鹅圆环病毒TB Green实时荧光定量PCR方法的建立及初步应用

Development of a TB Green real-time PCR assay for the detection of goose circovirus

鹅圆环病毒主要感染宿主的淋巴细胞,导致其免疫功能下降,造成继发感染和共感染。为建立鹅圆环病毒分子检测方法,本研究根据NCBI数据库中GoCV基因特征,设计特异性引物,建立检测GoCV的TB Green实时荧光定量PCR方法。结果显示,建立检测GoCV的TB Green实时荧光定量PCR方法,灵敏度高,最低检测限为54.10拷贝/μL;特异性好,与水禽常见传染病病原均无交叉反应,扩增产物的熔解曲线仅GoCV出现1个特异性单峰,Tm值为(84.46±0.09)℃;重复性好,组内和组间重复试验变异系数分别为0.26%~0.55%和0.23%~0.87%。利用建立的TB Green实时荧光定量PCR方法和常规PCR方法同时对64份临床样品进行GoCV感染的检测,结果显示,常规PCR方法检出阳性样品15份,阳性率为23.44%;TB Green实时荧光定量PCR方法检出阳性样品18份,阳性率28.13%,且15份PCR阳性样品经TB Green实时荧光定量PCR方法检测均为阳性,符合率为100%。本研究为后续开展GoCV感染的分子流行病学调查和致病机理研究提供技术支撑。

Goose circovirus(GoCV) can cause decreased lymphocytes in the sac, spleen and thymus, thus easy to follow multiple conditional pathogen secondary infections, causing great harm to goose industry. Here, we developed a TB Green real-time PCR assay(qPCR) for the detection of GoCV. The primers were designed the conserved within GoCV retrieve from the NCBI database. After the optimization of the qPCR conditions, the lower limit of detection for MDPV was 54.10 copies/μL. The assay was highly specific for GoCV and no cross-reactivity was observed with other waterfowl-derived pathogens, and the melting curve of the amplified GoCV products appeared a specific single peak, with Tm value was(84.46±0.09)℃. It had good reproducibility, and the coefficient of variation of repeated experiments within and between groups was 0.26%~0.55% and 0.23%~0.87%, respectively. Using the established q PCR and PCR method, 64 clinical samples were tested for GoCV infection. The results showed that the established PCR method detected 15 positives with a positive rate of 23.44%. While qPCR method only detected 18 positives with a positive rate of 28.13%. All PCR positive samples were tested positive by qPCR with agreenment rate was 100%. This study successfully established qPCR for the detection of GoCV, which can be used for epidemiological investigation and pathogenesis.