摘要
目的利用二聚体蝎型荧光探针技术,建立一种能用于临床的快捷、敏感、特异、价格低廉的HBV实时荧光定量PCR检测方法。方法根据HBV基因组保守区域precore/core设计二聚体蝎型荧光探针和引物,构建质粒标准品,优化荧光定量PCR条件,并进行方法学评价。结果所建方法的检测范围为101～108copies/μl,灵敏度达到10copies/μl,低拷贝样品的批内和日间重复性(CV)分别为1.71%和2.57%,高拷贝样品的批内和日间CV分别为3.00%和3.86%。与商品化TaqMan试剂盒相比,本法阳性检出率较高,且检测时间缩短了45min(约减少1/3),成本降低50%。结论成功建立了快速检测HBVDNA的二聚体蝎型荧光定量PCR方法,为研发适合临床应用的HBV基因定量检测试剂盒奠定了基础。
Objective To develop a novel real-time fluorescence PCR method using duplex scorpion probe for fast, sensitive, specific and cost-saving detection of hepatitis B virus （HBV） gene. Method Duplex scorpion probe was designed according to the sequence of cloned gene. The PCR reaction system was optimized and evaluated. Results The developed method showed a wide range of linearity from 101 to 10s copies/ml, high sensitivity （10 copies/ml）, good repeatability and specificity. In comparison with commercial Taqman kits, it exhibited higher sensitivity. The time for detection was decreased for 45 minute, as about one third as the commercial method, and the cost was decreased by 50%. Conclusion A novel real-time fluorescence quantitative PCR method for detection of HBV gene was established. It provided a basis for developing new diagnostic kits using the duplex scorpion probe for clinical application.
出处
《临床检验杂志》
CAS
CSCD
北大核心
2007年第4期244-246,共3页
Chinese Journal of Clinical Laboratory Science
基金
国家自然科学基金(30471838)
