Development and evaluation of real-time loop-mediated isothermal amplification for hepatitis B virus DNA quantification: a new tool for HBV management.
BACKGROUND
Accurate quantitation of hepatitis B viral load is a critical aspect in screening and monitoring HBV infection.


OBJECTIVE
We used loop-mediated isothermal amplification (LAMP) to develop a real-time fluorogenic (RtF-LAMP) protocol to quantitate HBV DNA. Quantitative analysis was obtained by measuring time-to-positive (TTP), a biomarker similar to cycle threshold (Ct) in real-time PCR.


METHODS
Sensitivity, specificity, reproducibility, and dynamic range for RtF-LAMP were evaluated using molecular and biological standards. Four hundred and two patient samples were then used to compare the performance of RtF-LAMP to a commercial real-time PCR assay (DaAn Gene Co, China).


RESULTS
The lower detection limit (LDL) of RtF-LAMP by Probit analysis at the 95% detection level was 210 copies/ml, and the dynamic range was 8 orders of magnitude. The conversion factor for results obtained with the RtF-LAMP assay was 1 IU/ml equals to 4.4 copies/ml. Coefficients of variation (CV) reflected low intra-assay and inter-assay variability (4.24-12.11%). In a large number of serum samples, there was excellent an correlation between RtF-LAMP and real-time PCR (R(2)=0.96). There was a good agreement between the two tests except at the detection cutoff of the real-time PCR assay.


CONCLUSIONS
Our RtF-LAMP protocol appears to be precise, accurate and rapid. It could be a valuable tool for the detection of HBV in large clinical and epidemiological studies.
Cai
Ting
T
Department of Microbiology, School of Medicine, Zhejiang University, 310031 Hangzhou, China.
Lou
Guoqiang
G
Yang
Jin
J
Xu
Dai
D
Meng
Zhonghua
Z
D000704:Analysis of Variance; D001483:Base Sequence; D002681:China; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000097:blood*; D006509:Hepatitis B / Q000821:virology*; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*
D017931:DNA Primers; D004279:DNA, Viral