A novel real-time PCR assay for determination of viral loads in person infected with hepatitis B virus.
A novel LUX (Light Upon eXtension) primer-based real-time PCR assay was developed and evaluated in this study, which was designed to provide a cost-effective, specific and highly sensitive method for viral load determination of hepatitis B virus (HBV). The assay employed an effective and rapid nucleic acid extraction system based on magnetic beads. To evaluate its efficacy, this new viral DNA preparation method was compared with QIAamp Blood Mini Kit and the results showed a good correlation (r=0.971; P<0.001). The performance of the LUX real-time assay was validated by testing serial dilutions of HBV plasmid DNA (5 to 5 x 10(8)copies/reaction) and a good linear relationship was obtained between the Ct values and the log(10) concentration of the HBV DNA. The assay possessed high sensitivity and the detection limit of this system was as few as 25 copies/ml of serum. A total of 91 positive serum samples were detected to evaluate further the assay and the high specificity was confirmed by melting curve analysis. This assay provides an ideal tool for monitoring the treatment efficacy and studying the relationship between HBV viral load and the stage of disease.
Li
Guimin
G
School of Life Sciences, Northeast Normal University, Changchun 130024, China.
Li
Wangfeng
W
Guo
Fengyao
F
Xu
Shoumin
S
Zhao
Ning
N
Chen
Shan
S
Liu
Lixia
L
D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D006509:Hepatitis B / Q000821:virology*; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008280:Magnetics; D008863:Microspheres; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D044967:Serum / Q000821:virology*; D019562:Viral Load / Q000379:methods*
D017931:DNA Primers; D004279:DNA, Viral