Abstract: Objective To establish and apply TaqMan probe fluorescent quantitative PCR method for pigeon circovirus( PiCV) . Method By comparing the sequences of various isolates of PiCV published by NCBI, selecting the conservative sequence of polymerase gene to design primers and probes, establishing a fluorescent quantitative PCR method for PiCV, and verifying the specificity and sensitivity of the method ; the plasmid standards which concentration was 3. 48×10 5 and 3. 48 ×10 4 copies/ μL, each concentration was tested in 6 parallels, the test was repeated 3 times, the coefficient of variation intra-assay and inter-assay was calculated to verify the repeatability and stability of the method ; The method was used to detect 40 samples of pigeon liver, spleen, lung, bursa of Fabricius and 36 samples of pigeon cecum collected from a breeding farm in Beijing, verifying the capability of the method in practical application. Result The established PiCV fluorescence quantitative PCR method has a linear relationship between the Ct value and the plasmid concentration in the range of 3. 48×10 7 -3. 48×10 1 copies/ μL. The standard curve Slope is - 3. 381, the R 2 value is 1, and the amplification efficiency is 97. 61%. The lowest detectable concentration of PiCV is 34. 8 copies/ μL. Compared with the conventional PCR method , the detection sensitivity is 2 orders of magnitude higher; There was no amplification curve for porcine circovirus type 2, avian adenovirus type I and III, avian leukemia virus, reticuloendotheliosis virus as templates; repeated detection of plasmid standards at 3. 48×10 5 and 3. 48×10 4 copies/ μL showed that the coefficient of variation intra-assay was less than 1. 63%, and the coefficient of variation inter-assay was less than 7. 23%. The established fluorescence quantitative PCR method was used to detect pigeon tissue samples. The positive rates of liver, spleen, lung, bursa of Fabricius and cecum were 97. 5%, 100%, 100%, 100%, and 97. 2% respectively. The chi-square test showed no significant difference ( P > 0. 05) in virus content of each tissue. Conclusion The established PiCV FQ-PCR method can effectively detect PiCV in pigeon tissues, providing technical support and basic data for the formulation of relevant microbial standards for experimental pigeons.

Key words: Pigeon circovirus, FQ-PCR

摘要： 目的 建立鸽圆环病毒 ( PiCV) TaqMan 探针荧光定量 PCR 方法并对其进行 初 步 应 用。 方 法 通 过 比 对 NCBI 发表的 PiCV 各分离株序列,选取其 polymerase 基因保守序列设计引物探针,建立 PiCV 的荧光定量 PCR 方 法,对方法的特异性及敏感性进行验证;取浓度为 3. 48×10 5 和 3. 48×10 4 copies/ μL 的质粒标准品,每个浓度做 6 个 平行,重复检测 3 次,计算组内和组间变异系数,验证方法的重复性和稳定性;用该方法检测采自北京某养殖场的 鸽肝、脾、肺、法式囊样本各 40 份及鸽盲肠样本 36 份,以验证此方法在实际应用中的检测能力。 结果 建立的 PiCV 荧光定量 PCR 方法在 3. 48×10 7 ~ 3. 48× 10 1 copies/ μL 范围 Ct 值与质粒浓度呈线性关系,标准曲线 Slope 为 -3. 381,R 2 值为 1,扩增效率为 97. 61%。 可检测到的 PiCV 最低浓度为 34. 8 copies/ μL,与常规 PCR 法相比,检测 灵敏度高 2 个数量级;以猪圆环病毒 2 型、禽腺病毒 I 型及 III 型、禽白血病病毒、禽网状内皮组织增生性病毒为模 板均无扩增曲线;重复检测 3. 48×10 5 和 3. 48×10 4 copies/ μL 两个浓度的质粒标准品,组内变异系数小于 1. 63%,组 间变异系数小于 7. 23%。 用建立的荧光定量 PCR 方法检测鸽组织样本,肝、脾、肺、法式囊及盲肠阳性率分别为 97. 5%、100%、100%、100%、97. 2%,经卡方检验,各组织病毒含量无显著差异( P> 0. 05) 。 结论 建立的鸽圆环病 毒荧光定量 PCR 方法可有效检测鸽组织中的 PiCV,为实验鸽相关微生物标准的制定提供了技术支持和基础数据。

关键词: 鸽圆环病毒, 荧光定量 PCR