GB/T 18646-2025 动物布鲁氏菌病诊断技术

ICS11.220

CCSB41

中华人民共和国国家标准

GB/T18646—2025

代替GB/T18646—2018

动物布鲁氏菌病诊断技术

Diagnostictechniquesforanimalbrucellosis

2025⁃05⁃30发布2025⁃12⁃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/T18646—2025

目次

前言··························································································································Ⅴ

引言··························································································································Ⅵ

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

5.1流行特点···········································································································2

5.2临床症状···········································································································2

5.3病理变化···········································································································3

5.4结果判定···········································································································3

6样品采集、保存、运输及处理·························································································3

6.1仪器设备···········································································································3

6.2试剂材料···········································································································3

6.3样品采集···········································································································3

6.4样品运输和保存··································································································4

6.5样品处理···········································································································4

7病原学诊断方法········································································································5

7.1改良萋⁃尼氏（Ziehl⁃Neelsen）抗酸染色·······································································5

7.2布鲁氏菌分离与鉴定····························································································6

7.3实时荧光PCR方法······························································································7

7.4荧光RAA方法···································································································9

7.5多重PCR方法··································································································11

8免疫学诊断方法······································································································12

8.1标准血清··········································································································12

8.2虎红平板凝集试验（RBT）····················································································13

8.3试管凝集试验（SAT）··························································································14

8.4补体结合试验（CFT）···························································································17

8.5间接ELISA抗体检测方法（iELISA）······································································22

8.6竞争ELISA抗体检测方法（cELISA）······································································24

8.7荧光偏振试验（FPA）···························································································26

8.8全乳环状试验（MRT）（仅适用于牛）········································································29

8.9免疫层析法（ICA）······························································································30

Ⅰ

GB/T18646—2025

9综合判定···············································································································30

9.1布鲁氏菌疑似感染动物判定···············································································30

9.2布鲁氏菌感染动物判定·····················································································31

附录A（资料性）检测方法的适用性················································································32

附录B（规范性）布鲁氏菌培养基的配制··········································································33

B.1基础培养基······································································································33

B.2血清葡萄糖培养基·····························································································33

B.3选择培养基······································································································33

附录C（规范性）生化反应试剂的配制·············································································34

C.1结晶紫储备液和工作液配制·················································································34

C.2氧化酶试验试剂配制··························································································34

C.3尿素酶活性试验培养基配方及制备········································································34

附录D（规范性）布鲁氏菌种和生物型的生化鉴定······························································35

附录E（规范性）实时荧光PCR、RAA及多重PCR引物和探针序列·······································37

附录F（规范性）多重PCR溶液配制及结果判定·······························································39

F.1TAE电泳缓冲液·······························································································39

F.21.5%琼脂糖凝胶·······························································································39

F.3多重PCR判定标准····························································································39

附录G（规范性）试管凝集试验（SAT）试剂的配制·····························································40

G.1含0.5%苯酚的生理盐水·····················································································40

G.2含0.5%苯酚的10%氯化钠溶液··········································································40

G.3参照比浊管的制备·····························································································40

附录H（规范性）补体结合试验（CFT）试剂的配制及效价测定··············································41

H.1稀释液配制······································································································41

H.2绵羊红细胞悬液的配制（2.5%）·············································································41

H.3溶血素效价测定································································································41

H.4补体制备及效价测定··························································································44

H.5抗原效价的测定································································································49

H.6溶血标准比色管的制备及补体结合试验结果判定方法················································50

H.7待检血清的稀释和灭活·······················································································51

附录I（规范性）酶联免疫吸附试验（ELISA）试剂的制备······················································52

I.1包被抗原LPS提取·····························································································52

I.2包被液（0.05mol/L的pH9.6碳酸盐缓冲液）····························································52

I.3封闭液（含2%BSA、0.05%吐温⁃20的pH7.4PBS）···················································52

I.4样品稀释液（含1%BSA的pH7.4PBS）··································································52

I.5洗涤液（含0.05%吐温⁃20的pH7.4PBS）································································52

Ⅱ

GB/T18646—2025

I.6显色液·············································································································52

I.7终止液（2mol/L硫酸）·························································································53

附录J（资料性）竞争酶联免疫吸附试验（cELISA）单克隆抗体的制备·····································54

附录K（规范性）荧光偏振试验（FPA）抗原和稀释液的制备··················································55

K.1标记抗原的制备································································································55

K.2样品稀释液的配制·····························································································55

参考文献····················································································································56

Ⅲ

GB/T18646—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/T18646—2018《动物布鲁氏菌病诊断技术》，与GB/T18646—2018相比，除结构

调整和编辑性改动外，主要技术变化如下：

a）更改了“范围”（见第1章，2018年版的第1章）；

b）更改了“临床诊断”，增加了流行病学、临床症状和病理变化内容（见第5章，2018年版的4.1、

4.2、4.3）；

c）增加了“实时荧光PCR方法”“荧光RAA方法”（见7.3和7.4）；

d）将“布鲁氏菌Bruce⁃Ladder检测方法”更改为“多重PCR方法”（见7.5，2018年版的4.13）；

e）增加了免疫学诊断方法中RBT、SAT、CFT、iELISA、cELISA和FPA方法的标准化内容（见

第8章）；

f）更改了“虎红平板凝集试验（RBT）”（见8.2，2018年版的4.4）、“间接ELISA抗体检测方法

（iELISA）”（见8.5，2018年版的4.8）、“竞争ELISA抗体检测方法（cELISA）”（见8.6，2018

年版的4.9）；

g）增加了“荧光偏振试验（FPA）”“免疫层析法（ICA）”（见8.7和8.9）。

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：中国动物卫生与流行病学中心、中国兽医药品监察所、中国动物疫病预防控制中

心、中国农业科学院哈尔滨兽医研究所、中国疾病预防控制中心、新疆生产建设兵团畜牧兽医工作总

站、辽宁省动物疫病预防控制中心、内蒙古自治区动物疫病预防控制中心、上海市动物疫病预防控制中

心、河南省动物疫病预防控制中心、重庆市动物疫病预防控制中心、浙江省动物疫病预防控制中心、

青岛市动物疫病预防控制中心。

本文件主要起草人：孙淑芳、李俊平、邵卫星、孙明军、姜海、南文龙、田莉莉、樊晓旭、焉鑫、孙雨、

胡森、李岩、朱良全、王建龙、闫若潜、张存瑞、李彦、顾贵波、赵洪进、董春霞、赵灵燕、郭宇、孙翔翔、刘蒙达、

张皓博、孙世雄、李嘉琪。

本文件及其所代替文件的历次版本发布情况为：

——2002年首次发布为GB/T18646—2002，2018年第一次修订；

——本次为第二次修订。

Ⅴ

GB/T18646—2025

引言

布鲁氏菌病（Brucellosis）是由布鲁氏菌（Brucella）感染动物和人而引起的一种人兽共患传染病。

目前已报道的布鲁氏菌属成员包括6个经典种、5个新鉴定种及部分未确定种型的非典型布鲁氏菌。

经典种包括羊种布鲁氏菌（Brucellamelitensis）、牛种布鲁氏菌（Brucellaabortus）、猪种布鲁氏菌（Bru⁃

cellasuis）、绵羊附睾种布鲁氏菌（Brucellaovis）、犬种布鲁氏菌（Brucellacanis）和沙林鼠种布鲁氏菌

（Brucellaneotomae）；新鉴定种包括鲸种布鲁氏菌（Brucellaceti）、鳍种布鲁氏菌（Brucellapinnipedia⁃

lis）、田鼠种布鲁氏菌（Brucellamicroti）、人源布鲁氏菌（Brucellainopinata，暂未确定其他动物宿主）和

狒狒种布鲁氏菌（Brucellapapionis）；未确定种型的包括分离自啮齿类、狐类和蛙类的非典型布鲁氏菌。

本文件主要规定了对畜牧业影响最大的羊种布鲁氏菌、牛种布鲁氏菌和猪种布鲁氏菌三种光滑型布鲁

氏菌引起的动物布鲁氏菌病相关诊断检测技术内容，仅在病原鉴定及PCR检测方法中涉及了粗糙型

的绵羊附睾种和犬种布鲁氏菌检测。

布鲁氏菌病在全世界范围内严重威胁动物和人类健康。世界动物卫生组织（WorldOrganization

ofAnimalHealth，WOAH）将布鲁氏菌病列入通报病种名录，我国农业农村部发布的《一、二、三类动物

疫病病种名录》将布鲁氏菌病列为二类动物疫病，国家卫生健康委员会将布鲁氏菌病列为法定报告乙

类传染病。从事布鲁氏菌病诊断相关的样品采集、包装运输、检测诊断等活动的人员，经过严格培训，

在相应级别的生物安全防护条件下进行试验活动。

本文件参考了WOAH《陆生动物诊断试验与疫苗手册》3.1.4章“布鲁氏菌病（牛种、羊种和猪种布

鲁氏菌感染）（2022版）”规定的诊断技术方法，包括临床诊断、病原学诊断方法（涂片镜检、分离与鉴

定、实时荧光PCR方法）及免疫学诊断方法［虎红平板凝集试验（RBT）、试管凝集试验（SAT）、补体结

合试验（CFT）、间接ELISA抗体检测方法（iELISA）、竞争ELISA抗体检测方法（cELISA）、荧光偏振

试验（FPA）和全乳环状试验（MRT）］；并根据我国技术发展增加了技术成熟的荧光RAA方法和免疫

层析法（ICA）。

本文件的发布机构提请注意，声明相关产品符合本文件时，可能涉及本文件7.3中引物和探针序

列与《一种区分布鲁氏菌A19疫苗株与野毒株的分子标记》（授权公告号：CN113186312B）、《鉴别布鲁

氏菌疫苗株S2和野毒株的SNP分子标记及其应用》（授权公告号：CN111793704B）相关专利的使用。

本文件的发布机构对于涉及专利的真实性、有效性和范围无任何立场。

该专利持有人已向本文件的发布机构承诺，同意在公平、合理、无歧视基础上，免费许可任何组织

或个人在实施该国家标准时实施专利。该专利持有人的声明已在本文件的发布机构备案。相关信息

可以通过以下联系方式获得：

专利（授权公告号：CN113186312B）持有人姓名：中国动物卫生与流行病学中心。

地址：山东省青岛市市北区南京路369号。

专利（授权公告号：CN111793704B）持有人姓名：中国疾病预防控制中心传染病预防控制所。

地址：北京市昌平区昌百路155号。

请注意，除上述2项专利外，本文件的某些内容仍可能涉及专利。本文件的发布机构不承担识别

专利的责任。

Ⅵ

GB/T18646—2025

动物布鲁氏菌病诊断技术

1范围

本文件描述了动物布鲁氏菌病的临床诊断方法、病原学诊断方法和免疫学诊断方法，并规定了综

合判定要求。

本文件适用于布鲁氏菌病畜群无疫检测、动物个体无疫检测、根除计划实施、疑似或临诊病例确

诊、群体流行率监测等。

注：附录A中详细列出了不同领域检测方法的选择。

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）适用于

本文件。

NY/T541兽医诊断样品采集、保存与运输技术规范

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。

cELISA：竞争酶联免疫吸附试验（CompetitiveEnzymeLinkedImmunosorbentAssay）

CFT：补体结合试验（ComplementFixationTest）

Ct：循环阈值（CycleThreshold）

DNA：脱氧核糖核酸（DeoxyribonucleicAcid）

FPA：荧光偏振试验（FluorescencePolarisationAssay）

HRP：辣根过氧化物酶（HorseradishPeroxidase）

ICA：免疫层析试验（ImmunochromatographyAssay）

iELISA：间接酶联免疫吸附试验（IndirectEnzymeLinkedImmunosorbentAssay）

LPS：脂多糖（Lipopolysaccharide）

MRT：全乳环状试验（MilkRingTest）

OD值：光密度值（OpticalDensity）

PCR：聚合酶链式反应（PolymeraseChainReaction）

RAA：重组酶介导等温扩增技术（RecombinaseAidedAmplification）

RBT：虎红平板凝集试验（RoseBengalTest）

SAT：试管凝集试验（SerumAgglutinationTest）

SDS：十二烷基硫酸钠（Sodiumdodecylsulfate）

1