布鲁菌双重TaqMan荧光定量PCR检测方法的建立及初步应用

doi:10.11843/j.issn.0366-6964.2026.01.032

摘要/Abstract

摘要：

旨在建立一种经济高效的布鲁菌双重TaqMan荧光定量PCR鉴别检测方法，用于布鲁菌感染的动物及动物产品的快速检测并区分牛种布鲁菌和其他种布鲁菌感染的动物，为临床常的疫苗和研发的新型疫苗提供科学有效的鉴别诊断方法。本研究根据GenBank公布的galU和Omp31的序列设计检测引物和探针，构建重组质粒，对方法特异性、灵敏性及重复性评价，检测实验室制备和临床动物布病血清及组织样品。结果表明，成功建立了布鲁菌双重TaqMan荧光定量PCR检测方法，该方法最低检测限为1.93×10⁰ copies·μL^-1，与其他革兰阴性菌无交叉反应，批内及批间变异系数均小于2%。利用本研究建立的方法检测实验室制备样品和临床采集样品共156份，与BCSP31-PCR相比，两者阳性符合率为69.75%；与PCR和虎红凝集试验相比，灵敏性更高，双重TaqMan荧光定量PCR的敏感性为100%，PCR的敏感性为69.75%，虎红凝集试验的敏感性为58.65%。本研究建立的布鲁菌双重TaqMan荧光定量PCR检测方法，为布鲁菌临床鉴别检测、流行病学调查及净化提供技术支持。

关键词: 布鲁菌, 双重Taq Man荧光定量PCR, 检测方法, 鉴别诊断

Abstract:

This study aimed to establish a cost-effective duplex TaqMan real-time quantitative PCR （qPCR） assay for the rapid detection of Brucella infection in animals and animal products， and to differentiate B.abortus from other Brucella species. This method provides a scientifically valid diagnostic tool for distinguishing currently used vaccine strains and novel vaccines under development. Specific primers and TaqMan probes targeting the galU and Omp31 genes were designed based on GenBank sequences. Recombinant plasmids were constructed. The assay's specificity， sensitivity， and repeatability were rigorously evaluated. Clinical serum and tissue samples from animals with brucellosis were tested. A duplex TaqMan qPCR assay for Brucella detection was successfully developed. The limit of detection （LOD） for recombinant plasmid standards was 1.93×10⁰copies·μL^-1. The assay exhibited no cross-reactivity with other Gram-negative bacteria. Both intra-assay and inter-assay reproducibility were excellent， with coefficients of variation （CV） consistently below 2%. Testing of 156 samples （including laboratory-prepared and clinical specimens） revealed a positive agreement rate of 69.75% compared to BCSP31-PCR. Furthermore， the duplex TaqMan qPCR demonstrated significantly higher sensitivity （100%） than conventional PCR （69.75%） and the RBPT （58.65%）. The established duplex TaqMan qPCR assay provides a robust technical foundation for the clinical differential diagnosis， epidemiological surveillance， and eradication programs of brucellosis.

Key words: Brucella, duplex TaqMan real-time PCR, detection method, differential diagnosis

中图分类号:

R378.5

彭海涛, 高阳, 刘雨欣, 顾德媛, 张东, 张如, 许会会, 陈思, 杨艳玲. 布鲁菌双重TaqMan荧光定量PCR检测方法的建立及初步应用[J]. 畜牧兽医学报, 2026, 57(1): 369-377.

PENG Haitao, GAO Yang, LIU Yuxin, GU Deyuan, ZHANG Dong, ZHANG Ru, XU Huihui, CHEN Si, YANG Yanling. Establishment and Preliminary Application of a Duplex TaqMan Real-time PCR Assay for Brucella[J]. Acta Veterinaria et Zootechnica Sinica, 2026, 57(1): 369-377.

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基因 Gene	引物和探针 Primer andprobe	序列 Sequence	产物大小/bp Product size
galU	galU-F	5’-GAACGGGGGAAAACCGAAGA-3’	149
	galU-R	5’-AGTGCAAAAGGTTCGTTGCC-3’
	galU-P	FAM-CAGCAGGTGCCGCTTGGCCTC-BHQ1
	P-galU-F	AGTTCTAAATGAGTTCTATTCGGAAAATCCGCAAGGCAG	917
	P-galU-R	GACGGCCAGTGAATTTCAGGCTGGCTGGATCGTCTTCAACAG	917
Omp31	Omp31-F	5’-TTCCGCCCCTACTGCTGCT-3’	138
	Omp31-R	5’-CCGGAAACCTGTTCGTTGTC-3’
	Omp31-P	HEX-ACGCCGGTTACGCAGGCGGCAAGTT-BHQ1
	P-Omp31-F	TCGACTCTAGAGGATATGAAATCCGTAATTTTGGCGTCCATCGCC	755
	P-Omp31-R	AACTCATTTAGAACTTGTAGTTCAGACCGACGCGAACAGT	755
pUC19	pUC19-F	ATCCTCTAGAGTCGACCTGCAGGCA	2 698
pUC19	pUC19-R	AATTCACTGGCCGTCGTTTTACAACGT	2 698

探针浓度/（nmol·L^-1） Probe concentration	引物 Primer	引物浓度/（nmol·L^-1） Primer concentration
探针浓度/（nmol·L^-1） Probe concentration	引物 Primer	500	250	125
250	Ct（galU）	20.20±0.13	20.50±0.17	22.40±0.18
250	Ct（Omp31）	22.20±0.22	22.50±0.17	22.90±0.18
125	Ct（galU）	19.80±0.08	20.90±0.11	22.50±0.12
125	Ct（Omp31）	21.80±0.15	21.90±0.12	22.30±0.16
62.5	Ct（galU）	22.00±0.13	21.80±0.16	20.90±0.10
62.5	Ct（Omp31）	21.80±0.20	21.80±0.21	22.20±0.14

样品类型 Sample type	双重qPCR检测结果 Duplex real-time PCR test results		PCR检测结果 PCR test results	虎红平板凝集试验 Rose Bengal plate test
样品类型 Sample type	galU	Omp31	PCR检测结果 PCR test results	虎红平板凝集试验 Rose Bengal plate test
免疫菌影疫苗羊血清 Serum of Sheep immunized with BG	0/16	0/16	0/16	0/16
羊种布鲁菌感染羊血清 Serum from Brucella melitensis-infected sheep	21/21	21/21	13/21	21/21
布病阴性羊血清 Sheep serum negative for Brucella antibody	0/8	0/8	0/8	0/8
免疫gaIU基因缺失弱毒疫苗豚鼠血清 Serum of guinea pig with galU deficient Brucella	0/9	9/9	5/9	5/9
羊种布鲁菌感染豚鼠血清 Serum from Brucella melitensis-infected guinea pig	5/5	5/5	5/5	5/5