鸭巴泰病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立

Establishment a SYBR Green Ⅰ-based Real-time PCR Method for Detection of Duck Batai Virus

为建立鸭巴泰病毒(Batai virus,BATV)SYBR Green Ⅰ实时荧光定量PCR(qRT-PCR)检测方法,研究根据鸭BATV非结构蛋白(NSs)基因特征,设计引物,经条件优化后建立检测BATV感染的qRT-PCR方法。用建立的qRT-PCR方法对临床72份疑似BATV感染的病料进行检测,并对阳性样品进行病毒分离,评价两种方法的符合率。结果表明,当病毒NS基因含量为3.59×10~2~3.59×10~7拷贝/μL时有良好的线性扩增,其扩增相关系数为0.9994,扩增效率为98%。最低检测限为3.59×10~2拷贝/μL;扩增产物的熔解曲线分析只出现1个单特异峰,无引物二聚体,Tm值为(81.60±0.16)℃,对鸭源常见病毒(如鸭甲肝病毒、禽坦布苏病毒、禽Ⅰ型副黏病毒、H9N2亚型禽流感病毒、新型鸭呼肠孤病毒和番鸭呼肠孤病毒)检测均为阴性,组内变异系数和组间变异系数分别为0.49%~1.99%和0.58%~2.18%。临床送检的72份病料SYBRⅠ实时荧光定量PCR方法的阳性率为4.17%(3/72),并分离到2株鸭源BATV,2种方法的阳性符合率为66.67%(2/3)。建立的基于SYBRⅠ检测BATV的qRT-PCR方法、特异性强、灵敏度高、重复性好,可用于BATV的分子流行病学研究。

A SYBR Green Ⅰ-based real-time PCR method（qRT-PCR） was developed for detection duck Batai virus（BATV） with specific primers targeting to the nonstructural（NSs） gene, also the specificity and repeatability were detected for the established method. 72 clinic samples were tested by the established qRT-PCR, then the coincidence rate were calculated compared with the virus isolation and identification. The results showed established qRT-PCR standard curve with the BATV NS gene positive plasmids had a wide dynamic range from 3.59×10^2 to 3.59×10^7 DNA copies/μL with a linear correlation（R～2） of 0.9994 and 98 percentage of efficiency between Ct value and logarithm of the plasmids copy number. The lowest limit of detection concentration was 3.59×10^2 copies/μL. The melting curve analysis using SYBR Green Ⅰ dye showed one specific peak with a melting temperature（Tm） was（81.60±0.16） ℃, and no primer-dimers peak was represent. No amplification was detected from common duck origin viruses samples by this method, such as, duck hepatitis virus type 1, avian Tembusu virus, avian paramyxovirus-1, H9N2 subtype avian influenza virus, novel duck reovirus and Muscovy duck reovirus. Reproducibility test showed that the intra-and inter-assay were ranged from 0.49% to 1.99% and 0.58% to 2.18%, respectively. The positive rate 72 clinical samples tested by established qRT-PCR was 4.17%（3/72）. Two of the three clinical samples were isolated duck BATV sufessfully, with the coincidence rate was 66.67%（2/3）. In conclusion, the establishment of a SYBR Green Ⅰ-based real-time PCR method could provide a useful tool for molecular epidemiology for duck Batai virus.