新型黄病毒NALSV检测方法的建立及应用

Establishment and Application of a New Detection Method for a Novel Flavivirus

蜱是多种病原体的储存宿主和传播媒介,近年来由蜱体内新发现了多种潜在的人兽共患病病原,因此对蜱所携带病原的早期识别和建立相应的检测与防范措施尤为重要。对辽宁省部分地区采集的长角血蜱样本进行宏转录组测序,发现其中存在一种新的黄病毒科病毒成员,是潜在的人兽共患病病原。为建立针对该病毒快速、灵敏的检测方法,选择其糖蛋白VP1基因进行引物探针设计,通过对反应条件及反应体系的优化,建立了基于TaqMan探针的实时荧光定量PCR检测方法,标准方程为y=-3.4436x+33.063,相关系数(R^(2))为0.9999;此方法可检出最低拷贝数为2.5×10copies·μL^(-1),与普通PCR相比,灵敏度提高约10^(4)倍,具有良好的灵敏度;与其他相近病毒的阳性质粒进行反应均无扩增,说明其具有良好的特异性;实验结果的变异系数均低于0.5%,重复性良好;对不同浓度质粒在不同时间进行反应,CT值无显著变化,稳定性良好。采用本方法对2020及2021年采集于辽宁省及内蒙古自治区的18个地区540头蜱进行检测,发现阳性样本数为234头,阳性率为43.3%。结果表明:所建立的实时荧光定量PCR检测方法具有特异性强,灵敏度高,线性关系、重复性及稳定性良好等特点,此检测方法的建立,填补了NALSV在实时荧光定量PCR检测方面的空白,为NALSV的病原检测提供了新的技术方法,对NALSV的分布监测、临床诊断及相关研究奠定了基础,具有一定的现实意义及应用价值。

A potential zoonotic pathogen, a new member of the family flaviviridae, was identified during macrotranscriptome sequencing of Haemaphysalis longicornis samples collected from parts of Liaoning Province. In order to detect this new virus rapidly and sensitively, we established a real-time qPCR detection method based on TaqMan probe, which was designed according to the VP1gene sequence encoding glycoprotein. After optimizing the reaction conditions and reaction system, the standard equation is y=-3.4436x+33.063, and the correlation coefficient(R^(2)) is 0.9999. We performed this method and found that the minimum detectable threshold is 2.5×10copies·μL^(-1), compared with PCR, the sensitivity was increased by about 104, there was no amplification when it reacted with the positive plasmid of other viruses, the coefficients of variation were all lower than 0.5% among experimental results,and there was no significant change in CT values when reacted with different concentrations of plasmids at different times. These phenomena indicated that the method we established is sensitive, specific, reproducible and stable. A total of 540 ticks collected from18 regions in Liaoning province and autonomous regions in 2020 and 2021 were detected by this method, 234 positive samples were detected, with a positive rate of 43.3%. In the present study, we developed the novel assay for pathogen detection, especially for the new virus. The establishment filled the void in real-time qPCR detection of NALSV, provided a new technical method for the pathogen detection of NALSV, and laid a foundation for the distribution monitoring, clinical diagnosis and related research of NALSV,which has great practical significance and application value.