Fang Shi-Song, Li Juan, Wang Xin, Liu Tao, Cheng Xiao-Wen, Lv Xing, Wu Chun-Li, Zheng Qing, Zhang Ren-Li, Cheng Jin-Quan
Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, China.
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2012 Oct;26(5):384-7.
To develop a novel sensitive duplex real-time PCR assay for accurately identifying B/Yamagata and B/Victoria lineages of influenza virus type B.
50 HA (hemagglutinin) gene sequences coding for B/Yamagata and B/Victoria lineage, respectively, were randomly downloaded for GenBank and analyzed by software MEGA. Primers and probes specific for HA gene of B/Yamagata and B/Victoria lineages were designed by Primer Primer and then applied in the duplex real-time RT-PCR method that was followed developed. Influenza virus B type and A type isolated in our laboratory and typing-confirmed by HAI method were used as reference strains to determine the specificity of this assay and the sensitivity of the duplex amplification was evaluated by viral load testing in terms of in vitro transcribed RNA copy number.
In 2006-2010, 793 influenza virus type B strains were isolated from 17 765 throat swab samples, among which 152 strains were differentiated as By lineage and 641 as Bv lineage by this assay. These results was agreement with that determined by HAI assay. This developed assay allows to accurately identify approximately 10(2) copies/microl for Bv and By lineage virus with intra- and inter-coefficient of variation (CV) < 3.5% and nearly 100% specificity.
This method provides sensitive and robust tool for routine diagnosis and on-time epidemiological examination of influenza virus, which could be applied in influenza surveillance laboratories for rapid molecular diagnosis.
开发一种新型灵敏的双重实时荧光定量PCR检测方法，用于准确鉴定乙型流感病毒的B/山形株系和B/维多利亚株系。
从GenBank中随机下载分别编码B/山形株系和B/维多利亚株系的50个血凝素（HA）基因序列，并用MEGA软件进行分析。通过Primer Primer软件设计针对B/山形株系和B/维多利亚株系HA基因的引物和探针，然后将其应用于随后开发的双重实时逆转录PCR方法中。以本实验室分离并经血凝抑制试验（HAI）方法进行分型确认的乙型和甲型流感病毒作为参考菌株，以确定该检测方法的特异性，并通过体外转录RNA拷贝数的病毒载量检测评估双重扩增的灵敏度。
2006 - 2010年期间，从17765份咽拭子样本中分离出793株乙型流感病毒，其中通过该检测方法鉴定出152株为B/山形株系，641株为B/维多利亚株系。这些结果与HAI试验的结果一致。该开发的检测方法能够准确鉴定出B/维多利亚株系和B/山形株系病毒，其检测下限约为10²拷贝/微升，组内和组间变异系数（CV）< 3.5%，特异性接近100%。
该方法为流感病毒的常规诊断和及时的流行病学检查提供了灵敏且可靠的工具，可应用于流感监测实验室进行快速分子诊断。
