Freezing complete polymerase chain reaction master mix reagents for routine molecular diagnostics.
The potential of storing complete frozen real-time polymerase chain reactions (PCRs) and real-time reverse transcription PCRs (RT-PCRs), which require only thawing and the addition of template nucleic acid before PCR cycling, was examined. Master mixes containing all necessary reagents at working concentration (except template nucleic acid) were aliquoted into single-reaction volumes and stored at -70 degrees C for periods of up to 8 months. Reactions were removed from storage and nucleic acid template was added and amplified using different real-time PCR instruments. Threshold cycle values were used to monitor changes in assay performance after storage. Results for hybridization probe and TaqMan probe assays showed that freezing complete real-time PCR and RT-PCR reaction mixes was possible without deterioration in assay performance. This approach has advantages for routine molecular diagnostics in areas such as convenience, test consistency, quality control, and ease of use by nonspecialist staff.
West
Donna Marie
DM
Technology Transfer Unit, Biotechnology Department, Veterinary Laboratories Agency-Weybridge, New Haw, Addlestone, Surrey, United Kingdom.
Sawyer
Jason
J
D000818:Animals; D002417:Cattle; D015925:Cryopreservation; D015342:DNA Probes / Q000592:standards*; D004269:DNA, Bacterial / Q000032:analysis; D004272:DNA, Mitochondrial / Q000032:analysis; D003851:Deoxyribonucleases; D007919:Leptospira / Q000235:genetics; D008322:Mammals; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004269:DNA, Bacterial; D004272:DNA, Mitochondrial; D012367:RNA, Viral; D003851:Deoxyribonucleases