[Detection of TT virus DNA by polymerase chain reaction-microplate hybridization].
OBJECTIVE
To establish a method for the detection of TT virus (TTV) DNA by polymerase chain reaction (PCR)-microplate hybridization and to find out various influential factors of the assay.


METHODS
TTV DNA extracted by guanidinium thiocyanate was amplified by PCR, the PCR primers were pre-labeled with biotin. By conjugation of biotin with streptavidin, the amplified products were coated to the microplate well, and hybridized with FITC- probe simultaneously. After being reacted with Anti-Fluorescein POD, the products that contained FITC probe were coloured by TMB-H2O2 and then measured the optical density using a microplate reader at 450 nm wavelength.


RESULTS
The optimal concentration of NaOH was 0.3 mol/L, and the optimal time of hybridization and enzymatic reaction was 60 minutes and 45 minutes, respectively. The results also showed that the positive rate of TTV infection in normal population, hepatitis A-G and hepatitis non A-G was 13.6%, 20.3% and 28.1%, respectively.


CONCLUSIONS
Comparing the PCR-microplate hybridization with the electrophoresis assay, the former is simple, rapid, sensitive and specific and is expected to be a new technique instead of electrophoresis assay as a routine method for TTV DNA detection.
Ma
D
D
The First People's Hospital of Yancheng City, Jiangsu 224001, China.
Guo
N
N
Wang
H
H
D004266:DNA Virus Infections / Q000821:virology; D004279:DNA, Viral / Q000097:blood*; D006525:Hepatitis, Viral, Human / Q000821:virology; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D022783:Torque teno virus / Q000235:genetics / Q000302:isolation & purification*
D004279:DNA, Viral