title: An optimized sensitive method for quantitation of DNA/RNA viruses in heparinized and cryopreserved plasma.
abstract: 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.affiliation: Graduate School of Public Health, Pittsburgh, PA, United States.
authors.forename: Ming
authors.initials: M
authors.lastname: Ding
authors.forename: Arlene
authors.initials: A
authors.lastname: Bullotta
authors.forename: Lori
authors.initials: L
authors.lastname: Caruso
authors.forename: Phalguni
authors.initials: P
authors.lastname: Gupta
authors.forename: Charles R
authors.initials: CR
authors.lastname: Rinaldo
authors.forename: Yue
authors.initials: Y
authors.lastname: Chen
mesh_terms: D015925:Cryopreservation / Q000379:methods*; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D003586:Cytomegalovirus Infections / Q000821:virology; D004267:DNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D004279:DNA, Viral / Q000097:blood / Q000302:isolation & purification; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D006493:Heparin; D006801:Humans; D010949:Plasma / Q000737:chemistry / Q000821:virology*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D012367:RNA, Viral / Q000097:blood / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019562:Viral Load
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral; D006493:Heparin