Several concerns about the primer design in the universal molecular beacon real-time PCR assay and its application in HBV DNA detection.
A universal hepatitis B virus (HBV) DNA detection kit is appealing for the worldwide diagnosis and monitoring of the treatment of different mutant types of hepatitis B virus. A sensitive and reproducible real-time PCR assay based on the universal molecular beacon (U-MB) technique was developed for the detection of HBV DNA in serum. The U-MB probe used in the assay has no interaction with the HBV DNA sequence. The U-MB technique not only reduced the cost of HBV detection but also had the potential for the development of a universal detection kit for different mutant HBV types and other DNA systems. To demonstrate its clinical utility, 90 serum samples were analyzed using the U-MB real-time PCR method. In the experiments we found that several crucial factors needed to be considered in the primer design, such as the avoidance of formation of severe primer-dimer and primer self-hairpin structure. With the optimized primer sets, satisfactory results were obtained for all the tested samples. We concluded that this assay would be an excellent candidate for a universal HBV DNA detection method.
Li
Xiaomin
X
The Key Laboratory of Bioorganic Chemistry & Molecular Engineering and Institute of Analytical Chemistry, College of Chemistry & Molecular Engineering, Peking University, Beijing, China.
Huang
Yong
Y
Song
Chen
C
Zhao
Meiping
M
Li
Yuanzong
Y
D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000097:blood*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic / Q000191:economics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004279:DNA, Viral; D011933:Reagent Kits, Diagnostic; D004259:DNA-Directed DNA Polymerase