基于 TaqMan 水解探针的实时 PCR 检测和定量分析皮肤痂皮中的骆驼痘病毒。
TaqMan hydrolysis probe based real time PCR for detection and quantitation of camelpox virus in skin scabs.
机构信息
Pox Virus Laboratory, Division of Virology, Indian Veterinary Research Institute, Nainital Distt., Mukteswar 263138, Uttarakhand, India.
出版信息
J Virol Methods. 2012 May;181(2):192-6. doi: 10.1016/j.jviromet.2012.02.002. Epub 2012 Feb 21.
The study describes the development of TaqMan hydrolysis probe based real time PCR (rt-PCR) assay targeting the ankyrin repeat protein (C18L) gene sequences for the detection and quantitation of camelpox virus (CMLV) nucleic acid and its comparison with established conventional and SYBR green rt-PCR assays. The assay was specific with an efficiency of 99.4%. The analytical sensitivity was 4 × 10¹ and 0.35 in terms of copy number and picogram of virus genomic DNA, respectively. The assay was linear with an acceptable intra (0.9-2.83% and 0.9-2.3%) and inter-assay (0.46-2.3% and 0.9-3.3) variations, when standard plasmid DNA and genomic DNA from purified CMLV respectively were tested. The assay was rapid, specific and sensitive as that of SYBR green and 1000 times more sensitive than the conventional PCR. It is suitable for the detection of CMLV nucleic acid directly from clinical samples. Further, the assay was evaluated using cell culture adapted CMLV isolates (n=11) and clinical samples (n=23) from camels and humans suspected of camelpox. This is an improved technique over conventional and SYBR green rt-PCR methods for the detection and quantitation of CMLV from skin scabs.
本研究描述了 TaqMan 水解探针实时 PCR（rt-PCR）检测方法的开发，该方法针对锚蛋白重复蛋白（C18L）基因序列，用于检测和定量骆驼痘病毒（CMLV）核酸，并与已建立的常规和 SYBR 绿色 rt-PCR 检测方法进行了比较。该检测方法具有特异性，效率为 99.4%。分析灵敏度分别为 4×10¹和 0.35，以拷贝数和病毒基因组 DNA 的皮克数量表示。当使用标准质粒 DNA 和从纯化的 CMLV 中提取的基因组 DNA 进行测试时，该检测方法在日内（0.9-2.83%和 0.9-2.3%）和日间（0.46-2.3%和 0.9-3.3）的变异均具有可接受的线性。该检测方法快速、特异、灵敏，与 SYBR 绿色实时 PCR 相当，比常规 PCR 灵敏 1000 倍。它适用于直接从临床样本中检测 CMLV 核酸。此外，该检测方法还评估了细胞培养适应的 CMLV 分离株（n=11）和骆驼及疑似患骆驼痘的人类的临床样本（n=23）。这是一种优于常规和 SYBR 绿色 rt-PCR 方法的改进技术，可用于从皮肤痂中检测和定量 CMLV。
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