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

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (12): 6316-6325.doi: 10.11843/j.issn.0366-6964.2025.12.034

J、K亚群禽白血病病毒血液核酸快速筛查技术的建立

王红梅¹(), 周婉怡¹, 罗鑫杰¹, 陈荟琳¹, 陈钦玺¹, 舒雪利¹, 张金玉¹, 苏桐¹, 廖明³, 曹伟胜¹^,²^,³^,⁴^,⁵^,*()

1. 华南农业大学兽医学院, 广州 510642
2. 广东省动物源性人兽共患病预防与控制重点实验室, 广州 510642
3. 人兽共患病防控制剂国家地方联合工程实验室, 广州 510642
4. 农业农村部兽用疫苗创制重点实验室, 广州 510642
5. 农业农村部人兽共患病重点实验室, 广州 510642

收稿日期:2025-03-18 出版日期:2025-12-23 发布日期:2025-12-24
通讯作者: 曹伟胜 E-mail:wanghongmei202202@163.com;caoweish@scau.edu.cn
作者简介:王红梅(1998-)，女，四川人，硕士生，主要从事动物传染病研究，E-mail: wanghongmei202202@163.com
基金资助:
广东省家禽产业技术体系(2023KJ128；2024CXTD20)；国家肉鸡产业技术体系(CARS-41)

Establishment of Blood Nucleic Acid Screening Technology for Subgroup J/K Avian Leukosis Virus

WANG Hongmei¹(), ZHOU Wanyi¹, LUO Xinjie¹, CHEN Huilin¹, CHEN Qinxi¹, SHU Xueli¹, ZHANG Jinyu¹, SU Tong¹, LIAO Ming³, CAO Weisheng¹^,²^,³^,⁴^,⁵^,*()

1. College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
2. Guangdong Provincial Key Laboratory of Prevention and Control for Animal Originated Zoonotic Diseases, Guangzhou 510642, China
3. National-Local Joint Engineering Laboratory for Zoonotic Disease Control and Prevention, Guangzhou 510642, China
4. Key Laboratory of Veterinary Vaccine Innovation of the Ministry of Agriculture and Rural Affairs, Guangzhou 510642, China
5. Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, Guangzhou 510642, China

Received:2025-03-18 Online:2025-12-23 Published:2025-12-24
Contact: CAO Weisheng E-mail:wanghongmei202202@163.com;caoweish@scau.edu.cn

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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引物/探针名称 Primers/Probes	序列(5′→3′) Sequences	产物长度/bp Product length
J-F	CAAGAAAGACCCGGAGAAG	92
J-R	CRACGGAAATAATAACCACG
J-P	(HEX)ACCCTTGCTGCCATCGAGAGGTTACT(BHQ1)
K-F	GCTTTCAGATTGGTCCAG	138
K-R	CTGTAACCATATGTACCGC
K-P	(FAM)TCCTCCTCCGCCGCAATTACACAGA(BHQ1)
H5	GGATGAGGTGACTAAGAAAG	545
H7	CGAACCAAAGGTAACACACG
K1	TCCAGGCCGCAACTCAC	1 214
K2	CATACCACCACCCACGTACT

标准质粒 Standard plasmid	浓度/(copies·μL^-1) Concentration	批内重复性试验 Intra-assay			批间重复性试验 Inter-assay
标准质粒 Standard plasmid	浓度/(copies·μL^-1) Concentration	平均Ct值 ${\bar x}$	标准差 s	变异系数 CV/%	平均Ct值 ${\bar x}$	标准差 s	变异系数 CV/%
ALV-J	10⁶	17.20	0.079	0.46	17.40	0.28	1.61
	10⁵	20.65	0.029	0.14	20.67	0.23	1.11
	10⁴	23.61	0.050	0.21	23.73	0.25	1.05
ALV-K	10⁶	18.43	0.045	0.24	18.50	0.13	0.70
	10⁵	21.70	0.097	0.45	22.13	0.43	1.94
	10⁴	25.26	0.041	0.16	25.52	0.30	1.18

模板 Templates	阳性检出率/% Positive detection rate	Ct值($\bar x \pm s$) Ct value ($\bar x \pm s$)
抗凝血DNA Anticoagulated whole blood DNA	100(16/16)	26.21±1.23
血细胞DNA Blood cell DNA	100(16/16)	27.39±1.30
血浆cDNA Plasma cDNA	62.5(10/16)	29.05±3.03

提取方法 Extraction method	检出样本数 Number of detected samples/pieces	未检出样本数 Number of undetected samples/pieces	检出率/% Detection rate	Ct值($\bar x \pm s$) Ct value ($\bar x \pm s$)
血液室温裂解试剂盒 Blood room-temperature lysis kit	17	4	81	29.47±2.97
通用型柱式基因组DNA提取试剂盒 Universal column-based genomic DNA extraction kit	21	0	100	24.92±1.48

Ct值范围 Ct value range	平均Ct值±标准差Mean Ct value±standard deviation
Ct值范围 Ct value range	单检	4倍	6倍	8倍	10倍
20＜Ct值≤25	23.30±0.91	24.46±0.88	25.32±0.89	25.99±0.99	26.48±0.92
25＜Ct值≤30	25.98±1.16	27.12±1.22	27.99±1.24	28.57±1.26	29.02±1.33
30＜Ct值≤33	30.80±1.71	31.97±1.71	32.67±1.70	33.35±1.63	33.90±1.68

J、K亚群禽白血病病毒血液核酸快速筛查技术的建立

Establishment of Blood Nucleic Acid Screening Technology for Subgroup J/K Avian Leukosis Virus

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参考文献 32

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ALV-J/K-B-qPCR	病毒分离法Viral isolation		合计 Total amounts
ALV-J/K-B-qPCR	反应性Reactivity	非反应性Non-reactivity	合计 Total amounts
反应性Reactivity	3	5	8
非反应性Non-reactivity	0	192	192
合计Total amounts	3	197	200

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