An insulated isothermal PCR method on a field-deployable device for rapid and sensitive detection of canine parvovirus type 2 at points of need

Abstract

Canine parvovirus type 2 (CPV-2), including subtypes 2a, 2b and 2c, causes an acute enteric disease in both domestic and wild animals. Rapid and sensitive diagnosis aids effective disease management at points of need (PON). A commercially available, field-deployable and user-friendly system, designed with insulated isothermal PCR (iiPCR) technology, displays excellent sensitivity and specificity for nucleic acid detection. An iiPCR method was developed for on-site detection of all circulating CPV-2 strains. Limit of detection was determined using plasmid DNA. CPV-2a, 2b and 2c strains, a feline panleukopenia virus (FPV) strain, and nine canine pathogens were tested to evaluate assay specificity. Reaction sensitivity and performance were compared with an in-house real-time PCR using serial dilutions of a CPV-2b strain and 100 canine fecal clinical samples collected from 2010 to 2014, respectively. The 95% limit of detection of the iiPCR method was 13 copies of standard DNA and detection limits for CPV-2b DNA were equivalent for iiPCR and real-time PCR. The iiPCR reaction detected CPV-2a, 2b and 2c and FPV. Non-targeted pathogens were not detected. Test results of real-time PCR and iiPCR from 99 fecal samples agreed with each other, while one real-time PCR-positive sample tested negative by iiPCR. Therefore, excellent agreement (k = 0.98) with sensitivity of 98.41% and specificity of 100% in detecting CPV-2 in feces was found between the two methods. In conclusion, the iiPCR system has potential to serve as a useful tool for rapid and accurate PON, molecular detection of CPV-2.

A multiplex TaqMan real-time PCR for detection and differentiation of four antigenic types of canine parvovirus in China

“…CPV causes acute hemorrhagic enteritis and myocarditis in dogs [5], and the mortality rate of the disease is high (up to 70%) in puppies [11,14]. The main method for controlling the disease in domestic animals is by vaccination.…”

“…Several assays have been reported for the detection or quantitation of CPV DNA [12][13][14][15][16][17][18], including PCR, nested PCR, iiPCR, RPA, LAMP-ELISA, LAMP-LFD, LAMP, polymerase spiral reaction, and SYBR Green based real-time PCR, but none of these assays enable differentiation CPV antigenic types and CPV-like viruses (MEV and FPV). Several other assays have been reported for the detection and differentiation of type 2-based vaccines and field strains of CPV [19] or typing of three antigenic types of CPV [20] or CPV and MEV [21] assay and MGB probe real-time RT-PCR, but none of these assays enables simultaneous detection and differentiation of four antigenic types of CPV.…”

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.…”

New frontiers in applied veterinary point‐of‐capture diagnostics: Toward early detection and control of zoonotic influenza

“…The downscaling in size of nucleic acid extraction and high‐speed real‐time PCR platforms is opening new opportunities for utility at or near the source of collection . In many cases, advancements in developing rapid, point‐of‐capture real‐time PCR platforms have been pioneered for veterinary applications, facilitating diagnosis and treatment in companion animal medicine as well as management and control of infectious disease affecting livestock production …”