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

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, making them possible for rapid and accurate detection of disease-causing agents from WHB.

SARS-CoV-2 Direct real-time Polymerase Chain Reaction Testing in Laboratories With Shortage Challenges

“…The direct RT-PCR in the application of DNA viral detection has shown promising results in hepatitis B and Mycoplasma organisms [ 21 , 22 ]. Other studies using direct RT-PCR on detecting viral RNA suggested that the use of PCR enhancers could lead to early RNAase release, which compromises the RNA integrity [ 23 ]. PCR inhibition is the leading cause of reporting low testing sensitivity in various fluids, in which some are reported as resistant to heat and demand the extraction step with sample dilution to eliminate the inhibition [ 24 , 25 ].…”

PCR inhibition in qPCR, dPCR and MPS—mechanisms and solutions

“…The risk for detection inhibition should be considered when analysing challenging samples such as those containing soil or blood. There are several studies where SYBR Green I detection has been applied for analysis of matrices that contain blood or humic acid [141][142][143], which may have skewed the results and conclusions. Using hydrolysis probes for detection is a much better alternative than dsDNA-binding dyes in order to avoid fluorescence inhibition when humic acid or haemoglobin is present in the reactions.…”

Pre-analytical Practices in the Molecular Diagnostic Tests, A Concise Review

“…There is controversy about the effect of heparin on the molecular assays. There are some reports implying that heparin, if not completely removed during extraction, can inhibit enzyme-based molecular tests (46,(60)(61)(62)(63)(64). It has been shown that even low concentrations of heparin may suppress DNA amplification although the inhibition of heparin on PCR using large amounts of DNA may be not significant (64)(65)(66).…”