A nonradioactive method for rapid and sensitive detection of polymerase chain reaction products by use of bromo-deoxyuridine.
A highly sensitive and nonradioactive method that allows for rapid detection of polymerase chain reaction (PCR) products, without the need for hybridization with oligonucleotide probes, is described. In this method, a 410-bp sequence of the human respiratory syncytial virus nucleocapsid cDNA was amplified by PCR in the presence of bromo-deoxyuridine-triphosphate, an analog of deoxythymidine-triphosphate. After agarose gel electrophoresis and Southern blotting, the PCR product was directly identified by immunoenzyme-chemiluminescent reaction after binding with an antibromodeoxyuridine antibody. The results show that substitution of bromo-deoxyuridine-triphosphate for deoxythymidine-triphosphate does not affect the efficiency of PCR, and as low as one copy-equivalent of the target DNA sequence could be detected within 2 hours, whereas it required 1 to 5 days to reach comparable sensitivity level after hybridization with a 32P-labeled oligonucleotide probe and autoradiography. Compared with the use of digoxygenin-11-deoxyuridine-triphosphate, the sensitivity of detection was 100-fold higher with the use of Bromo-deoxyuridine-triphosphate. When applied to the diagnosis by use of reverse transcription-PCR of respiratory syncytial virus infections in nasopharyngeal washes from children with symptoms of acute upper respiratory tract infection, the current method detected respiratory syncytial virus genome in 29 of 100 specimens, and there was a complete concordance with the results of hybridization of reverse transcription-PCR products by use of a radiolabeled oligonucleotide probe. Thus, in addition to its rapidity of detection and high sensitivity, this method provides safety of use and can be readily applied to the clinical diagnosis of viral respiratory tract infections.
Dakhama
A
A
University of British Columbia, Pulmonary Research Laboratory, St. Paul's Hospital, Vancouver, Canada.
Hegele
R G
RG
D001973:Bromodeoxyuridine; D002648:Child; D002675:Child, Preschool; D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D007223:Infant; D008163:Luminescent Measurements; D008297:Male; D009305:Nasopharynx / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D018357:Respiratory Syncytial Virus Infections / Q000175:diagnosis* / Q000473:pathology; D018113:Respiratory Syncytial Virus, Human / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D001973:Bromodeoxyuridine