Transcriptional enhancement of RT-PCR for rapid and sensitive detection of Noroviruses.
Previously reported nucleic acid sequence-based amplification (NASBA) primers specific for the GII Noroviruses were adapted for reverse transcriptase-polymerase chain reaction (RT-PCR), and detection sensitivity was then enhanced by a subsequent in vitro transcription of the RT-PCR amplicons. The NASBA-derived primers performed comparably to other broadly reactive GII Norovirus primers with respect to detection limits (i.e. 1 RT-PCR amplifiable unit (RT-PCRU) per reaction). Detection limits improved by approximately 1 log(10) to 0.3 RT-PCRU per reaction when transcriptional enhancement and electrochemiluminescence (ECL) hybridization followed RT-PCR. The method shows promise for improved detection sensitivity in instances where very low levels of virus contamination might be anticipated.
Jean
Julie
J
North Carolina State University, Department of Food Science, Raleigh, NC 27695-7624, USA. jjean@unity.ncsu.edu
D'Souza
Doris
D
Jaykus
Lee-Ann
LA
D004783:Environmental Microbiology; D005516:Food Microbiology; D029322:Norovirus / Q000235:genetics* / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D021161:Self-Sustained Sequence Replication / Q000379:methods; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic