Validation of a loop-mediated isothermal amplification assay for visualised detection of wild-type classical swine fever virus
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).
References (17)
- et al.
Rapid pre-clinical detection of classical swine fever by reverse transcription loop-mediated isothermal amplification
Mol. Cell Probes
(2009) - et al.
Differentiation of C-strain “Riems” or CP7_E2alf vaccinated animals from animals infected by classical swine fever virus field strains using real-time RT-PCR
J. Virol. Methods
(2009) - et al.
A multiplex nested RT-PCR for the detection and differentiation of wild-type viruses from C-strain vaccine of classical swine fever virus
J. Virol. Methods
(2007) - et al.
Development of a primer-probe energy transfer real-time PCR assay for improved detection of classical swine fever virus
J. Virol. Methods
(2009) - et al.
Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases
J. Infect. Chemother.
(2009) - et al.
Negative impact of porcine reproductive and respiratory syndrome virus infection on the efficacy of classical swine fever vaccine
Vaccine
(2006) - et al.
Phylogenetic comparison of classical swine fever virus in China
Virus Res.
(2001) Vaccinology of classical swine fever: from lab to field
Vet. Microbiol.
(2003)
Cited by (18)
Veterinary Medicine, Eleventh Edition
2016, Veterinary Medicine, Eleventh EditionClassical swine fever in China: A minireview
2014, Veterinary MicrobiologyCitation Excerpt :Further large-range surveillance is needed in the future. Different assays (Table 1), such as RT-PCR, RT-nested PCR, RT-nested PCR based restriction fragment length polymorphism (RFLP), real-time RT-PCR, and RT-LAMP have been developed in China for detection of CSFV or/and differentiation of wild-type CSFV and C-strain (Li et al., 2007b; Chen et al., 2010; Zhao et al., 2008; Zhang et al., 2010). A triplex TaqMan real-time RT-PCR assay has been established for differential detection of wild-type and vaccine strains (C-strain) of CSFV and BVDV-1 (Zhang et al., 2012).
Development of a loop-mediated isothermal amplification assay combined with a lateral flow dipstick for rapid and simple detection of classical swine fever virus in the field
2014, Journal of Virological MethodsCitation Excerpt :In addition, the LFD readout also improved RT-LAMP detection of viral RNA in certain samples. Compared with previous studies (Chen et al., 2009; Yin et al., 2010; Zhang et al., 2010), the new assay was evaluated with a broad range of CSFV genotypes and a well-defined RNA panel of different pestiviruses. Thus, the new RT-LAMP-LFD assay has a great potential to be used in the field for the rapid and sensitive detection of CSFV.
Development of a rapid and specific loop-mediated isothermal amplification detection method that targets Marek's disease virus meq gene
2012, Journal of Virological MethodsCitation Excerpt :LAMP assay has been reported to be highly specific, sensitive, rapid, and cost-effective (Han and Ge, 2008; Hara et al., 2005; Parida et al., 2004; Yeh et al., 2006). This assay has been used previously for the identification of numerous viruses that include Newcastle disease virus (NDV) (Pham et al., 2005), pseudorabies virus (En et al., 2008), porcine reproductive and respiratory syndrome virus (Li et al., 2009), infectious bursal disease virus (Xu et al., 2009), infectious laryngotracheitis virus (ILTV) (Qing et al., 2010), reticuloendotheliosis virus (REV) (Deng et al., 2010), wild-type classical swine fever virus (Zhang et al., 2010) and influenza A (H1N1) virus 2009 (Ma et al., 2010). However, the use of LAMP for detection of MDV has not been reported.
Evaluation of a real-time RT-PCR assay using minor groove binding probe for specific detection of Chinese wild-type classical swine fever virus
2011, Journal of Virological MethodsCitation Excerpt :However, these viruses have never been isolated in China, suggesting that they would have little effect on the performance of this assay in China. To minimize the impact of such limitations, the samples should be co-detected by the wt-rRT-PCR and another differential diagnostic assay, such as loop-mediated isothermal amplification assay (Zhang et al., 2010), by which these viruses can be detected. Although virus isolation is considered to be the diagnostic standard for CSF (De Smit et al., 1999; Pearson, 1992), it is unable to differentiate between wild-type CSFV and vaccine strains.
Development of a loop-mediated isothermal amplification for visual detection of the HCLV vaccine against classical swine fever in China
2011, Journal of Virological MethodsCitation Excerpt :Rapid and cost-effective LAMP assays have been described for the preclinical detection of CSFV (Chen et al., 2009; Yin et al., 2010; Chen et al., 2010). To avoid misdiagnosis of CSF in vaccinated herds, a LAMP assay was developed for the specific detection of wild-type CSFV (Zhang et al., 2010a). The objective of this study was to develop an accompanying HCLV-specific LAMP assay as a simple tool for detection of the vaccine virus from clinical samples in less well-equipped laboratories and under field conditions, and for quality control of HCLV vaccines.