title: 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.
authors.affiliation: Biomedical and Diagnostic Sciences, University of Tennessee, Knoxville, TN, United States. Electronic address: beckpen@utk.edu.
authors.forename: Rebecca P
authors.initials: RP
authors.lastname: Wilkes
authors.affiliation: GeneReach USA, Lexington, MA, United States.
authors.forename: Pei-Yu A
authors.initials: PY
authors.lastname: Lee
authors.affiliation: GeneReach USA, Lexington, MA, United States.
authors.forename: Yun-Long
authors.initials: YL
authors.lastname: Tsai
authors.affiliation: GeneReach USA, Lexington, MA, United States.
authors.forename: Chuan-Fu
authors.initials: CF
authors.lastname: Tsai
authors.affiliation: GeneReach USA, Lexington, MA, United States.
authors.forename: Hsiu-Hui
authors.initials: HH
authors.lastname: Chang
authors.affiliation: GeneReach USA, Lexington, MA, United States.
authors.forename: Hsiao-Fen G
authors.initials: HF
authors.lastname: Chang
authors.affiliation: GeneReach USA, Lexington, MA, United States.
authors.forename: Hwa-Tang T
authors.initials: HT
authors.lastname: Wang
mesh_terms: D000818:Animals; D004283:Dog Diseases / Q000175:diagnosis* / Q000821:virology*; D004285:Dogs; D005243:Feces / Q000821:virology; D010322:Parvoviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D017993:Parvovirus, Canine / Q000302:isolation & purification*; D019095:Point-of-Care Systems; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
keywords: Canine parvovirus; Insulated isothermal PCR; Point-of-need detection