Dataset Information

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.

SUBMITTER: Cai D

PROVIDER: S-EPMC5821888 | biostudies-other | 2018 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

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

Cai Dongyang D Behrmann Ole O Hufert Frank F Dame Gregory G Urban Gerald G

Scientific reports 20180221 1

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 inhibito ...[more]

PMID: 29467420

Similar Datasets

Project description:Rapid identification of pathogens is critical for early and appropriate treatment of bloodstream infections. The various culture-independent assays that have been developed often have long turnaround times, low sensitivity and narrow pathogen coverage. Here, we propose a new multiplex PCR assay, MeltArray, which uses intact microbial cells as the source of genomic DNA (gDNA). The successive steps of the MeltArray assay, including selective lysis of human cells, microbial cell sedimentation, microbial cellular DNA extraction, target-specific pre-amplification and multiplex PCR detection, allowed the detection of 35 major bloodstream infectious pathogens in whole blood within 5.5 h. The limits of detection varied depending on the pathogen and ranged from 1 to 5 CFU/mL. Of 443 blood culture samples, including 373 positive blood culture samples and 70 negative blood culture samples, the MeltArray assay showed a sensitivity of 93.8% (350/373, 95% confidence interval [CI] = 90.7%-96.0%), specificity of 98.6% (69/70, 95% CI = 91.2%-99.9%), positive predictive value of 99.7% (95% CI = 98.1%-99.9%), and negative predictive value of 75.0% (95% CI = 64.7%-83.2%). The MeltArray detection results of 16 samples differed from MALDI-TOF and were confirmed by Sanger sequencing. Further testing of 110 whole blood samples from patients with suspected bloodstream infections using blood culture results revealed that the MeltArray assay had a clinical sensitivity of 100% (9/9, 95% CI = 62.8%-100.0%), clinical specificity of 74.5% (70/94, 95% CI = 64.2%-82.7%), positive predictive value of 27.3% (95% CI = 13.9%-45.8%), and negative predictive value of 100.0% (95% CI = 93.5%-100.0%). Compared with metagenomic next-generation sequencing, the MeltArray assay displayed a positive agreement of 85.7% (6/7, 95% CI = 42.0%-99.2%) and negative agreement of 100.0% (4/4, 95% CI = 39.6%-100.0%). We conclude that the MeltArray assay can be used as a rapid and reliable tool for direct identification of pathogens in bloodstream infections.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets