Document Detail


An optimized sensitive method for quantitation of DNA/RNA viruses in heparinized and cryopreserved plasma.
MedLine Citation:
PMID:  21645549     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Sodium heparin, an anticoagulant used widely for blood collection, has been known to inhibit DNA polymerase activity in polymerase chain reaction (PCR) assays. However, all cryopreserved plasma samples collected in the 1980s and early 1990s at the Multicenter AIDS Cohort Study were from heparin-treated blood, which poses a problem in quantifying the target nucleic acids contained in those samples by PCR assay. In this study, a nucleic acid extraction procedure was optimized to remove the heparin from extracted nucleic acids. Using this optimized method, similar human immunodeficiency virus 1 (HIV-1) and cytomegalovirus (CMV) loads of these viruses that were added to normal donor blood from ethylenediaminetetraacetic acid (EDTA), acid citrate dextrose (ACD) or sodium heparin tubes were detected by reverse transcriptase (RT) real-time PCR and real-time PCR. Comparable HIV-1 and CMV loads were also detected in the blood of persons with active HIV-1 and CMV infections collected in EDTA-, ACD- or sodium heparin-treated tubes by RT real-time and real-time PCR. The findings showed that the optimized nucleic acid extraction procedure efficiently removes the heparin inhibition effect on the performance of real-time PCR. This method could be used to extract nucleic acids from archived, heparinized plasma for PCR based quantitation of target molecules.
Authors:
Ming Ding; Arlene Bullotta; Lori Caruso; Phalguni Gupta; Charles R Rinaldo; Yue Chen
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Publication Detail:
Type:  Evaluation Studies; Journal Article; Research Support, N.I.H., Extramural     Date:  2011-05-17
Journal Detail:
Title:  Journal of virological methods     Volume:  176     ISSN:  1879-0984     ISO Abbreviation:  J. Virol. Methods     Publication Date:  2011 Sep 
Date Detail:
Created Date:  2011-07-25     Completed Date:  2011-11-21     Revised Date:  2013-06-28    
Medline Journal Info:
Nlm Unique ID:  8005839     Medline TA:  J Virol Methods     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  1-8     Citation Subset:  IM    
Copyright Information:
Copyright © 2011 Elsevier B.V. All rights reserved.
Affiliation:
Graduate School of Public Health, Pittsburgh, PA, United States.
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MeSH Terms
Descriptor/Qualifier:
Cryopreservation / methods*
Cytomegalovirus / genetics,  isolation & purification,  physiology
Cytomegalovirus Infections / virology
DNA Viruses / genetics,  isolation & purification*
DNA, Viral / blood,  isolation & purification
HIV Infections / virology
HIV-1 / genetics,  isolation & purification,  physiology
Heparin*
Humans
Plasma / chemistry,  virology*
RNA Viruses / genetics,  isolation & purification*,  physiology
RNA, Viral / blood,  isolation & purification
Real-Time Polymerase Chain Reaction / methods*
Reverse Transcriptase Polymerase Chain Reaction / methods*
Sensitivity and Specificity
Viral Load
Grant Support
ID/Acronym/Agency:
U01 AI035041/AI/NIAID NIH HHS; U01 AI035041-17/AI/NIAID NIH HHS; U01AI035041/AI/NIAID NIH HHS; UL1 RR024153/RR/NCRR NIH HHS; UL1 TR000005/TR/NCATS NIH HHS
Chemical
Reg. No./Substance:
0/DNA, Viral; 0/RNA, Viral; 9005-49-6/Heparin
Comments/Corrections

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