Insulated Isothermal Reverse Transcriptase PCR (iiRT-PCR) for Rapid and Sensitive Detection of Classical Swine Fever Virus

Abstract

Classical swine fever (CSF) is an OIE-listed disease that can have a severe impact on the swine industry. User-friendly, sensitive, rapid diagnostic tests that utilize low-cost field-deployable instruments for CSF diagnosis can be useful for disease surveillance and outbreak monitoring. In this study, we describe validation of a new probe-based insulated isothermal reverse transcriptase PCR (iiRT-PCR) assay for rapid detection of classical swine fever virus (CSFV) on a compact, user-friendly device (POCKIT(™) Nucleic Acid Analyzer) that does not need data interpretation by the user. The assay accurately detected CSFV RNA from a diverse panel of 33 CSFV strains representing all three genotypes plus an additional in vitro-transcribed RNA from cloned sequences representing a vaccine strain. No cross-reactivity was observed with a panel of 18 viruses associated with livestock including eight other pestivirus strains (bovine viral diarrhoea virus type 1 and type 2, border disease virus, HoBi atypical pestivirus), African swine fever virus, swine vesicular disease virus, swine influenza virus, porcine respiratory and reproductive syndrome virus, porcine circovirus 1, porcine circovirus 2, porcine respiratory coronavirus, vesicular exanthema of swine virus, bovine herpes virus type 1 and vesicular stomatitis virus. The iiRT-PCR assay accurately detected CSFV as early as 2 days post-inoculation in RNA extracted from serum samples of experimentally infected pigs, before appearance of clinical signs. The limit of detection (LOD95% ) calculated by probit regression analysis was 23 copies per reaction. The assay has a sample to answer turnaround time of less than an hour using extracted RNA or diluted or low volume of neat serum. The user-friendly, compact device that automatically analyses and displays results could potentially be a useful tool for surveillance and monitoring of CSF in a disease outbreak.

Classical Swine Fever—An Updated Review

“…Recently, field applicable RT-PCRs [184] but also alternatives have been designed such as loop-mediated isothermal amplification (LAMP) assays [185,186,187,188,189,190], primer-probe energy transfer RT-qPCR [191,192] or recently insulated isothermal RT-qPCR [193]. Moreover, CSFV can be isolated on different permanent cell lines such as porcine kidney cell lines PK15 or SK6 (Technical Annex to Commission Decision 2002/106/EC).…”

A Pan-Dengue Virus Reverse Transcription-Insulated Isothermal PCR Assay Intended for Point-of-Need Diagnosis of Dengue Virus Infection by Use of the POCKIT Nucleic Acid Analyzer

“…Thus, the simplicity and efficiency of the RT- iiPCR coupled with the POCKIT nucleic acid analyzer (RT-iiPCR/ POCKIT) to rapidly amplify nucleic acids under isothermal conditions suggested that this assay could be a potential alternative for detection of DENV especially in remote field settings. This system has been shown to offer sensitivity, specificity, and clinical performance comparable to those of reference real-time PCR or nested PCR methods for various microbial pathogens in various clinical sample types (36)(37)(38)(39)(40)(41)(42)(43)(44)(45). One of the major advantages of RT-iiPCR is its simple protocol.…”

A multiplex reverse transcription PCR and automated electronic microarray assay for detection and differentiation of seven viruses affecting swine

“…Quantified plasmids were restriction digested with HindIII (10 μ/μl) or other appropriate restriction enzymes when HindIII sites were found within the amplicon (Thermo Scientific‐Fermentas, Ottawa, ON), purified using the Zymo DNA Clean & Concentrator™‐25 column kit (Zymo Research, Irvine, CA) and transcribed using the MEGAscript ® kit (Life Technologies). For FMDV, the desired gene regions were synthesized by IDT, cloned into the pGEM‐3Zf(+) vector, digested with EcoRI (Life Technologies, Thermo Scientific‐Fermentas, Ottawa, ON), amplified under the same cycling conditions by the described RT‐PCR in single‐plex and transcribed as described previously (Lung et al., ). RNA was quantified using the Qubit ® 2.0 Fluorometer and RNA broad range assay kit (Life Technologies).…”

“…Conventional and real‐time RT‐PCR assays have been developed for CSFV (Wernike, Hoffmann, & Beer, ), ASFV (Fernández‐Pinero et al., ) and FMDV (Hole, Clavijo, & Pineda, ; King et al., ; Reid et al., ) and its differentials that include vesicular stomatitis virus (VSV; Hole et al., ; Rasmussen, Uttenthal, & Agüero, ), SVDV (Fernández et al., ; Núñez et al., ), and VESV (Reid et al., ). User‐friendly reverse transcription insulated isothermal PCR (RT‐iiPCR) assays performed on compact, field‐deployable instruments with automatic display of “+” or “−” results have also been described for CSFV (Lung et al., ) and FMDV (Ambagala et al., ). Real‐time multiplex PCR assays have been reported for the detection of FMDV and CSFV (Shi et al., ; Wernike et al., ) and other targets (Haines, Hofmann, King, Drew, & Crooke, ; Li et al., ; Rasmussen et al., ; Wernike et al., ).…”