Use of modified oligonucleotides to compensate for sequence polymorphisms in the real-time detection of norovirus.
Noroviruses are major causative agents of sporadic and outbreak-associated nonbacterial gastroenteritis worldwide. Real-time RT-PCR has rapidly become the principle means for norovirus detection due to its sensitivity and specificity; however sequence variations in the Norovirus genome can cause problems for real-time chemistries. Using a combination of modified bases and minor groove binder-conjugated Eclipse hybridization probes, a one step real-time RT-PCR assay was designed and optimized to detect norovirus genogroups I and II. Additionally, an RNA internal control was incorporated into the assay to monitor nucleic acid extraction and amplification inhibition. As part of this study, a combination of 36 stool and RNA samples were tested and results were compared to a previously described TaqMan assay. An overall correlation of 97% was obtained. After 7 months of clinical testing, a percent positive rate of 39% was determined, with genogroup II accounting for 98% of the positives. Overall, the Eclipse assay is a sensitive and robust method for detecting and typing norovirus genogroups I and II as well as useful for routine clinical testing. Incorporating modified bases helps overcome design limitations due to single nucleotide polymorphisms.
Hymas
Weston
W
Associated Regional and University Pathologists (ARUP), Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108, USA. hymasw@aruplab.com
Atkinson
Annette
A
Stevenson
Jeffery
J
Hillyard
David
D
D001483:Base Sequence; D017250:Caliciviridae Infections / Q000175:diagnosis* / Q000821:virology; D015342:DNA Probes; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D029322:Norovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D011110:Polymorphism, Genetic; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D015342:DNA Probes; D012367:RNA, Viral