Simultaneous detection and quantitation of Chikungunya, dengue and West Nile viruses by multiplex RT-PCR assays and dengue virus typing using high resolution melting

J Virol Methods. 2009 Dec;162(1-2):1-7. doi: 10.1016/j.jviromet.2009.03.006. Epub 2009 Mar 17.

Abstract

Chikungunya (CHIKV), Dengue (DENV) and West Nile (WNV) viruses are arthropod-borne viruses that are able to emerge or re-emerge in many regions due to climatic changes and increase in travel. Since these viruses produce similar clinical signs it is important for physicians and epidemiologists to differentiate them rapidly. A molecular method was developed for their detection and quantitation in plasma samples and a DENV typing technique were developed. The method consisted in performing two multiplex real-time one-step RT-PCR assays, to detect and quantify the three viruses. Both assays were conducted in a single run, from a single RNA extract containing a unique coextracted and coamplified composite internal control. The quantitation results were close to the best detection thresholds obtained with simplex RT-PCR techniques. The differentiation of DENV types was performed using a High Resolution Melting technique. The assays enable the early diagnosis of the three arboviruses during viremia, including cases of coinfection. The method is rapid, specific and highly sensitive with a potential for clinical diagnosis and epidemiological surveillance. A DENV positive sample can be typed conveniently using the High Resolution Melting technique using the same apparatus.

Publication types

  • Evaluation Study

MeSH terms

  • Alphavirus Infections* / diagnosis
  • Alphavirus Infections* / virology
  • Chikungunya virus / genetics
  • Chikungunya virus / isolation & purification*
  • Dengue Virus* / classification
  • Dengue Virus* / genetics
  • Dengue Virus* / isolation & purification
  • Dengue* / diagnosis
  • Dengue* / virology
  • Humans
  • RNA, Viral / blood
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction* / methods
  • Reverse Transcriptase Polymerase Chain Reaction* / standards
  • Sensitivity and Specificity
  • Time Factors
  • Transition Temperature
  • West Nile Fever* / diagnosis
  • West Nile Fever* / virology
  • West Nile virus / genetics
  • West Nile virus / isolation & purification*

Substances

  • RNA, Viral