Chen Weijun, He Bo, Li Changgui, Zhang Xiaowei, Wu Weili, Yin Xuyang, Fan Baoxing, Fan Xingliang, Wang Jian
Beijing Genomics Institute, Chinese Academy of Sciences, Beijing, China.
National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China.
J Med Microbiol. 2007 May;56(Pt 5):603-607. doi: 10.1099/jmm.0.47014-0.
The recent recurrence of highly pathogenic avian influenza virus A H5N1 was firstly reported in mid-December 2003 and continued through 2005. This study describes a sensitive and specific real-time RT-PCR method for the detection of influenza A subtype H5 and for monitoring virus loads. Using serial dilutions of influenza A H5N1 cultures, this assay reproducibly determined the lowest detection limit to be approximately 5 x 10(-2) 50 % egg infective doses (EID(50)). In contrast, the minimum detection limit was approximately 3 EID(50) in conventional RT-PCR with WHO primers and 10 EID(50) in antigen-capture ELISA. In tests of serial dilutions of in vitro-transcribed influenza A H5 gene RNA, there was linear amplification from 40 copies to 4 x 10(8) copies of target RNA per reaction and approximately six copies, and sometimes even as few as three copies, of target RNA tested positive in our assay. Thirty-five throat swabs from ill birds were tested: 33 samples tested positive using this assay. In comparison, 27, 13 and 19 samples tested positive using conventional RT-PCR, antigen-capture ELISA and virus isolation, respectively. To evaluate further the sensitivity of this real-time RT-PCR, a standard panel and 60 H5N1 isolates that contained different clades of influenza virus A/H5N1 were tested and all tested positive. To evaluate the specificity of the assay, 60 throat swabs from patients infected with influenza virus A H1 were tested; all were negative. Thirteen other viruses were also tested and all tested negative.
高致病性甲型H5N1禽流感病毒最近一次复发于2003年12月中旬首次报告，并持续至2005年。本研究描述了一种灵敏且特异的实时逆转录聚合酶链反应（RT-PCR）方法，用于检测甲型H5流感病毒亚型并监测病毒载量。通过对甲型H5N1培养物进行系列稀释，该检测方法可重复性地确定最低检测限约为5×10⁻² 50%鸡胚感染剂量（EID₅₀）。相比之下，使用世界卫生组织（WHO）引物的传统RT-PCR最低检测限约为3 EID₅₀，抗原捕获酶联免疫吸附测定（ELISA）的最低检测限为10 EID₅₀。在对体外转录的甲型H5流感病毒基因RNA进行系列稀释测试中，每个反应中靶标RNA从40拷贝到4×10⁸拷贝呈线性扩增，在我们的检测中，约6个拷贝，有时甚至低至3个拷贝的靶标RNA检测呈阳性。对35份病禽咽喉拭子进行了检测：使用该检测方法33份样本检测呈阳性。相比之下，使用传统RT-PCR、抗原捕获ELISA和病毒分离法检测呈阳性的样本分别为27份、13份和19份。为进一步评估该实时RT-PCR的灵敏度，对一个标准样本组以及60株包含不同甲型H5N1流感病毒进化分支的分离株进行了检测，所有样本检测均呈阳性。为评估该检测方法的特异性，对60份感染甲型H1流感病毒患者的咽喉拭子进行了检测；所有样本均为阴性。还对其他13种病毒进行了检测，所有检测结果均为阴性。
