A visual DNA chip for simultaneous detection of hepatitis B virus, hepatitis C virus and human immunodeficiency virus type-1.
For the simultaneously visual detection of hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus type-1 (HIV-1), a qualitative DNA chip method, combining multiplex and nested polymerase chain reaction (PCR) with arrayed anchored primer PCR and a biotin-avidin alkaline phosphatase (Av-AP) indicator system, was developed. After pretreatment of infected blood samples and reverse transcription of the RNA virus genome, PCR was performed in a single tube by using the outer primer pairs. Second round nested multiplex PCR was performed on the DNA chip, on which the primers array had already been prepared. During the arrayed anchored multiplex PCR, 5[N-(N-biotinylaminocaproyl)-epsilon-3-aminoallyl]-2-deoxy-uridine-5-triphosphate (biotin-11-dUTP) was incorporated into the extended DNA chains in order to bind avidin alkaline phosphatase via avidin and biotin. To produce purple precipitates on the chips, the enzyme substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP) was used in conjunction with the enhancer, nitro blue tetrazolium (NBT). Blood samples containing the three viruses were tested using this DNA chip and about 1 pg of specific viral DNA fragments were detected on the chip wells after nested PCR.
Wen
Ji-Kai
JK
Wuhan Institution of Virology, Chinese Academy of Science, Wuhan 430071, China.
Zhang
Xian-En
XE
Cheng
Zhi
Z
Liu
Hong
H
Zhou
Ya-Feng
YF
Zhang
Zhi-Ping
ZP
Yang
Ji-Hong
JH
Deng
Jiao-Yu
JY
D003124:Colorimetry / Q000295:instrumentation* / Q000379:methods; D004279:DNA, Viral / Q000097:blood; D004867:Equipment Design; D019544:Equipment Failure Analysis; D015658:HIV Infections / Q000097:blood / Q000235:genetics; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D006509:Hepatitis B / Q000097:blood / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006526:Hepatitis C / Q000097:blood / Q000235:genetics; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004279:DNA, Viral