Use of armored RNA as a standard to construct a calibration curve for real-time RT-PCR.
Armored Enterovirus RNA was used to standardize a real-time reverse transcription (RT)-PCR for environmental testing. Armored technology is a system to produce a robust and stable RNA standard, trapped into phage proteins, to be used as internal control. The Armored Enterovirus RNA protected sequence includes 263 bp of highly conserved sequences in 5' UTR region. During these tests, Armored RNA has been used to produce a calibration curve, comparing three different fluorogenic chemistry: TaqMan system, Syber Green I and Lux-primers. The effective evaluation of three amplifying commercial reagent kits, in use to carry out real-time RT-PCR, and several extraction procedures of protected viral RNA have been carried out. The highest Armored RNA recovery was obtained by heat treatment while chemical extraction may decrease the quantity of RNA. The best sensitivity and specificity was obtained using the Syber Green I technique since it is a reproducible test, easy to use and the cheapest one. TaqMan and Lux-primer assays provide good RT-PCR efficiency in relationship to the several extraction methods used, since labelled probe or primer request in these chemistry strategies, increases the cost of testing.
Donia
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Department Public Health, University Tor Vergata, Faculty of Medicine, Via Montpellier 1, 00133 Rome, Italy. donia@med.uniroma2.it
Divizia
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Pana'
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D020121:5' Untranslated Regions / Q000032:analysis / Q000235:genetics; D002138:Calibration; D004770:Enterovirus / Q000235:genetics*; D017955:Poliovirus / Q000302:isolation & purification; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D012680:Sensitivity and Specificity
D020121:5' Untranslated Regions; D012367:RNA, Viral