Rapid quantification of hepatitis B virus DNA by direct real-time PCR from serum without DNA extraction.
The purpose of this study was to quantify hepatitis B virus DNA by direct real-time PCR from serum without the need for DNA extraction. Crossing point (Cp) values were determined automatically using the second derivative maximum mode. Since serum samples from patients are inevitably haemolysed, lipaemic or icteric, the interference of endogenous substances from the serum in real-time PCR was evaluated. The result showed that, although serum protein quenched the intensity of fluorescence, the Cp value adopted to calculate the quantity of DNA copies remained unchanged. Importantly, real-time PCR from serum with or without DNA extraction reached a high level of concordance. This direct serum PCR method without the DNA extraction and gel electrophoresis allows for substantial labour and cost savings. In addition, it is also suitable for rapid DNA quantification during clinical diagnosis.
Cheng
Zheng-Jiang
ZJ
Clinical Laboratory Medicine Department, Xiangfan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Xiangfan 441021, People's Republic of China.|Clinical Laboratory Medicine Department, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, People's Republic of China.
Hu
Li-Hua
LH
Clinical Laboratory Medicine Department, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, People's Republic of China.
Fu
Wen-Rong
WR
Pathology Department, Xiangfan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Xiangfan 441021, People's Republic of China.
Li
Yi-Rong
YR
Clinical Laboratory Medicine Department, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, People's Republic of China.
D004279:DNA, Viral / Q000097:blood* / Q000302:isolation & purification; D006515:Hepatitis B virus / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D044967:Serum / Q000821:virology*; D019562:Viral Load / Q000379:methods*
D004279:DNA, Viral