两种血清鸭乙型肝炎病毒DNA定量检测方法的建立与评估

Establishment and assessment of two methods for quantitative detection of serum duck hepatitis B virus DNA.

作者信息

Chen Ya-Xi, Huang Ai-Long, Qi Zhen-Yuan, Guo Shu-Hua

机构信息

Institute of Viral Hepatitis, Chongqing University of Medical Sciences, Chongqing, 400010, China.

出版信息

World J Gastroenterol. 2004 Sep 15;10(18):2666-9. doi: 10.3748/wjg.v10.i18.2666.

DOI:10.3748/wjg.v10.i18.2666

PMID:15309716

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4572190/

Abstract

AIM

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.

CONCLUSION

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.

摘要

目的

建立并评估使用碱性磷酸酶直接标记的鸭乙型肝炎病毒（DHBV）DNA探针进行定量膜杂交以及荧光定量PCR（qPCR）定量检测血清中DHBV DNA的方法。

方法

本实验采用碱性磷酸酶直接标记的DHBV DNA探针及化学发光底物CDP-star。通过放射自显影检测DHBV DNA，然后进行DNA斑点杂交扫描。此外，设计了源自DHBV DNA S基因的三条引物。半巢式引物用AmpliSensor标记。经过不对称预扩增、半巢式扩增及在线检测后，建立DHBV DNA阳性标准品的标准曲线。分别用碱性磷酸酶直接标记的DHBV DNA探针斑点杂交法和地高辛标记的DHBV DNA探针杂交法检测100份样本。用荧光定量PCR和地高辛标记的DHBV DNA探针斑点杂交法检测70份鸭血清样本，并分析结果的相关性。

结果

碱性磷酸酶直接标记的DHBV DNA探针灵敏度为10 pg。与地高辛标记的DHBV DNA探针检测法相比，符合率为100%。30个循环后，20 g/L琼脂糖凝胶电泳显示扩增产物有两条约180 bp和70 bp的条带。扩增产物浓度与阳性标准品初始浓度成正比。检测指数与当前循环中积累的扩增产物量成正比。阳性标准品初始浓度与获得足够量扩增产物所需的循环数成反比。荧光定量PCR与斑点杂交结果的相关系数为（0.97，P<0.01）。