Validation of a loop-mediated isothermal amplification assay for visualised detection of wild-type classical swine fever virus

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Abstract

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method applied and adapted to the detection of a number of pathogens. A LAMP assay was developed for the specific detection of wild-type classical swine fever virus (CSFV). Based on an alignment of genomic sequences of pestiviruses available in GenBank, four primers were selected targeting six positions in the NS5B gene region of the viral genome. The assay was performed in a simple water bath at a constant temperature of 62 °C, and after adding SYBR Green I, the results were visualised by the naked eye. This assay allowed easy applicability in a laboratory that is equipped simply, or even on site, close to the outbreaks and under field conditions. For confirmation, the results could also be visualised under UV light or by separation of the amplified products on 2% agarose gels. The detection limit of the assay was 2.5 medium tissue culture infectious dose (TCID50). The assay was validated with 116 clinical samples from vaccinated swineherds and 53 blood samples from experimental infections, and the results were comparable to a real-time RT-PCR assay. In summary, the LAMP assay provides a simple, rapid, and sensitive tool for the detection of wild-type CSFV under field conditions.

Introduction

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious disease that requires notification to the World Organisation for Animal Health (OIE). CSFV, together with Bovine viral diarrhoea virus types 1 and 2 (BVDV-1 and BVDV-2), and Border disease virus (BDV), belongs to the genus Pestivirus within the family Flaviviridae (Thiel et al., 2005). Pestiviruses are a group of small, enveloped, single-stranded RNA viruses. The pestiviral genome is approximately 12.5 kb in length and encodes a single, large open reading frame (ORF) flanked by two highly conserved untranslated regions (UTRs) at the 5′- and 3′-ends. The host range of CSFV is narrow; this virus is restricted to its natural hosts, domestic pigs and wild boar.

The control of CSF is based on stamping out policies and/or on vaccination. One of the vaccines used most frequently is the lapinised live vaccine HCLV strain, which was developed in the mid-1950s in China. Subsequently, the HCLV-strain-based vaccines have been used widely to control CSF in both China and many other countries. Generally, it is able to induce virtually complete immunity. However, failed immunisation against CSF has been reported, mainly due to interference from maternal antibodies and other infections (Kaden et al., 2006, Suradhat et al., 2006). Although this vaccine strain causes generally short-term viremia and is barely detectable in tissue samples other than in tonsils, viral antigens and RNA are detectable at least 2–3 weeks post-vaccination (Terpstra, 1978, Jemersic et al., 2001). There is therefore a need for developing assays for specific identification of pigs infected with wild-type virus in herds vaccinated with a lapinised live vaccine (van Oirschot, 2003).

In 2000, a novel method of nucleic acid amplification, termed loop-mediated isothermal amplification (LAMP), for rapid, specific and efficient amplification of DNA under isothermal conditions was described (Notomi et al., 2000). LAMP uses a set of four specific primers that recognise six distinct sequences on the target DNA. An inner primer containing sequences of sense and antisense strands of the target DNA initiates LAMP through a DNA polymerase. Subsequent strand displacement DNA synthesis, primed by an outer primer, releases a single-stranded DNA that serves as template for DNA synthesis primed by the second inner and outer primers, which hybridise to the other end of the target and produce a stem–loop DNA structure. In subsequent LAMP cycling, one inner primer hybridises to the loop on the product and initiates displacement DNA synthesis, yielding the original stem–loop DNA and a new stem–loop DNA with a stem that is twice as long. The cycling reaction continues with accumulation of 109 copies of the target in less than an hour. The final products are stem–loop DNA with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing alternately inverted repeats of the target in the same strand (Notomi et al., 2000). Initially, LAMP recognises the target by six distinct sequences, and then by four distinct sequences; thus, it is expected to amplify the target sequence with high selectivity. LAMP or reverse transcription LAMP (RT-LAMP) has detected a variety of pathogens, including DNA or RNA viruses, especially in resource-limited laboratories in developing countries (reviewed by Mori and Notomi, 2009).

Rapid and cost-effective RT-LAMP assays for the pre-clinical detection of CSFV are described (Chen et al., 2009, Yin et al., 2010). As these assays target both wild-type CSFV and the HCLV vaccine strain, the interpretation of the results may be complicated in terms of eradication and control where a vaccination policy is practiced.

A specific LAMP assay was therefore developed and evaluated for simple and specific detection and rapid identification of wild-type CSFV. The assay provides a powerful diagnostic tool, which can be applied easily in less well-equipped laboratories, including under field conditions.

Section snippets

Viruses and field samples

Virus strains were maintained in the Harbin Veterinary Research Institute, Harbin, China. These strains included: CSFV Shimen strain and HCLV strain; BVDV BA strain; transmissible gastroenteritis virus (TGEV) Huadu strain; porcine epidemic diarrhoea virus (PEDV) CV777 strain; porcine rotavirus (PRV) Gottfried strain; Suid herpesvirus type 1 (SHV-1, Aujeszky's disease virus; pseudorabies virus) HLJ strain; porcine parvovirus (PPV) BQ strain; porcine reproductive and respiratory syndrome virus

Performance of the LAMP assay

With the serial dilutions of the Shimen strain, the assay detected 2.5 TCID50 of CSFV per reaction, as observed from gel electrophoresis (Fig. 2A), visualised directly by the naked eye (Fig. 2B), and under UV light (Fig. 2C). The assay detected eight strains of wild-type CSFV, revealing many bands of different sizes on the agarose electrophoresis, as the LAMP products consisted of several inverted-repeat structures, but presenting negative results for HCLV strain, BVDV, and seven non-CSFV swine

Discussion

Mass vaccination of domestic pigs with HCLV strain-based vaccines is practiced in China and other developing countries. In order to perform the vaccination programmes successfully, there is a basic requirement for methods that are able to discriminate between wild-type CSFV and the vaccine strains. Currently, several assays for the detection and/or differentiation of wild-type CSFV from HCLV strain (or its derivatives) are documented (Zaberezhny et al., 1999, Li et al., 2007, Pan et al., 2008,

Acknowledgements

This study was supported by the National 973 Program (No. 2005CB523202) and National Key Project of Scientific and Technical Supporting Programs (Nos. 2006BAD06A03 and 2006BAD06A00) funded by Ministry of Science and Technology of China. SB wishes to acknowledge the support of the Award of Excellence of the Swedish University of Agriculture, SLU (2007).

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