Simultaneous detection and quantitation of Chikungunya, dengue and West Nile viruses by multiplex RT-PCR assays and dengue virus typing using high resolution melting.
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.
Naze
F
F
Service de Bactériologie-Parasitologie-Virologie et Hygiène, Groupe Hospitalier Sud Réunion, BP 350, Saint-Pierre Cedex 97448, Reunion.
Le Roux
K
K
Schuffenecker
I
I
Zeller
H
H
Staikowsky
F
F
Grivard
P
P
Michault
A
A
Laurent
P
P
D018354:Alphavirus Infections / Q000175:diagnosis / Q000821:virology; D002646:Chikungunya virus / Q000235:genetics / Q000302:isolation & purification*; D003715:Dengue / Q000175:diagnosis / Q000821:virology; D003716:Dengue Virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D012367:RNA, Viral / Q000097:blood; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012680:Sensitivity and Specificity; D013997:Time Factors; D044366:Transition Temperature; D014901:West Nile Fever / Q000175:diagnosis / Q000821:virology; D014902:West Nile virus / Q000235:genetics / Q000302:isolation & purification*
D012367:RNA, Viral