为缩短J亚群禽白血病病毒(subgroup J avian leukosis virus,ALV-J)检测周期,进一步加快禽白血病净化进程,本研究结合SYBR Green I qPCR和混样检测,建立了一种适用于低ALV-J流行率场景下的快速筛检ALV-J 的血液核酸筛查技术(ALV-J blood quantitative polymerase chain reaction,ALV-J-B-qPCR).根据 GenBank 中ALV-J pol及env基因序列,设计ALV-J的特异性引物,并优化反应条件,建立了 ALV-J SYBR Green I qPCR检测方法.以ALV-J病毒分离为阳性的鸡抗凝血为实验材料,分别制备抗凝血 DNA、血细胞DNA、外周血淋巴细胞DNA、外周血淋巴细胞cDNA和血浆cDNA 5类血液检测模板进行ALV-J的PCR检测,筛选最佳检测模板;进一步比较蒸馏水破裂红细胞法提取混合血液DNA的混样方法,血液混样总体积为200 μL的混样方法,血液混样总体积为10μL的混样方法共3种混样方法的检测准确性,筛选最佳混样方法.综合上述qPCR方法、检测模板与混样方法,成功建立了 ALV-J-B-qPCR.运用ALV-J-B-qPCR分别进行预期流行率为1％～2％、4％～5％场景下的模拟筛查试验,并与病毒分离法比较.qPCR方法的特异性、灵敏性和重复性试验显示,该方法仅特异性扩增ALV-J,对标准质粒的最低检测限度为1×102 copies·μL-1,批内与批间变异系数均＜1％.对90份临床送检血液样品的检测结果显示,该qPCR方法对ALV-J的检出率(15.6％)高于p27抗原ELISA和普通PCR的检出率(12.2％).检测模板筛选试验中,抗凝血DNA最符合检测准确度高、操作复杂度和成本低的要求,为最佳检测模板.混样方法摸索试验中,混样总体积为10 μL(混样规模＜12份)的混样方法检测准确性最高,为最佳混样方法.在样本数为400份,预期流行率为4％～5％场景的模拟筛查中,ALV-J-B-qPCR检出率为6.25％,比病毒分离法高2.00％;在样本数为400份,预期流行率为1％～2％场景的模拟筛查中,ALV-J-B-qPCR检出率为2.25％,比病毒分离法高0.75％.本研究建立的ALV-J-B-qPCR技术具有灵敏性高、检测快速和节约成本的优势,为种禽场加快ALV净化进程提供了新的思路和方法.
Establishment of Blood Nucleic Acid Screening Technology for Subgroup J Avian Leukosis Virus
To shorten the detection cycle of subgroup J avian leukosis virus(ALV-J)and further accelerate the process of avian leukosis decontamination,this study combined SYBR Green]qPCR and pooling sample assay to establish a blood nucleic acid screening technique(ALV-J-B-qPCR)for rapid screening of ALV-J in low prevalence.Based on the sequences of ALV-J pol and env genes in GenBank,the specific primers for ALV-J were designed and the reaction conditions were optimized to establish the ALV-J SYBR Green Ⅰ qPCR assay.Anticoagulant DNA,blood cell DNA,peripheral blood lymphocyte DNA,peripheral blood lymphocyte cDNA and plasma cDNA were respectively prepared to perform PCR detection of ALV-J,and the best assay templates were screened.Further,the accuracy of the three mixing methods of extracting DNA from mixed blood by red blood cell cracking method,the mixed method with 200 μL total volume of blood mixed sample and the mixed method with 10 μL total volume of blood mixed sample were compared,and the best mixing method was selected.The ALV-J-B-qPCR was established by combining the above qPCR method,assay template and mixing method.Simulated screening tests with expected prevalence scenarios of 1％-2％and 4％-5％were performed using ALV-J-B-qPCR respectively,and compared with the virus isolation method.Specificity,sensitivity and reproducibility tests of the qPCR method showed that the method specifically amplifies only ALV-J and has a minimum detection limit of 1×102 copies·μL-1 for standard plasmids,with intra-and inter-batch coefficients of variation＜1％.Results on 90 clinical samples showed that the detection rate of ALV-J by this qPCR method(15.6％)was higher than that of p27 antigen ELISA and PCR(12.2％).In the assay template screening test,anticoagulated blood DNA best met the requirements of high assay accuracy,operational complexity and low cost,and was the best assay template.In the mixing method screening test,the mixing method with a total mixing volume of 10 μL(mixing size＜12)had the highest detection accuracy and was the best mixing method.In a simulated screening with a sample size of 400 and an expected prevalence of 4％-5％,the detection rate of ALV-J-B-qPCR was 6.25％,which was 2.00％higher than that of the virus isolation method;in a simulated screening with a sample size of 400 and an expected prevalence of 1％-2％,the detection rate of ALV-J-B-qPCR was 2.25％,which was 0.75％higher than that of the virus isolation method.The ALV-J-B-qPCR technique established in this study has the advantages of high sensitivity,rapid detection and cost saving,which provides a new idea and method to accelerate the ALV purification process in breeding poultry farms.
万方数据
维普
