Titration of varicella-zoster virus DNA in throat swabs from varicella patients by combined use of PCR and microplate hybridization.
We devised a simple procedure for titration of varicella-zoster virus (VZV) DNA in throat swabs from varicella patients. DNA which was extracted from throat swabs, together with known copy numbers of a cloned VZV DNA fragment, were 10-fold serially diluted and used as template in PCR. The PCR products, after heat denaturation, again serially diluted in 1.5 M NaCl and adsorbed to microplate wells. Then, biotin-labeled DNA probes were hybridized with the immobilized DNA. The hybridization signal was produced by streptavidin-conjugated beta-galactosidase and a fluorogenic enzyme substrate. By comparing the titration curves of a clinical specimen with those of the cloned fragment, of which detection limit was about 10 copies, we estimated the copy numbers of VZV DNA in the specimen. With this technique, we evaluated the degree of potential contagiousness of the patient along the course of infection: we found that varicella patients possessed highest quantity of VZV DNA in the throat on the first day of illness.
Hondo
R
R
Department of Microbiology, the Institute of Public Health, Tokyo.
Ito
S
S
Inouye
S
S
D002644:Chickenpox / Q000175:diagnosis* / Q000821:virology; D002648:Child; D002675:Child, Preschool; D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D014645:Herpesvirus 3, Human / Q000235:genetics*; D006801:Humans; D008297:Male; D008846:Micromanipulation; D009693:Nucleic Acid Hybridization; D010614:Pharynx / Q000821:virology*; D016133:Polymerase Chain Reaction; D012015:Reference Standards; D017186:Titrimetry / Q000379:methods*
D004279:DNA, Viral