Detection of PCR products using self-reporting duplex mutation primers.
A novel oligonucleotide probe, with duplex mutation primers, which comprised a normal reverse primer labelled with a fluorophore at its 5'-end and a single-base mismatched complementary oligonucleotide labelled with a quencher at its 3'-end, was designed. Its application in the detection of hepatitis B virus (HBV) DNA of serum was investigated; results were compared with those obtained using a commercial TaqMan kit. There was a good linear correlation in the range of 10(2) approximately 10(7) copies/ml (r(2) = 0.999) with the method. Intra-experimental coefficients of variation (CVs) were 0.70% approximately 7.80%, and inter-experimental CVs were 0.78% approximately 9.02%, respectively. There was no significant difference of HBV genome number tested by the two methods (p<0.05) in 132 hepatitis B patients; HBV DNA was not detected in any serum samples of 20 healthy volunteers by the two methods.
Xia
Q-F
QF
Hainan Medical College, Haikou, PR China. xiaqianfeng@sina.com
Tu
Z-G
ZG
Liu
J-B
JB
Qin
X
X
Qian
S-Y
SY
Li
P
P
D017931:DNA Primers; D004279:DNA, Viral / Q000097:blood*; D020131:Genes, Duplicate; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D019694:Hepatitis B, Chronic / Q000175:diagnosis*; D006801:Humans; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results
D017931:DNA Primers; D004279:DNA, Viral; D015345:Oligonucleotide Probes; D011933:Reagent Kits, Diagnostic