A duplex real-time reverse transcriptase polymerase chain reaction assay for the detection of California serogroup and Cache Valley viruses.
A duplex TaqMan real-time reverse transcriptase polymerase chain reaction (PCR) assay was developed for the detection of California (CAL) serogroup viruses and Cache Valley virus (CVV), for use in human surveillance. The targets selected for the assay were the sequences encoding the nucleocapsid protein of CAL and the G1 glycoprotein of CVV. Conserved regions were selected by aligning genetic sequences from various strains available in the GenBank database. Primers and probes were selected in conserved regions. The assay sensitivity was 75 gene copies (gc)/reaction for CAL serogroup viruses and 30 gc/reaction for CVV. The performance of the assay was linear over at least 6 log(10) gc. The assay was specific, given that it did not cross-react with a variety of pathogens. It did, however, detect 11 viruses within the CAL serogroup and 12 CVV isolates. The use of an internal control ensured that possible inefficiency in nucleic acid extraction or PCR inhibition would be detected.
Wang
Heng
H
Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.
Nattanmai
Seela
S
Kramer
Laura D
LD
Bernard
Kristen A
KA
Tavakoli
Norma P
NP
D002042:Bunyamwera virus / Q000235:genetics / Q000302:isolation & purification*; D002044:Bunyaviridae Infections / Q000175:diagnosis*; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D019590:Nucleocapsid Proteins / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D014759:Viral Envelope Proteins / Q000235:genetics
D017931:DNA Primers; D019590:Nucleocapsid Proteins; D014759:Viral Envelope Proteins