ICS11.220

CCSB41

中華人民共和國(guó)國(guó)家標(biāo)準(zhǔn)

GB/T18646—2025

代替GB/T18646—2018

動(dòng)物布魯氏菌病診斷技術(shù)

Diagnostictechniquesforanimalbrucellosis

2025?05?30發(fā)布2025?12?01實(shí)施

國(guó)家市場(chǎng)監(jiān)督管理總局

國(guó)家標(biāo)準(zhǔn)化管理委員會(huì)發(fā)布

GB/T18646—2025

目次

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

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

1范圍·······················································································································1

2規(guī)范性引用文件········································································································1

3術(shù)語(yǔ)和定義··············································································································1

4縮略語(yǔ)····················································································································1

5臨床診斷·················································································································2

5.1流行特點(diǎn)···········································································································2

5.2臨床癥狀···········································································································2

5.3病理變化···········································································································3

5.4結(jié)果判定···········································································································3

6樣品采集、保存、運(yùn)輸及處理·························································································3

6.1儀器設(shè)備···········································································································3

6.2試劑材料···········································································································3

6.3樣品采集···········································································································3

6.4樣品運(yùn)輸和保存··································································································4

6.5樣品處理···········································································································4

7病原學(xué)診斷方法········································································································5

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

7.2布魯氏菌分離與鑒定····························································································6

7.3實(shí)時(shí)熒光PCR方法······························································································7

7.4熒光RAA方法···································································································9

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

8免疫學(xué)診斷方法······································································································12

8.1標(biāo)準(zhǔn)血清··········································································································12

8.2虎紅平板凝集試驗(yàn)（RBT）····················································································13

8.3試管凝集試驗(yàn)（SAT）··························································································14

8.4補(bǔ)體結(jié)合試驗(yàn)（CFT）···························································································17

8.5間接ELISA抗體檢測(cè)方法（iELISA）······································································22

8.6競(jìng)爭(zhēng)ELISA抗體檢測(cè)方法（cELISA）······································································24

8.7熒光偏振試驗(yàn)（FPA）···························································································26

8.8全乳環(huán)狀試驗(yàn)（MRT）（僅適用于牛）········································································29

8.9免疫層析法（ICA）······························································································30

Ⅰ

GB/T18646—2025

9綜合判定···············································································································30

9.1布魯氏菌疑似感染動(dòng)物判定···············································································30

9.2布魯氏菌感染動(dòng)物判定·····················································································31

附錄A（資料性）檢測(cè)方法的適用性················································································32

附錄B（規(guī)范性）布魯氏菌培養(yǎng)基的配制··········································································33

B.1基礎(chǔ)培養(yǎng)基······································································································33

B.2血清葡萄糖培養(yǎng)基·····························································································33

B.3選擇培養(yǎng)基······································································································33

附錄C（規(guī)范性）生化反應(yīng)試劑的配制·············································································34

C.1結(jié)晶紫儲(chǔ)備液和工作液配制·················································································34

C.2氧化酶試驗(yàn)試劑配制··························································································34

C.3尿素酶活性試驗(yàn)培養(yǎng)基配方及制備········································································34

附錄D（規(guī)范性）布魯氏菌種和生物型的生化鑒定······························································35

附錄E（規(guī)范性）實(shí)時(shí)熒光PCR、RAA及多重PCR引物和探針序列·······································37

附錄F（規(guī)范性）多重PCR溶液配制及結(jié)果判定·······························································39

F.1TAE電泳緩沖液·······························································································39

F.21.5%瓊脂糖凝膠·······························································································39

F.3多重PCR判定標(biāo)準(zhǔn)····························································································39

附錄G（規(guī)范性）試管凝集試驗(yàn)（SAT）試劑的配制·····························································40

G.1含0.5%苯酚的生理鹽水·····················································································40

G.2含0.5%苯酚的10%氯化鈉溶液··········································································40

G.3參照比濁管的制備·····························································································40

附錄H（規(guī)范性）補(bǔ)體結(jié)合試驗(yàn)（CFT）試劑的配制及效價(jià)測(cè)定··············································41

H.1稀釋液配制······································································································41

H.2綿羊紅細(xì)胞懸液的配制（2.5%）·············································································41

H.3溶血素效價(jià)測(cè)定································································································41

H.4補(bǔ)體制備及效價(jià)測(cè)定··························································································44

H.5抗原效價(jià)的測(cè)定································································································49

H.6溶血標(biāo)準(zhǔn)比色管的制備及補(bǔ)體結(jié)合試驗(yàn)結(jié)果判定方法················································50

H.7待檢血清的稀釋和滅活·······················································································51

附錄I（規(guī)范性）酶聯(lián)免疫吸附試驗(yàn)（ELISA）試劑的制備······················································52

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

I.2包被液（0.05mol/L的pH9.6碳酸鹽緩沖液）····························································52

I.3封閉液（含2%BSA、0.05%吐溫?20的pH7.4PBS）···················································52

I.4樣品稀釋液（含1%BSA的pH7.4PBS）··································································52

I.5洗滌液（含0.05%吐溫?20的pH7.4PBS）································································52

Ⅱ

GB/T18646—2025

I.6顯色液·············································································································52

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

附錄J（資料性）競(jìng)爭(zhēng)酶聯(lián)免疫吸附試驗(yàn)（cELISA）單克隆抗體的制備·····································54

附錄K（規(guī)范性）熒光偏振試驗(yàn)（FPA）抗原和稀釋液的制備··················································55

K.1標(biāo)記抗原的制備································································································55

K.2樣品稀釋液的配制·····························································································55

參考文獻(xiàn)····················································································································56

Ⅲ

GB/T18646—2025

前言

本文件按照GB/T1.1—2020《標(biāo)準(zhǔn)化工作導(dǎo)則第1部分：標(biāo)準(zhǔn)化文件的結(jié)構(gòu)和起草規(guī)則》的規(guī)

定起草。

本文件代替GB/T18646—2018《動(dòng)物布魯氏菌病診斷技術(shù)》，與GB/T18646—2018相比，除結(jié)構(gòu)

調(diào)整和編輯性改動(dòng)外，主要技術(shù)變化如下：

a）更改了“范圍”（見第1章，2018年版的第1章）；

b）更改了“臨床診斷”，增加了流行病學(xué)、臨床癥狀和病理變化內(nèi)容（見第5章，2018年版的4.1、

4.2、4.3）；

c）增加了“實(shí)時(shí)熒光PCR方法”“熒光RAA方法”（見7.3和7.4）；

d）將“布魯氏菌Bruce?Ladder檢測(cè)方法”更改為“多重PCR方法”（見7.5，2018年版的4.13）；

e）增加了免疫學(xué)診斷方法中RBT、SAT、CFT、iELISA、cELISA和FPA方法的標(biāo)準(zhǔn)化內(nèi)容（見

第8章）；

f）更改了“虎紅平板凝集試驗(yàn)（RBT）”（見8.2，2018年版的4.4）、“間接ELISA抗體檢測(cè)方法

（iELISA）”（見8.5，2018年版的4.8）、“競(jìng)爭(zhēng)ELISA抗體檢測(cè)方法（cELISA）”（見8.6，2018

年版的4.9）；

g）增加了“熒光偏振試驗(yàn)（FPA）”“免疫層析法（ICA）”（見8.7和8.9）。

本文件由中華人民共和國(guó)農(nóng)業(yè)農(nóng)村部提出。

本文件由全國(guó)動(dòng)物衛(wèi)生標(biāo)準(zhǔn)化技術(shù)委員會(huì)（SAC/TC181）歸口。

本文件起草單位：中國(guó)動(dòng)物衛(wèi)生與流行病學(xué)中心、中國(guó)獸醫(yī)藥品監(jiān)察所、中國(guó)動(dòng)物疫病預(yù)防控制中

心、中國(guó)農(nóng)業(yè)科學(xué)院哈爾濱獸醫(yī)研究所、中國(guó)疾病預(yù)防控制中心、新疆生產(chǎn)建設(shè)兵團(tuán)畜牧獸醫(yī)工作總

站、遼寧省動(dòng)物疫病預(yù)防控制中心、內(nèi)蒙古自治區(qū)動(dòng)物疫病預(yù)防控制中心、上海市動(dòng)物疫病預(yù)防控制中

心、河南省動(dòng)物疫病預(yù)防控制中心、重慶市動(dòng)物疫病預(yù)防控制中心、浙江省動(dòng)物疫病預(yù)防控制中心、

青島市動(dòng)物疫病預(yù)防控制中心。

本文件主要起草人：孫淑芳、李俊平、邵衛(wèi)星、孫明軍、姜海、南文龍、田莉莉、樊曉旭、焉鑫、孫雨、

胡森、李巖、朱良全、王建龍、閆若潛、張存瑞、李彥、顧貴波、趙洪進(jìn)、董春霞、趙靈燕、郭宇、孫翔翔、劉蒙達(dá)、

張皓博、孫世雄、李嘉琪。

本文件及其所代替文件的歷次版本發(fā)布情況為：

——2002年首次發(fā)布為GB/T18646—2002，2018年第一次修訂；

——本次為第二次修訂。

Ⅴ

GB/T18646—2025

引言

布魯氏菌?。˙rucellosis）是由布魯氏菌（Brucella）感染動(dòng)物和人而引起的一種人獸共患傳染病。

目前已報(bào)道的布魯氏菌屬成員包括6個(gè)經(jīng)典種、5個(gè)新鑒定種及部分未確定種型的非典型布魯氏菌。

經(jīng)典種包括羊種布魯氏菌（Brucellamelitensis）、牛種布魯氏菌（Brucellaabortus）、豬種布魯氏菌（Bru?

cellasuis）、綿羊附睪種布魯氏菌（Brucellaovis）、犬種布魯氏菌（Brucellacanis）和沙林鼠種布魯氏菌

（Brucellaneotomae）；新鑒定種包括鯨種布魯氏菌（Brucellaceti）、鰭種布魯氏菌（Brucellapinnipedia?

lis）、田鼠種布魯氏菌（Brucellamicroti）、人源布魯氏菌（Brucellainopinata，暫未確定其他動(dòng)物宿主）和

狒狒種布魯氏菌（Brucellapapionis）；未確定種型的包括分離自嚙齒類、狐類和蛙類的非典型布魯氏菌。

本文件主要規(guī)定了對(duì)畜牧業(yè)影響最大的羊種布魯氏菌、牛種布魯氏菌和豬種布魯氏菌三種光滑型布魯

氏菌引起的動(dòng)物布魯氏菌病相關(guān)診斷檢測(cè)技術(shù)內(nèi)容，僅在病原鑒定及PCR檢測(cè)方法中涉及了粗糙型

的綿羊附睪種和犬種布魯氏菌檢測(cè)。

布魯氏菌病在全世界范圍內(nèi)嚴(yán)重威脅動(dòng)物和人類健康。世界動(dòng)物衛(wèi)生組織（WorldOrganization

ofAnimalHealth，WOAH）將布魯氏菌病列入通報(bào)病種名錄，我國(guó)農(nóng)業(yè)農(nóng)村部發(fā)布的《一、二、三類動(dòng)物

疫病病種名錄》將布魯氏菌病列為二類動(dòng)物疫病，國(guó)家衛(wèi)生健康委員會(huì)將布魯氏菌病列為法定報(bào)告乙

類傳染病。從事布魯氏菌病診斷相關(guān)的樣品采集、包裝運(yùn)輸、檢測(cè)診斷等活動(dòng)的人員，經(jīng)過嚴(yán)格培訓(xùn)，

在相應(yīng)級(jí)別的生物安全防護(hù)條件下進(jìn)行試驗(yàn)活動(dòng)。

本文件參考了WOAH《陸生動(dòng)物診斷試驗(yàn)與疫苗手冊(cè)》3.1.4章“布魯氏菌病（牛種、羊種和豬種布

魯氏菌感染）（2022版）”規(guī)定的診斷技術(shù)方法，包括臨床診斷、病原學(xué)診斷方法（涂片鏡檢、分離與鑒

定、實(shí)時(shí)熒光PCR方法）及免疫學(xué)診斷方法［虎紅平板凝集試驗(yàn)（RBT）、試管凝集試驗(yàn)（SAT）、補(bǔ)體結(jié)

合試驗(yàn)（CFT）、間接ELISA抗體檢測(cè)方法（iELISA）、競(jìng)爭(zhēng)ELISA抗體檢測(cè)方法（cELISA）、熒光偏振

試驗(yàn)（FPA）和全乳環(huán)狀試驗(yàn)（MRT）］；并根據(jù)我國(guó)技術(shù)發(fā)展增加了技術(shù)成熟的熒光RAA方法和免疫

層析法（ICA）。

本文件的發(fā)布機(jī)構(gòu)提請(qǐng)注意，聲明相關(guān)產(chǎn)品符合本文件時(shí)，可能涉及本文件7.3中引物和探針序

列與《一種區(qū)分布魯氏菌A19疫苗株與野毒株的分子標(biāo)記》（授權(quán)公告號(hào)：CN113186312B）、《鑒別布魯

氏菌疫苗株S2和野毒株的SNP分子標(biāo)記及其應(yīng)用》（授權(quán)公告號(hào)：CN111793704B）相關(guān)專利的使用。

本文件的發(fā)布機(jī)構(gòu)對(duì)于涉及專利的真實(shí)性、有效性和范圍無(wú)任何立場(chǎng)。

該專利持有人已向本文件的發(fā)布機(jī)構(gòu)承諾，同意在公平、合理、無(wú)歧視基礎(chǔ)上，免費(fèi)許可任何組織

或個(gè)人在實(shí)施該國(guó)家標(biāo)準(zhǔn)時(shí)實(shí)施專利。該專利持有人的聲明已在本文件的發(fā)布機(jī)構(gòu)備案。相關(guān)信息

可以通過以下聯(lián)系方式獲得：

專利（授權(quán)公告號(hào)：CN113186312B）持有人姓名：中國(guó)動(dòng)物衛(wèi)生與流行病學(xué)中心。

地址：山東省青島市市北區(qū)南京路369號(hào)。

專利（授權(quán)公告號(hào)：CN111793704B）持有