Rapid and sensitive detection of canine distemper virus by one-tube reverse transcription-insulated isothermal polymerase chain reaction

Abstract

Background: Canine distemper virus (CDV) has been associated with outbreaks of canine infectious respiratory disease in shelters and boarding kennel environments. POCKIT TM Nucleic Acid Analyzer is a field-deployable device capable of generating automatically interpreted insulated isothermal polymerase chain reaction (iiPCR) results from extracted nucleic acid within one hour. In this study, reverse transcription iiPCR (RT-iiPCR) was developed to facilitate point-of-need diagnosis of CDV infection. Results: Analytical sensitivity (limit of detection 95%) of the established CDV RT-iiPCR was about 11 copies of in vitro transcribed RNA per reaction. CDV RT-iiPCR generated positive signals from CDV, but not Bordetella bronchiseptica, canine parvovirus, canine herpesvirus, canine adenovirus 2, canine influenza virus (subtype H3N8), canine parainfluenza virus, and canine respiratory coronavirus. To evaluate accuracy of the established reaction in canine distemper clinical diagnosis, 110 specimens from dogs, raccoons, and foxes suspected with CDV infection were tested simultaneously by CDV RT-iiPCR and real-time RT-PCR. CDV RT-iiPCR demonstrated excellent sensitivity (100%) and specificity (100%), compared to real-time RT-PCR.

A Rapid Field-Deployable Reverse Transcription-Insulated Isothermal Polymerase Chain Reaction Assay for Sensitive and Specific Detection of Bluetongue Virus

“…The RT‐iiPCR assay was performed on the POCKIT ™ instrument as described previously (Balasuriya et al., ; Wilkes et al., ; Lung et al., ). Briefly, 50 μ l of Premix Buffer B (GeneReach USA) was added to individual Premix tubes containing custom lyophilized reagents [primers, probe, dNTPs, reverse transcriptase and Taq polymerase (all from GeneReach USA)], and the tubes were briefly spun in a Cubee ™ mini‐centrifuge.…”

“…The POCKIT ™ nucleic acid analyzer is a component of the POCKIT ™ Xpress mobile laboratory (GeneReach USA), which also includes a mini‐centrifuge (Cubee ™ , GeneReach USA) and two micropipettes, all the equipment necessary to run an RT‐iiPCR assay. This technology has been proven to be successful in amplification of both DNA and RNA viruses, and as sensitive as laboratory‐based nested (Tsai et al., ) and real‐time PCR assays (Balasuriya et al., ; Tsai et al., ; Wilkes et al., ; Lung et al., ).…”

Rapid detection of equine infectious anaemia virus nucleic acid by insulated isothermal RT‐PCR assay to aid diagnosis under field conditions

“…The equipment including an automated nucleic acid extraction system (taco TM mini) required for iiPCR (POCKIT TM Nucleic Acid Analyzer a , POCKIT TM ) is specifically designed with the option of using batteries as an electrical power source and lyophilised reagents avoiding the need for a cold chain to be fully field-deployable [26]. This enables it to be used for point of need diagnosis plus results are obtainable within 1 h. Moreover, a number of assays have been successfully developed for detection of pathogens associated with humans, companion animals, livestock and farmed aquatic species based on the iiPCR/POCKIT TM system and all have been shown to have equivalent specificity and sensitivity to RT-qPCR [27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45]. In this report, an EIAV RT-iiPCR based on the POCKIT TM system designed to aid in the diagnosis of EIAV-infected equids was evaluated by comparison with a conventional EIAV RT-qPCR assay, where both assays targeted the same region of the viral genome although not using identical primers and probes.…”

Rapid and sensitive detection of Mycoplasma synoviae by an insulated isothermal polymerase chain reaction-based assay on a field-deployable device

“…The reaction worked on a portable POCKIT™ Nucleic Acid Analyzer (POCKIT™; GeneReach, Taichung, Taiwan), which detected and processed reaction signals automatically to generate easy final positive and negative readouts. Carried out in a capillary tube and driven by the Rayleigh-Bènard convention (Krishnan et al., 2002), the reaction could generate detectable signals in less than 1 h. The iiPCR/POCKIT™ system has been demonstrated to achieve specific and sensitive detection of several bacterial and viral infections of veterinary interest in companion animals, livestock animals, and aquaculture animals (Tsai et al., 2012; Tsen et al., 2013; Balasuriya et al., 2014; Tsai et al., 2014; Wilkes et al., 2014; Ambagala et al., 2015; Lung et al., 2015; Wilkes et al., 2015a,b). In addition, a nucleic acid extraction step is generally required before PCR to remove reaction inhibitors in the sample matrix (Schrader et al., 2012).…”