Comparison of three methods for extraction of viral nucleic acids from blood cultures.
Reliable nucleic acid extraction techniques for blood specimens are required for the sensitive detection of viral DNA. Standardized procedures for processing blood specimens for the molecular detection of herpesviruses (cytomegalovirus [CMV], herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus [EBV]) have not been established. Three methods were used to extract DNA from blood specimens from healthy donors and asymptomatic immunocompromised patients: (i) IsoQuick treatment of whole blood, (ii) extraction of the peripheral blood leukocytes by lysis (lysis buffer and proteinase K), and (iii) extraction of peripheral blood leukocytes with phenol-chloroform (sodium docecyl sulfate solution and proteinase K). All blood specimens from 25 healthy blood donors were negative for CMV, herpes simplex virus and varicella-zoster virus nucleic acid sequences, regardless of the extraction method, while three samples (12%) extracted by the lysis technique were positive for EBV DNA. Of 25 blood samples from asymptomatic immunocompromised patients, CMV and EBV each were detected in nine specimens by lysis extraction, four each by IsoQuick and four (CMV) and six (EBV) by the phenol-chloroform method. Our results indicate that the lysis method is optimal for the detection of CMV and EBV DNA sequences by PCR from the leukocytic fraction of blood specimens. DNA of these viruses is frequently present in blood specimens from asymptomatic immunocompromised patients and occasionally from healthy donors.
Espy
M J
MJ
Division of Clinical Microbiology, Mayo Clinic, Rochester, Minnesota 55905.
Patel
R
R
Paya
C V
CV
Smith
T F
TF
D001483:Base Sequence; D001769:Blood / Q000821:virology*; D002478:Cells, Cultured; D004279:DNA, Viral / Q000302:isolation & purification*; D006566:Herpesviridae Infections / Q000175:diagnosis* / Q000453:epidemiology; D016867:Immunocompromised Host; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015995:Prevalence; D011933:Reagent Kits, Diagnostic; D014776:Virus Cultivation
D004279:DNA, Viral; D011933:Reagent Kits, Diagnostic