Use of heat labile UNG in an RT-PCR assay for enterovirus detection.
A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to replace the Roche AMPLICOR Enterovirus Test used in our laboratory from 1996 to 1999. The new assay design was optimized to match or exceed the performance of the Roche AMPLICOR Enterovirus test kit with respect to analytical sensitivity and specificity, contamination control, ease of use and availability of reagents. This new assay uses a heat labile form of the enzyme uracil DNA glycosylase (UNG) for amplicon contamination control and an RT-PCR enzyme mixture, enabling a one tube/one step amplification. RNA preparation was undertaken using a commercial extraction kit. End detection was accomplished using a probe-capture enzyme immuno assay (EIA) plate format. This EV RT-PCR assay exceeds the performance of the Roche AMPLICOR Enterovirus assay in a direct comparison. The combined enzymological approach has potential application to a wide variety of assays requiring sensitive RNA detection and stringent contamination control, including those utilizing real time detection methods.
Taggart
Edward W
EW
Associated Regional and University Pathologists (ARUP), Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108, USA. taggarew@aruplab.com
Carroll
Karen C
KC
Byington
Carrie L
CL
Crist
Gwen A
GA
Hillyard
David R
DR
D016477:Artifacts; D002555:Cerebrospinal Fluid / Q000821:virology; D045647:DNA Glycosylases; D004770:Enterovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004769:Enterovirus Infections / Q000821:virology*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D006358:Hot Temperature; D006801:Humans; D007124:Immunoenzyme Techniques; D008587:Meningitis, Viral / Q000821:virology*; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D012367:RNA, Viral / Q000032:analysis; D011933:Reagent Kits, Diagnostic; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D051981:Uracil-DNA Glycosidase; D014776:Virus Cultivation
D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase