Direct DNA and RNA detection from large volumes of whole human blood

Scientific Reports
Dongyang CaiGerald Urban

Abstract

PCR inhibitors in clinical specimens negatively affect the sensitivity of diagnostic PCR and RT-PCR or may even cause false-negative results. To overcome PCR inhibition, increase the sensitivity of the assays and simplify the detection protocols, simple methods based on quantitative nested real-time PCR and RT-PCR were developed to detect exogenous DNA and RNA directly from large volumes of whole human blood (WHB). Thermus thermophilus (Tth) polymerase is resistant to several common PCR inhibitors and exhibits reverse transcriptase activity in the presence of manganese ions. In combination with optimized concentrations of magnesium ions and manganese ions, Tth polymerase enabled efficient detection of DNA and RNA from large volumes of WHB treated with various anticoagulants. The applicability of these methods was further demonstrated by examining WHB specimens collected from different healthy individuals and those stored under a variety of conditions. The detection limit of these methods was determined by detecting exogenous DNA, RNA, and bacteria spiked in WHB. To the best of our knowledge, direct RNA detection from large volumes of WHB has not been reported. The results of the developed methods can be obtained within 4 hours,...Continue Reading

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Citations

Feb 14, 2020·Analytical and Bioanalytical Chemistry·Maja SidstedtJohannes Hedman
May 26, 2020·The Journal of Molecular Diagnostics : JMD·Andrew T BenderJonathan D Posner
Apr 7, 2020·ACS Sensors·Ayokunle O OlanrewajuJonathan D Posner
Sep 29, 2020·Ophthalmology·Satoko NakanoUNKNOWN Direct Strip Polymerase Chain Reaction Project Groups

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Methods Mentioned

BETA
PCR
electrophoresis

Software Mentioned

Primer3plus
Roche LightCycler

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