Reverse transcription-PCR-enzyme-linked immunosorbent assay for rapid detection and differentiation of alphavirus infections.
Due to the lack of a rapid, simple, and inexpensive assay for detecting alphavirus infections, we combined a reverse transcription-PCR with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) to identify human pathogenic alphaviruses that are endemic in the New World. By combining the sensitivity of PCR, the detection simplicity of ELISA, and the specificities of DNA probes, this method rapidly detected and differentiated closely related species and subtypes of several medically important alphaviruses. After an amplification using RT-PCR with primers targeting conserved sequences in the nonstructural protein 1 gene, sequence-specific, biotin-labeled probes targeted against Venezuelan, eastern, and western equine encephalitis or Mayaro virus genes were used for the detection of amplicons using ELISA. The assay is simple, fast, and easy to perform in an ordinary diagnostic laboratory or clinical setting. Nucleic acid derived from cell cultures infected with several alphaviruses, clinical specimens, and mosquito pools as well as frozen and paraffin-embedded animal tissues were detected and identified within 6 to 7 h in a sensitive and specific manner.
Wang
Eryu
E
Center for Biodefense and Emerging Infectious Diseases, Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0609, USA.
Paessler
Slobodan
S
Aguilar
Patricia V
PV
Carrara
Anne-Sophie
AS
Ni
Haolin
H
Greene
Ivorlyne P
IP
Weaver
Scott C
SC
D018354:Alphavirus Infections / Q000175:diagnosis*; D003429:Cross Reactions; D004666:Encephalitis Virus, Venezuelan Equine / Q000145:classification; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity