Detection of PCR products using self-reporting duplex mutation primers

• Published in: Journal of clinical pathology Vol. 62; no. 11; p. 1046
• Main Authors: Xia, Q-F, Tu, Z-G, Liu, J-B, Qin, X, Qian, S-Y, Li, P
• Format: Journal Article
• Language: English
• Published: England 01-11-2009
• Subjects: DNA Primers; DNA, Viral - blood; Genes, Duplicate; Hepatitis B virus - genetics; Hepatitis B virus - isolation & purification; Hepatitis B, Chronic - diagnosis; Humans; Oligonucleotide Probes; Polymerase Chain Reaction - methods; Reagent Kits, Diagnostic; Reproducibility of Results
• ISSN: 1472-4146
• DOI: 10.1136/jcp.2009.064402

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.