Direct DNA and RNA detection from large volumes of whole human blood.
Sci Rep
; 8(1): 3410, 2018 02 21.
Article
en En
| MEDLINE
| ID: mdl-29467420
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ADN
/
ARN
Tipo de estudio:
Diagnostic_studies
/
Guideline
Límite:
Humans
Idioma:
En
Revista:
Sci Rep
Año:
2018
Tipo del documento:
Article
País de afiliación:
Alemania
Pais de publicación:
Reino Unido