Development of Quantitative PCR Method based on SYBR Green Ⅰ Fluorescence for Detecting Subgroup F Avian Leukosis Virus
PAN Honyan
LI Jinqun
LI Qi
YUAN Yilin
CAO Weisheng
Abstract：To accurately detect subgroup F avian leukosis virus(ALV-F),a pair of specific primers F1/F2 were designed according to the env gene of the ALV-F epidemic strains,and a SYBR GreenⅠfluorescence quantitative PCR detection method for ALV-F was established.The condition optimization experiment results showed that the optimal annealing temperature was 64.5℃,and the optimal primer concentration was 0.4 μM.The standard curve equation established using the optimal reaction conditions was y=-3.303x+42.525,R2=0.997,amplification efficiency E=100.8%,and the copy number of the standard plasmid had a good linear relationship with the Ct value.The specificity results showed that this method can only specifically detect ALV-F,and has no cross-reaction to ALV-A,ALV-B,ALV-J,ALV-K,avian influenza virus(AIV),Marek's virus(MDV),reticuloendotheliosis virus(REV)and chicken infectious anemia virus(CIAV).The sensitivity results showed that the minimum detection limit of standard plasmid was 4.67×102 copies/μL,and the sensitivity was 1000 times that of routine PCR.The reproducibility results showed that the coefficient of variation of intra-assay and inter-assay reproducibility was less than 2%.23 primary cell(PEF)samples prepared from pheasant embryos were detected by using this method,ALV p27 ELISA and routine PCR respectively,and the results showed that the positive detection rates of this method and PCR method were both 95.65%(22/23),while the positive detection rate of ALV p27 ELISA was only 39.13%(9/23).The total consistency rate of this method with routine PCR was 100%,while the total consistency rate with ALV p27 ELISA was 43.48%.20 anticoagulated blood samples of pheasant were further detected by using this method,virus isolation method through ALV p27 ELISA and routine PCR method,and the results showed that the positive detection rates of this method and ordinary PCR method were 100.00%(20/20)and 75.00%(15/20),respectively,with an overall compliance rate of 75.00%,while the positive detection rate of the virus isolation method was 0.00%(0/20).All results indicated that the method established in this study has good specificity,sensitivity and stability to provide technical support for the accurate detection of ALV-F.
Keywords：Avian leukosisALV-FSYBR Green Ⅰ fluorescence quantitative PCR
Publication Date：2025-04-18
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：7( 23-29 )