Evaluation of a Field-Deployable Insulated Isothermal Polymerase Chain Reaction Nucleic Acid Analyzer for Influenza A Virus Detection at Swine Exhibitions

Vector Borne Zoonotic Dis. 2019 Mar;19(3):212-216. doi: 10.1089/vbz.2018.2345. Epub 2018 Sep 5.


Rapid detection of influenza A virus (IAV) at swine exhibitions, where zoonotic transmission has occurred, can allow exhibition officials to quickly implement mitigation strategies and reduce public health risk. While laboratory diagnostic methods using PCR exist, pen-side detection of IAV can reduce lag time between sample collection and results. Portable insulated isothermal PCR (RT-iiPCR) has been used for point-of-care pathogen detection in veterinary medicine. This study compared laboratory methods of real-time reverse transcription PCR (rRT-PCR) to RT-iiPCR to determine the potential effectiveness of RT-iiPCR for detection of IAV in swine in the field. Two methods of extraction (magnetic bead and spin-column) and the two PCR platforms were used in a crossover study design to detect IAV in nasal wipes of 150 individual swine from one exhibition. Magnetic bead extraction is considered the laboratory gold standard while spin-column purification is considered the field-deployable method. IAV RNA was detected in 17 samples using Mag/rRT-PCR (reference assay) and 16 samples using Mag/RT-iiPCR (Sensitivity-S 76.5%), whereas only 14 samples using Spin/rRT-PCR (S 88.2%) and 12 samples using Spin/RT-iiPCR (field method) (S 58.8%) were positive, demonstrating a reduction in detection of viral RNA using column purification. There is moderate agreement (Cohen's kappa = 0.6575) between Mag/rRT-PCR and Spin/RT-iiPCR. There is good agreement between both PCR assays when using the same method of extraction (Mag: Cohen's kappa = 0.8203, Spin: Cohen's kappa = 0.7642). RT-iiPCR requires testing of 10 more samples than the rRT-PCR to detect disease at the 95% confidence level in a population of 300 animals with a disease prevalence of 20%. In conclusion, although there is some reduction in sensitivity, RT-iiPCR used in conjunction with spin-column purification is an acceptable method of IAV in swine detection at exhibitions where it may help reduce lag time and allow for rapid control of an IAV outbreak.

Keywords: RT-iiPCR; influenza A virus; point-of-care systems; real-time polymerase chain reaction; sensitivity and specificity; swine.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Influenza A virus / genetics
  • Influenza A virus / isolation & purification*
  • Orthomyxoviridae Infections / diagnosis
  • Orthomyxoviridae Infections / veterinary*
  • Orthomyxoviridae Infections / virology
  • Reverse Transcriptase Polymerase Chain Reaction / instrumentation*
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Swine
  • Swine Diseases / diagnosis
  • Swine Diseases / epidemiology
  • Swine Diseases / virology*