J、K亚群禽白血病病毒血液核酸快速筛查技术的建立

doi:10.11843/j.issn.0366-6964.2025.12.034

摘要/Abstract

摘要： 为了缩短J、K亚群禽白血病病毒(subgroup J/K avian leukosis virus, ALV-J/K)净化检测周期，加快禽白血病(avian leukosis，AL)净化进程，本研究建立了一种适用于ALV-J/K低流行率场景下的血液核酸快速筛查技术(ALV-J/K blood quantitative polymerase chain reaction, ALV-J/K-B-qPCR)。选取ALV-J、ALV-K保守区域设计特异性引物和TaqMan探针，并优化反应条件，建立了ALV-J/K TaqMan qPCR方法。以细胞病毒分离鉴定为ALV-J/K的阳性血液为实验材料，制备抗凝血DNA、血细胞DNA和血浆cDNA，进行ALV-J/K TaqMan qPCR检测，筛选最佳检测模板；使用血液室温裂解试剂盒和通用型柱式基因组DNA提取试剂盒提取核酸，筛选核酸提取方法；探索阳性样品不同稀释倍数对检测结果的影响，评估混合样本检测的可行性。运用ALV-J/K-B-qPCR进行预期流行率为1%~2%的血液核酸筛查模拟试验，并与病毒分离法比较。结果显示：ALV-J/K TaqMan qPCR特异性、灵敏性和重复性试验结果显示，该方法仅能特异性扩增ALV-J和ALV-K，对阳性标准品的检测下限分别为8.95×10¹ copies·μL^-1、8.16×10¹ copies·μL^-1，比普通PCR灵敏性高100倍，批内和批间变异系数均＜2%。96份临床样本检测结果显示，ALV-J/K TaqMan qPCR检出率为21.9%，高于普通PCR(15.6%)和ALV p27 ELISA(18.8%)。检测模板筛选试验中，抗凝血DNA为最佳检测模板。核酸提取方法筛选试验中，通用型柱式基因组DNA提取试剂盒提取核酸检测效果最好。混合样本检测可行性评估试验中，本研究建立的检测方法混合样本数量＜10时，可检测低病毒载量样本。预期流行率为1%~2%的血液核酸筛查模拟试验结果显示，ALV-J/K-B-qPCR检出率为4%，比病毒分离法高2.5%。本研究建立的ALV-J/K-B-qPCR技术具有同时检测并鉴别ALV-J和ALV-K、检测效率高和节约成本的优势，以期为种禽场AL净化提供新技术。

关键词: J亚群禽白血病病毒, K亚群禽白血病病毒, qPCR, 核酸筛查, 混样检测

Abstract: This study aimed to shorten the detection cycle for subgroup J/K avian leukosis virus (ALV-J/K) purification and accelerate the avian leukosis (AL) purification process, this study established a rapid blood nucleic acid screening technique (ALV-J/K blood quantitative polymerase chain reaction, ALV-J/K-B-qPCR) suitable for low-prevalence scenarios of ALV-J/K. By selecting conserved regions of ALV-J and K to design specific primers and TaqMan probes, and optimizing the reaction conditions, a TaqMan qPCR detection method for ALV-J/K was established. Using blood samples identified as ALV-J/K positive through cell culture virus isolation as experimental materials, anticoagulated whole blood DNA, blood cell DNA, and plasma cDNA were prepared and subjected to ALV-J/K TaqMan qPCR detection to identify the optimal detection template. Nucleic acids were extracted using a blood room-temperature lysis kit and a universal column-based genomic DNA extraction kit to screen for the optimal nucleic acid extraction method. The effect of varying dilution factors of positive samples on detection results was investigated to assess the feasibility of mixed sample detection. A simulated blood nucleic acid screening test with an expected prevalence rate of 1% to 2% was conducted using ALV-J/K-B-qPCR, and the results were compared with those obtained by the virus isolation method. The specificity, sensitivity, and repeatability tests of ALV-J/K TaqMan qPCR demonstrated that this method specifically amplifies only ALV-J and ALV-K, with detection limits of 8.95×10¹ copies·μL^-1, 8.16×10¹ copies·μL^-1 for positive standards, respectively. The sensitivity was 100 times higher than that of conventional PCR, and both intra- and inter-assay coefficients of variation <2%. The detection results of 96 clinical samples revealed that the ALV-J/K TaqMan qPCR had a detection rate of 21.9%, which was higher than that of conventional PCR (15.6%) and ALV p27 ELISA (18.8%). In the detection template screening test, anticoagulated whole blood DNA was identified as the optimal detection template. In the nucleic acid extraction method screening test, the universal column-based genomic DNA extraction kit yielded the best nucleic acid detection results. In the feasibility assessment test for mixed sample detection, the method established in this study was capable of detecting samples with low viral loads when the number of mixed samples <10. The simulated nucleic acid screening test for blood with an expected prevalence rate of 1% to 2% showed that the ALV-J/K-B-qPCR had a detection rate of 4%, which was 2.5% higher than that of the virus isolation method. The ALV-J/K-B-qPCR technology established in this study offers the advantages of simultaneous detection and differentiation of ALV-J and ALV-K, high sensitivity, strong specificity, high detection efficiency, and cost-effectiveness, aiming to provide a novel technique for AL purification in breeding poultry farms.

Key words: subgroup J avian leukosis virus, subgroup K avian leukosis virus, qPCR, nucleic acid screening, pooling sample assay

中图分类号:

S852.659.3

王红梅, 周婉怡, 罗鑫杰, 陈荟琳, 陈钦玺, 舒雪利, 张金玉, 苏桐, 廖明, 曹伟胜. J、K亚群禽白血病病毒血液核酸快速筛查技术的建立[J]. 畜牧兽医学报, 2025, 56(12): 6316-6325.

WANG Hongmei, ZHOU Wanyi, LUO Xinjie, CHEN Huilin, CHEN Qinxi, SHU Xueli, ZHANG Jinyu, SU Tong, LIAO Ming, CAO Weisheng. Establishment of Blood Nucleic Acid Screening Technology for Subgroup J/K Avian Leukosis Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6316-6325.

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