GB/T 26436-2025 禽白血病诊断技术

ICS11.220

CCSB41

中华人民共和国国家标准

GB/T26436—2025

代替GB/T26436—2010

禽白血病诊断技术

Diagnostictechniquesforavianleukosis

2025⁃01⁃24发布2025⁃08⁃01实施

国家市场监督管理总局

国家标准化管理委员会发布

GB/T26436—2025

目次

前言··························································································································Ⅲ

引言··························································································································Ⅳ

1范围·······················································································································1

2规范性引用文件········································································································1

3术语和定义··············································································································1

4缩略语····················································································································1

5实验室生物安全措施··································································································2

6临床诊断·················································································································2

6.1流行病学···········································································································2

6.2临床症状···········································································································2

6.3病理变化···········································································································3

6.4鉴别诊断···········································································································3

6.5临床判定···········································································································3

7样品采集与处理········································································································4

7.1样品采集···········································································································4

7.2样品处理···········································································································4

8实验室诊断··············································································································4

8.1病毒分离和鉴定··································································································4

8.2病毒亚群的鉴定··································································································7

8.3Real⁃timeRT⁃PCR扩增ALV⁃J···············································································7

8.4血清学诊断········································································································7

9综合判定·················································································································8

9.1疑似·················································································································8

9.2确诊·················································································································8

附录A（规范性）病毒分离相关溶液的配制········································································9

A.10.01mol/LPBS（pH7.4）······················································································9

A.250%甘油⁃PBS保存液（pH7.4）··············································································9

A.3细胞完全营养液和维持液······················································································9

附录B（资料性）CEF（C/E品系）的制备··········································································10

附录C（资料性）抗ALV单因子鸡血清的制备··································································11

附录D（规范性）p27抗原ELISA检测相关溶液的配制·······················································12

D.1包被液············································································································12

D.2洗涤液············································································································12

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GB/T26436—2025

D.3样品稀释液······································································································12

D.4底物溶液·········································································································12

D.5终止液············································································································12

附录E（资料性）ALV亚群鉴定程序···············································································13

E.1克隆载体和宿主菌·····························································································13

E.2病毒模板的制备································································································13

E.3囊膜蛋白env基因的扩增·····················································································13

E.4PCR产物的克隆·······························································································14

E.5质粒DNA的提取和鉴定····················································································15

E.6克隆序列的测定································································································15

附录F（资料性）Real⁃timeRT⁃PCR扩增ALV⁃J································································17

F.1引物和探针序列································································································17

F.2样品采集和前处理·····························································································17

F.3核酸抽提·········································································································17

F.4扩增试剂准备···································································································17

F.5加样检测·········································································································18

F.6反应条件设置···································································································18

F.7荧光素设定······································································································18

F.8分析条件设定及结果判定····················································································18

附录G（资料性）ALV⁃J一步法Real⁃timeRT⁃PCR检测试剂盒的组成及使用···························19

G.1试剂盒组成······································································································19

G.2说明···············································································································19

G.3使用时的注意事项·····························································································19

附录H（资料性）IFA法抗体检测用ALV感染细胞的制备··················································20

参考文献····················································································································21

Ⅱ

GB/T26436—2025

前言

本文件按照GB/T1.1—2020《标准化工作导则第1部分：标准化文件的结构和起草规则》的规

定起草。

本文件代替GB/T26436—2010《禽白血病诊断技术》，与GB/T26436—2010相比，除结构调整和

编辑性改动外，主要技术变化如下：

——增加了“缩略语”（见第4章）；

——增加了ALV⁃p27抗原免疫胶体金试纸卡检测方法（见8.1.4.3）；

——更改了抗体检测结果对确诊的判定标准（见8.4.5，2010年版的4.3.2.3）。

请注意本文件的某些内容可能涉及专利。本文件的发布机构不承担识别专利的责任。

本文件由中华人民共和国农业农村部提出。

本文件由全国动物卫生标准化技术委员会（SAC/TC181）归口。

本文件起草单位：山东农业大学、中华人民共和国拱北海关技术中心、华南农业大学、扬州大学、

中国农业科学院哈尔滨兽医研究所、灵羽生工（北京）医药科技有限公司、中国农业大学、普莱柯生物工

程股份有限公司、中国动物疫病预防控制中心。

本文件主要起草人：赵鹏、崔治中、孙淑红、杨素、沙才华、廖明、曹伟胜、秦爱建、钱琨、王一新、

高玉龙、陈西钊、苏敬良、田克恭、吴清民、王传彬。

本文件及其所代替文件的历次版本发布情况为：

——2010年首次发布为GB/T26436—2010；

——本次为第一次修订。

Ⅲ

GB/T26436—2025

引言

禽白血病（Avianleukosis，AL）是由禽白血病病毒（Avianleukosisvirus，ALV）感染引起的禽类多

种肿瘤性疾病的统称，主要引起鸡的恶性肿瘤和亚临床感染，导致鸡只生长迟缓、产蛋下降和免疫抑

制，是一种能够通过种蛋垂直传播的免疫抑制性疾病，我国将其列为三类动物疫病。

ALV属于反转录病毒科正反录病毒亚科甲型反转录病毒属，根据其gp85蛋白的特性目前将

ALV分为A、B、C、D、E、F、G、H、I、J和K共11个亚群。ALV分为内源性ALV和外源性ALV两大

类。内源性ALV是指整合进宿主细胞染色体基因组因而能通过染色体垂直传播的ALV，它可能只是

病毒基因组的不完全片段不产生传染性病毒粒子，也可能是病毒全基因组因而能产生传染性病毒粒

子，不过这类病毒通常致病性很低或无致病性，主要是E亚群ALV。外源性ALV是与内源性ALV

相对应的，指不通过宿主细胞染色体传递给下一代的ALV，包括A、B、C、D、J和K亚群，致病性强的

ALV均属于外源性病毒。内源性ALV的干扰是开展实验室检测，特别是进行核酸检测时首要排除

因素。

本病发生没有明显的季节性或地域性，通常因为饲养种源未净化ALV的鸡苗而发病，以及在育雏

期与感染鸡群混养或者使用存在外源性ALV污染的生物制品而感染发病。

本文件诊断技术内容包括临床诊断、病原学诊断和血清学诊断方法。本文件的修订参考了世界动

物卫生组织（WOAH）《陆生动物诊断试验和疫苗手册》，并结合了我国相关技术研究新成果。

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GB/T26436—2025

禽白血病诊断技术

1范围

本文件描述了禽白血病的临床诊断、样品采集与处理，以及病毒分离和鉴定、病毒亚群的鉴定、

Real⁃timeRT⁃PCR、扩增ALV⁃J、血清学诊断等实验室诊断方法。

本文件适用于鸡群中禽白血病的诊断、检测、检疫、监测和流行病学调查。

2规范性引用文件

下列文件中的内容通过文中的规范性引用而构成本文件必不可少的条款，其中，注日期的引用文

件，仅该日期对应的版本适用于本文件；不注日期的引用文件，其最新版本（包括所有的修改单）适用本

文件。

GB/T6682分析实验室用水规格和试验方法

GB/T18088出入境动物检疫采样

GB/T18643鸡马立克氏病诊断技术

GB19489实验室生物安全通用要求

NY/T541兽医诊断样品采集、保存与运输技术规范

NY/T1247禽网状内皮组织增殖症诊断技术

3术语和定义

本文件没有需要界定的术语和定义。

4缩略语

下列缩略语适用于本文件。

AL：禽白血病（Avianleukosis）

ALV：禽白血病病毒（Avianleukosisvirus）

CPE：细胞病变效应（Cytopathiceffect）

Ct值：循环阈值（Cyclethreshold）

CEF：鸡胚成纤维细胞（Chickembryofibroblast）

DEPC：焦碳酸二乙酯（Diethylpyrocarbonate）

DMEM：杜氏改良Eagle培养基（Dulbecco’smodificationofEagle’smedium）

dNTP：脱氧核苷三磷酸（Deoxyribonucleosidetriphosphate）

EDTA：乙二胺四乙酸（Ethylenediaminetetraaceticacid）

ELISA：酶联免疫吸附试验（Enzyme⁃linkedimmunosorbentassay）

IFA：间接免疫荧光试验（Indirectimmunofluorescenceassay）

MDV：鸡马立克氏病病毒（Marek’sdiseasevirus）

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