An alternative method for the synthesis of competitor RNA transcripts useful for specific detection and quantitation of dengue virus serotype 2 genome and replicative intermediate RNA.
The development of a quantitative-competitive reverse transcription-PCR (RT-PCR) assay to quantify dengue virus (DEN) genome (vRNA) and its replicative intermediate RNA (vRI) is described. A highly conserved region located on the DEN capsid-premembrane genes was used to produce a competitor RNA molecule which contains an internal deletion of 70 nucleotides. The competitor provides a suitable internal control useful to quantify viral RNA from all four dengue virus (DEN 1-4) serotypes. The detection limit of the assay was found to be 100 copies per reaction. This is a rapid, simple, sensitive, inexpensive and easy method for quantitation of DEN RNA species.
Vaughan
Gilberto
G
Department of Immunology Research, Institute for Epidemiological Diagnosis and Reference, Secretariat of Health, Carpio 470, Col. Santo Tomas, Mexico City 11340, Mexico.
Gonzalez-Hernandez
Yolanda
Y
Gudino
Jose-Carmen
JC
Olivera
Hiram
H
Landa-Piedra
Abraham
A
Escobar-Gutierrez
Alejandro
A
D000818:Animals; D002460:Cell Line; D003715:Dengue / Q000175:diagnosis / Q000821:virology*; D003716:Dengue Virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016679:Genome, Viral; D006801:Humans; D012367:RNA, Viral / Q000032:analysis* / Q000097:blood / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012703:Serotyping
D012367:RNA, Viral