Reverse transcription of an RNA genome from databasing paper (FTA(R)).
The instability of RNA in solutions during storage and travel is an impediment to its utilization in routine diagnostics. A robust and simple approach to the problem of RNA protection and processing is offered by storage of RNA desiccated with processing procedures that do not solublize the RNA until the beginning of reverse transcription. The feasibility of this general approach was tested with coxsackievirus B4 (CVB-4) from blood or culture fluid held on a storage and transport medium (FTA(R)) and analysed by reverse transcriptase PCR (RT-PCR) without removing the RNA from the FTA(R) until reverse transcription. Phase-trapping techniques based on water-miscible solvents such as ethanol or phenol were compared with simple buffers and concentrated lithium chloride solutions. RT-PCR detection of viral RNA reached a sensitivity of approximately 0.1 fg, which is comparable with other non-nested PCR techniques. Whole blood as a virus vehicle significantly interfered with CVB-4 detection, but to an acceptable degree. Desiccation-storage of the RNA of CVB-4 appears to be unaffected by weeks on the storage medium under ambient conditions. These characteristics indicate that this approach forms a credible developmental base for RNA-based pathogen diagnostics with particular application to the problem of transporting potentially infectious body fluids to a centralized laboratory for analysis.
Rogers
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School of Biological Sciences, Flinders University of South Australia, Sturt Road, Bedford Park, South Australia 5042, Australia.
Burgoyne
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LA
D001800:Blood Specimen Collection / Q000295:instrumentation / Q000379:methods; D017931:DNA Primers; D029822:Enterovirus B, Human / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D012367:RNA, Viral / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000295:instrumentation / Q000379:methods*
D017931:DNA Primers; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase