An improved reverse transcription loop-mediated isothermal amplification assay for sensitive and specific detection of serotype O foot-and-mouth disease virus

J Virol Methods. 2018 Oct:260:6-13. doi: 10.1016/j.jviromet.2018.06.017. Epub 2018 Jun 30.

Abstract

A sensitive and specific swarm primer-based reverse transcription loop-mediated isothermal amplification (sRT-LAMP) assay for the detection of serotype O foot-and-mouth disease virus (FMDV) was developed and evaluated. The assay specifically amplified the VP3 gene of serotype O FMDV, but did not amplify the VP3 gene of other serotype FMDVs or any other viruses. The limit of detection of the assay was 102 TCID50/mL or 103 RNA copies/μL, which is 100 times lower than that of the RT-LAMP assay without swarm primers. The new assay is 10 times more sensitive than reverse transcription-polymerase chain reaction (RT-PCR) and is comparable to the sensitivity of real time RT-PCR (qRT-PCR). Evaluation of the assay using different serotypes of FMDV strains showed 100% agreement with the RT-PCR results. The previously reported serotype O FMDV-specific RT-LAMP assay did not detect 20 out of 22 strains of serotype O FMDVs, probably due to multiple mismatches between the primer and template sequences, showing that it is not suitable for detecting the serotype O FMDVs circulating in Pool 1 region countries, including Korea. In contrast, the developed sRT-LAMP assay with improved primers can rapidly and accurately diagnose serotype O FMDVs circulating in Pool 1 region countries and will be a useful alternative to RT-PCR and qRT-PCR.

Keywords: Foot-and-mouth disease virus; RT-LAMP; Serotype-specific; Swarm primer.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Pair Mismatch
  • Capsid Proteins / genetics*
  • DNA Primers
  • Foot-and-Mouth Disease / diagnosis
  • Foot-and-Mouth Disease / virology*
  • Foot-and-Mouth Disease Virus / classification
  • Foot-and-Mouth Disease Virus / genetics
  • Foot-and-Mouth Disease Virus / isolation & purification*
  • Limit of Detection
  • Nucleic Acid Amplification Techniques / veterinary*
  • Republic of Korea
  • Reverse Transcriptase Polymerase Chain Reaction
  • Reverse Transcription
  • Sensitivity and Specificity
  • Serogroup

Substances

  • Capsid Proteins
  • DNA Primers
  • VP3 protein, Foot-and-mouth disease virus