Development of an astrovirus RT-PCR detection assay for use with conventional, real-time, and integrated cell culture/RT-PCR.
Astrovirus is a common cause of gastroenteritis in humans that has been determined to be responsible for outbreaks of illness in several countries. Since astrovirus can be waterborne, it is important to be able to identify this virus in environmental water. We have developed and optimized a reverse transcription - polymerase chain reaction (RT-PCR) method that was able to amplify all eight astrovirus serotypes in a single reaction. In addition, a positive control construct was designed so that any inhibitors of this astrovirus assay could be detected. The assay was adapted for use in a real-time PCR assay and the sensitivity of these two methods was compared. The real-time assay was then combined with CaCo2 cell culture to produce an integrated cell culture/RT-PCR (ICC/RT-PCR) assay that was able to detect low levels of astrovirus after an incubation of 7 days or less. Also, the sensitivity of the ICC/RT-PCR assay was compared with RT-PCR alone. The methods were used to detect astrovirus in acute phase illness stool samples as well as in a water sample spiked with astrovirus.
Grimm
Ann C
AC
Biohazard Assessment Research Branch, national Exposure Research laboratory, US Environmental Protection Agency, Cincinnati, OH 45268, USA. grimm.ann@epa.gov
Cashdollar
Jennifer L
JL
Williams
Frederick P
FP
Fout
G Shay
GS
D018938:Caco-2 Cells; D002478:Cells, Cultured; D006801:Humans; D001257:Mamastrovirus / Q000145:classification / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D010802:Phylogeny; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D012367:RNA, Viral