Detection of Middle East respiratory syndrome coronavirus using reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Virol J. 2014 Aug 8;11:139. doi: 10.1186/1743-422X-11-139.

Abstract

Background: The first documented case of Middle East Respiratory Syndrome coronavirus (MERS-CoV) occurred in 2012, and outbreaks have continued ever since, mainly in Saudi Arabia. MERS-CoV is primarily diagnosed using a real-time RT-PCR assay, with at least two different genomic targets required for a positive diagnosis according to the case definition of The World Health Organization (WHO) as of 3 July 2013. Therefore, it is urgently necessary to develop as many specific genetic diagnostic methods as possible to allow stable diagnosis of MERS-CoV infections.

Methods: Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) is a genetic diagnostic method used widely for the detection of viral pathogens, which requires only a single temperature for amplification, and can be completed in less than 1 h. This study developed a novel RT-LAMP assay for detecting MERS-CoV using primer sets targeting a conserved nucleocapsid protein region.

Results: The RT-LAMP assay was capable of detecting as few as 3.4 copies of MERS-CoV RNA, and was highly specific, with no cross-reaction to other respiratory viruses. Pilot experiments to detect MERS-CoV from medium containing pharyngeal swabs inoculated with pre-titrated viruses were also performed. The RT-LAMP assay exhibited sensitivity similar to that of MERS-CoV real-time RT-PCR.

Conclusions: These results suggest that the RT-LAMP assay described here is a useful tool for the diagnosis and epidemiologic surveillance of human MERS-CoV infections.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Coronavirus Infections / diagnosis*
  • Coronavirus Infections / virology*
  • Genes, Viral
  • Humans
  • Middle East Respiratory Syndrome Coronavirus / genetics*
  • Middle East Respiratory Syndrome Coronavirus / isolation & purification
  • Molecular Sequence Data
  • Nucleic Acid Amplification Techniques*
  • Nucleocapsid Proteins / genetics
  • Pilot Projects
  • Real-Time Polymerase Chain Reaction
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Sequence Alignment
  • Sequence Analysis, DNA

Substances

  • Nucleocapsid Proteins