【作者】 肖跃强； 刘吉山； 杨慧； 沈志强；

【Author】 Xiao Yue-qiang;Yang Hui;Shen Zhi-qiang;Liu Ji-shan;Shandong Binzhou Animal Science & Veterinary Medicine Academy;Shandong Lvdu Bio-Sciences and Technology Co.Ltd.;

【机构】 山东省滨州畜牧兽医研究院； 山东绿都生物科技有限公司；

【摘要】 【目的】建立能快速、特异、敏感的检测并临床应用的兔出血热病毒RT-PCR方法。【方法】根据GenBaank公布的RHDV序列,设计了3条引物,对引物组合进行了筛选,优化PCR体系各反应条件,测定该方法的敏感性与特异性,并进行临床应用对比实验。【结果】筛选出的引物组合能够特异性扩增331bp产物,Mg²⁺离子浓度在1.0¹.5mmol/L扩增效果最好,退火温度在50⁶0℃之间扩增无差异性;该方法可以扩增10²拷贝以上的模板,对兔轮状病毒与水泡性口炎病毒检测结果均为阴性;临床应用对比实验显示,该方法自27份临床采集样本较HA方法多检出3例RHDV阳性样本,检出阳性率由66.7%提升至77.8%。【结论】本研究建立的RT-PCR诊断方法具有特异、灵敏、快速等特点,能够用于该病的临床检测,为防控该病提供了技术保障。更多还原

【Abstract】 [Objective]A reverse transcription-polymerase chain reaction（RT-PCR） was developed for detection of rabbit haemorrhagic disease virus（RHDV）.【Method】3 primers were designed according to the sequences published in GenBank,then the different combination of primers were screened.The amplification conditions,including concentration of Mg²⁺ and annealing temperature,were optimized.The sensitivity test and the specificity test were performed,and this method was also used to detect the clinical samples comparing to the HA test.【Result】A target fragment of 331 bp could be amplified with the screened primers,the best concentration of Mg²⁺ was from 1.0 mmol/L to 1.5 mmol/L,and there were no differences from 50℃to 60℃ range of annealing temperature.The minimum detectable copy number of the template was 102,and the detection results of rabbit rotavirus and rabbit vesicular stomatitis virus were negative.The clinical diagnosis results of RT-PCR method showed that 3 more samples were positive than that of diagnosed with HA test,the positive rate increased from 66.7%to 77.8%.【Conclusion】The RT-PCR established in this study is a specific,sensitive and rapid diagnostic method,which can be used for clinical diagnosis and will provide technical support for prevention and control of rabbit haemorrhagic disease（RHD）.更多还原