GB/T 18646-2025動物布魯氏菌病診斷技術

ICS11.220

CCSB41

中華人民共和國國家標準

GB/T18646—2025

代替GB/T18646—2018

動物布魯氏菌病診斷技術

Diagnostictechniquesforanimalbrucellosis

2025?05?30發布2025?12?01實施

國家市場監督管理總局

國家標準化管理委員會發布

GB/T18646—2025

目次

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

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

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

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

3術語和定義··············································································································1

4縮略語····················································································································1

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

5.1流行特點···········································································································2

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

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

5.4結果判定···········································································································3

6樣品采集、保存、運輸及處理·························································································3

6.1儀器設備···········································································································3

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

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

6.4樣品運輸和保存··································································································4

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

7病原學診斷方法········································································································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實時熒光PCR方法······························································································7

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

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

8免疫學診斷方法······································································································12

8.1標準血清··········································································································12

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

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

8.4補體結合試驗（CFT）···························································································17

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

8.6競爭ELISA抗體檢測方法（cELISA）······································································24

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

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

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

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

9.1布魯氏菌疑似感染動物判定···············································································30

9.2布魯氏菌感染動物判定·····················································································31

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

附錄B（規范性）布魯氏菌培養基的配制··········································································33

B.1基礎培養基······································································································33

B.2血清葡萄糖培養基·····························································································33

B.3選擇培養基······································································································33

附錄C（規范性）生化反應試劑的配制·············································································34

C.1結晶紫儲備液和工作液配制·················································································34

C.2氧化酶試驗試劑配制··························································································34

C.3尿素酶活性試驗培養基配方及制備········································································34

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

附錄E（規范性）實時熒光PCR、RAA及多重PCR引物和探針序列·······································37

附錄F（規范性）多重PCR溶液配制及結果判定·······························································39

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

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

F.3多重PCR判定標準····························································································39

附錄G（規范性）試管凝集試驗（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（規范性）補體結合試驗（CFT）試劑的配制及效價測定··············································41

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

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

H.3溶血素效價測定································································································41

H.4補體制備及效價測定··························································································44

H.5抗原效價的測定································································································49

H.6溶血標準比色管的制備及補體結合試驗結果判定方法················································50

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

附錄I（規范性）酶聯免疫吸附試驗（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

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

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

附錄J（資料性）競爭酶聯免疫吸附試驗（cELISA）單克隆抗體的制備·····································54

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

K.1標記抗原的制備································································································55

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

參考文獻····················································································································56

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GB/T18646—2025

前言

本文件按照GB/T1.1—2020《標準化工作導則第1部分：標準化文件的結構和起草規則》的規

定起草。

本文件代替GB/T18646—2018《動物布魯氏菌病診斷技術》，與GB/T18646—2018相比，除結構

調整和編輯性改動外，主要技術變化如下：

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、臨床癥狀和病理變化內容（見第5章，2018年版的4.1、

4.2、4.3）；

c）增加了“實時熒光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）增加了免疫學診斷方法中RBT、SAT、CFT、iELISA、cELISA和FPA方法的標準化內容（見

第8章）；

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

（iELISA）”（見8.5，2018年版的4.8）、“競爭ELISA抗體檢測方法（cELISA）”（見8.6，2018

年版的4.9）；

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

本文件由中華人民共和國農業農村部提出。

本文件由全國動物衛生標準化技術委員會（SAC/TC181）歸口。

本文件起草單位：中國動物衛生與流行病學中心、中國獸醫藥品監察所、中國動物疫病預防控制中

心、中國農業科學院哈爾濱獸醫研究所、中國疾病預防控制中心、新疆生產建設兵團畜牧獸醫工作總

站、遼寧省動物疫病預防控制中心、內蒙古自治區動物疫病預防控制中心、上海市動物疫病預防控制中

心、河南省動物疫病預防控制中心、重慶市動物疫病預防控制中心、浙江省動物疫病預防控制中心、

青島市動物疫病預防控制中心。

本文件主要起草人：孫淑芳、李俊平、邵衛星、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、董春霞、趙靈燕、郭宇、孫翔翔、劉蒙達、

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布情況為：

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

——本次為第二次修訂。

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GB/T18646—2025

引言

布魯氏菌病（Brucellosis）是由布魯氏菌（Brucella）感染動物和人而引起的一種人獸共患傳染病。

目前已報道的布魯氏菌屬成員包括6個經典種、5個新鑒定種及部分未確定種型的非典型布魯氏菌。

經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。

本文件主要規定了對畜牧業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

氏菌引起的動物布魯氏菌病相關診斷檢測技術內容，僅在病原鑒定及PCR檢測方法中涉及了粗糙型

的綿羊附睪種和犬種布魯氏菌檢測。

布魯氏菌病在全世界范圍內嚴重威脅動物和人類健康。世界動物衛生組織（WorldOrganization

ofAnimalHealth，WOAH）將布魯氏菌病列入通報病種名錄，我國農業農村部發布的《一、二、三類動物

疫病病種名錄》將布魯氏菌病列為二類動物疫病，國家衛生健康委員會將布魯氏菌病列為法定報告乙

類傳染病。從事布魯氏菌病診斷相關的樣品采集、包裝運輸、檢測診斷等活動的人員，經過嚴格培訓，

在相應級別的生物安全防護條件下進行試驗活動。

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

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

定、實時熒光PCR方法）及免疫學診斷方法［虎紅平板凝集試驗（RBT）、試管凝集試驗（SAT）、補體結

合試驗（CFT）、間接ELISA抗體檢測方法（iELISA）、競爭ELISA抗體檢測方法（cELISA）、熒光偏振

試驗（FPA）和全乳環狀試驗（MRT）］；并根據我國技術發展增加了技術成熟的熒光RAA方法和免疫

層析法（ICA）。

本文件的發布機構提請注意，聲明相關產品符合本文件時，可能涉及本文件7.3中引物和探針序

列與《一種區分布魯氏菌A19疫苗株與野毒株的分子標記》（授權公告號：CN113186312B）、《鑒別布魯

氏菌疫苗株S2和野毒株的SNP分子標記及其應用》（授權公告號：CN111793704B）相關專利的使用。

本文件的發布機構對于涉及專利的真實性、有效性和范圍無任何立場。

該專利持有人已向本文件的發布機構承諾，同意在公平、合理、無歧視基礎上，免費許可任何組織

或個人在實施該國家標準時實施專利。該專利持有人的聲明已在本文件的發布機構備案。相關信息

可以通過以下聯系方式獲得：

專利（授權公告號：CN113186312B）持有人姓名：中國動物衛生與流行病學中心。

地址：山東省青島市市北區南京路369號。

專利（授權公告號：CN111793704B）持有