Comparison of a microtiter plate system to Southern blot for detection of human herpesvirus 8 DNA amplified from blood and saliva.
The recent discovery of human herpesvirus 8 (HHV-8) as the etiologic agent of Kaposi's sarcoma (KS) has led to the interest in the development of PCR for this virus that is accurate, rapid, and convenient. We developed a sensitive PCR assay for HHV-8 with microtiter plate detection of amplimers. DNA was purified from white blood cells and saliva from HIV-infected men with and without Kaposi's sarcoma and one-step PCR was undertaken with primer sets specific for the N-terminal region of the glycoprotein B gene and open reading frame (orf) 26 of HHV-8. PCR was performed on 40 clinical specimens, followed by Southern blot and microtiter plate detection of amplimers. Results from the two methods of detection were nearly identical. Sensitivity for both methods based on serial dilution of a known standard was five to ten copies of HHV-8 per 400 ng of cellular DNA. In conclusion, microtiter plate detection of HHV-8 PCR amplimers is as sensitive and specific as Southern blot with much faster turnaround time at comparable cost, and utilizes common laboratory equipment.
Stamey
F R
FR
Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
DeLeon-Carnes
M
M
Patel
M M
MM
Pellett
P E
PE
Dollard
S C
SC
D001483:Base Sequence; D015139:Blotting, Southern / Q000379:methods* / Q000706:statistics & numerical data; D015342:DNA Probes / Q000235:genetics; D004279:DNA, Viral / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000706:statistics & numerical data; D015658:HIV Infections / Q000150:complications / Q000821:virology; D019288:Herpesvirus 8, Human / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods / Q000706:statistics & numerical data; D015203:Reproducibility of Results; D012463:Saliva / Q000821:virology; D012514:Sarcoma, Kaposi / Q000150:complications / Q000821:virology; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods* / Q000706:statistics & numerical data
D015342:DNA Probes; D004279:DNA, Viral