关键词: 鸽圆环病毒, 荧光定量 PCR

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