Ultrasensitive detection of serum hepatitis B virus by coupling ultrafiltration DNA extraction with real-time PCR.
BACKGROUND
A simple and reliable DNA extraction of hepatitis B virus (HBV) is critical in developing an ultrasensitive detection method for HBV infection. Current commercially available serum Hepatitis B Virus (HBV) DNA extraction methods are time-consuming, expensive and/or require specialized equipment, which hinders wide adoption of clinical laboratories. This study offers a report on an ultrasensitive HBV DNA detection method by coupling serum HBV DNA extraction by ultrafiltration (UF) with real-time PCR (qPCR) detection.


METHODS
Serum proteins were precipitated by phenol to release HBV DNA in the supernatant which was then transferred to the UF devices. The resultant DNA concentrate was eluted and released into qPCR pre-mixture. The UF-qPCR assay performance, including recovery rate, linearity, detection sensitivity, precision and diagnostic accuracy that compared to the CAP-CTM V2.0 assay by analyzing batched low viral load clinical samples was evaluated.


RESULTS
The recovery rate of the UF-based HBV DNA extraction method was above 80%. The assay linearity was demonstrated with a slope of 0.95 and R2 values of 0.99. Limit-of-detection (LOD) of the UF-qPCR assay was determined to be 12.1IU/ml. The coefficient of variation (CV) of HBV quantitation for high, low and limit titer samples was 2.28%, 5.77% and 25.59%, respectively. Accuracy of the UF-qPCR assay was confirmed with the reference panel, and there was a strong correlation between these two methods (R2 = 0.55, p < 0.01).


CONCLUSIONS
The UF-qPCR assay is reliable, highly sensitive, affordable and time-saving, and the method can be used for ultrasensitive detection of serum HBV.
Department of Laboratory Medicine, Guangzhou First People's Hospital, Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.
Bin
B
Wu
Department of Laboratory Medicine, Guangzhou First People's Hospital, Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.
Feng
F
Xiao
Department of Laboratory Medicine, Guangzhou First People's Hospital, Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.
Peiwen
P
Li
Department of Laboratory Medicine, Guangzhou First People's Hospital, Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.
Yan
Y
Du
Department of Laboratory Medicine, Guangzhou First People's Hospital, Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.
Jinqiong
J
Lin
Department of Laboratory Medicine, Guangzhou First People's Hospital, Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.|Clinical Molecular Medicine and Molecular Diagnosis Key Laboratory of Guangdong Province, Guangzhou, China.
Kaihua
K
Ming
Department of Laboratory Medicine, Guangzhou First People's Hospital, Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.|Clinical Molecular Medicine and Molecular Diagnosis Key Laboratory of Guangdong Province, Guangzhou, China.
Bin
B
Chen
Department of Laboratory Medicine, Guangzhou First People's Hospital, Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.|Clinical Molecular Medicine and Molecular Diagnosis Key Laboratory of Guangdong Province, Guangzhou, China.
Xiuxia
X
Lei
Department of Laboratory Medicine, Guangzhou First People's Hospital, Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.|Clinical Molecular Medicine and Molecular Diagnosis Key Laboratory of Guangdong Province, Guangzhou, China.
Banglao
B
Xu
Department of Laboratory Medicine, Guangzhou First People's Hospital, Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.|Clinical Molecular Medicine and Molecular Diagnosis Key Laboratory of Guangdong Province, Guangzhou, China.
Dayu
D
Liu
D004279:DNA, Viral / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D006509:Hepatitis B / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008297:Male; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014462:Ultrafiltration
D004279:DNA, Viral