rates of 32.56% and 27.91%. This result confirmed that the method described is a rapid, accurate, and sensitive method for universal type II PRRSV detection. Also, this method can be used for the rapid detection of type II PRRSV during the early phase of an outbreak, especially for rapid veterinary diagnosis on the spot and in rural areas.

Introduction

Porcine reproductive and respiratory syndrome (PRRS) leads to reproductive failure in pregnant sows and respiratory diseases in piglets and thus tremendously affects the global swine industry (Rossow, 1998). The transmissible PRRS virus (PRRSV) is an enveloped positive-strand RNA virus. The 15.4-kb genome of this virus codes for at least 10 open reading frames (ORFs), including ORFs 1a, 1b, 2a, 2b, and 3–7. The ORFs 5, 6, and 7 encodes for three major structural proteins of PRRSV, namely, the GP5 protein, M protein, and N protein, respectively. The M protein is highly antigenic and the most conserved structural protein among the North American and European isolates (Dokland, 2010, Meulenberg et al., 1993). According to the characteristics of a genome, PRRSV can be divided into two genotypes: European (type I) and the North American (type II) (Nelsen et al., 1999). Although these two genotypes differ greatly in their antigenic properties and genetic content, both cause almost similar syndromes (Shi et al., 2010). In China, type II PRRSV is the main pathogen that causes PRRS. Notably, a highly pathogenic virus severely damaged the swine industry in China in 2006; this specific strain was identified to be a variant of type II PRRSV (Tian et al., 2007).

To reduce the economic losses caused by type II PRRSV in China, it is important to develop a rapid and effective detection method. Conventional diagnostic techniques for PRRS include direct virus isolation, immunohistochemistry, and immunofluorescence, which can detect PRRSV antigens in tissues, or an indirect immunofluorescence assay to test for anti-PRRSV antibodies (Halbur et al., 1994, Larochelle et al., 1996, Lyoo et al., 2005, Yoon et al., 1992). In recent years, several polymerase chain reaction (PCR)-based methods that are more specific and sensitive than the conventional methods have been established to detect the highly variable PRRSV genome (Egli et al., 2001, Kleiboeker et al., 2005, Suarez et al., 1994). However, false-negative results have been reported to be a major problem in testing for PRRSV by RT-PCR. Therefore, to achieve maximum accuracy in the molecular diagnosis of PRRSV, the combined use of different assays or kits is highly recommended (Wernike et al., 2012). Moreover, the time-consuming procedures and expensive equipment and reagents limit the application of RT-PCR in veterinary diagnostics, especially in rural areas. Thus, the development of a simple, rapid, and cost-effective method to detect PRRSV with high specificity and sensitivity is imperative.

Loop-mediated isothermal amplification (LAMP) is a new DNA amplification method (Notomi et al., 2000). Reverse transcription-LAMP (RT-LAMP) with high specificity and sensitivity has been used to diagnose different PRRSV variants (Gao et al., 2012, Li et al., 2009). This diagnosis method has several advantages such as allowing amplification of nucleic acids even in a normal water bath and enabling visual inspection of the results with a fluorescent dye. Notably, incubation of non-specific dyes with the reagents may give false-positive results and the SYBR Green I mixing step after the reaction may lead to subsequent contamination once the tubes are opened. Therefore, LAMP combined with a lateral flow dipstick (LFD) was developed for the detection of several viruses (Jaroenram et al., 2012, Njiru, 2011, Tsai et al., 2012). This assay exhibited high specificity, albeit the limitation that the tubes have to be opened during the probe hybridization process.

Recently, a vertical flow (VF) nucleic acid detection strip housed in a sealed plastic device for the prevention of the leakage of amplifications was introduced for isothermal helicase-dependent amplification (Chow et al., 2008). In this study, using loop primers labeled with biotin and fluorescein isothiocyanate (FITC), respectively, at the 5′-end, the RT-LAMP assay was first combined with the VF visualization strip to develop a universal assay to detect type II PRRSV. After the optimization process, the sensitivity and specificity of RT-LAMP-VF assay were compared with those of the conventional LAMP and PCR methods. Furthermore, the potential application of this assay was tested by analyzing 43 clinical samples.