Establishment and assessment of two methods for quantitative detection of serum duck hepatitis B virus DNA.
OBJECTIVE
To establish and assess the methods for quantitative detection of serum duck hepatitis B virus (DHBV) DNA by quantitative membrane hybridization using DHBV DNA probe labeled directly with alkaline phosphatase and fluorescence quantitative PCR (qPCR).


METHODS
Probes of DHBV DNA labeled directly with alkaline phosphatase and chemiluminescent substrate CDP-star were used in this assay. DHBV DNA was detected by autoradiography, and then scanned by DNA dot-blot. In addition, three primers derived from DHBV DNA S gene were designed. Semi-nested primer was labeled by AmpliSensor. Standard curve of the positive standards of DHBV DNA was established after asymmetric preamplification, semi-nested amplification and on-line detection. Results from 100 samples detected separately by alkaline phosphatase direct-labeled DHBV DNA probe with dot-blot hybridization and digoxigenin-labeled DHBV DNA probe hybridization. Seventy samples of duck serum were tested by fluorescent qPCR and digoxigenin-labeled DHBV DNA probe in dot-blot hybridization assay and the correlation of results was analysed.


RESULTS
Sensitivity of alkaline phosphatase direct-labeled DHBV DNA probe was 10 pg. The coincidence was 100% compared with digoxigenin-labeled DHBV DNA probe assay. After 30 cycles, amplification products showed two bands of about 180 bp and 70 bp by 20 g/L agarose gel electrophoresis. Concentration of amplification products was in direct proportion to the initial concentration of positive standards. The detection index was in direct proportion to the quantity of amplification products accumulated in the current cycle. The initial concentration of positive standards was in inverse proportion to the number of cycles needed for enough quantities of amplification products. Correlation coefficient of the results was (0.97, P<0.01) between fluorescent qPCR and dot-blot hybridization.


CONCLUSIONS
Alkaline phosphatase direct-labeled DHBV DNA probe in dot-blot hybridization and fluorescent qPCR can be used as valuable means to quantify DHBV DNA in serum.
Institute of Viral Hepatitis, Chongqing University of Medical Sciences, Chongqing, 400010, China. zlcyxi@sina.com
Ya-Xi
YX
Chen
Ai-Long
AL
Huang
Zhen-Yuan
ZY
Qi
Shu-Hua
SH
Guo
D000469:Alkaline Phosphatase; D000818:Animals; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis / Q000097:blood*; D004076:Digoxigenin; D004372:Ducks; D018347:Hepadnaviridae Infections / Q000097:blood / Q000175:diagnosis* / Q000821:virology; D015611:Hepatitis B Virus, Duck / Q000235:genetics / Q000302:isolation & purification*; D006524:Hepatitis, Viral, Animal / Q000097:blood / Q000175:diagnosis* / Q000821:virology; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004279:DNA, Viral; D000469:Alkaline Phosphatase; D004076:Digoxigenin