猪支气管败血波氏杆菌、副猪格拉瑟菌和猪链球菌TaqMan三重荧光定量PCR方法的建立与初步应用

doi:10.11843/j.issn.0366-6964.2025.11.048

摘要/Abstract

摘要：

本研究旨在建立一种可快速同时诊断猪支气管败血波氏杆菌(Bordetela bronchiseptica，Bb)、副猪格拉瑟菌(Glaesserella parasuis，GPS)和猪链球菌(Streptococcus suis，SS)的多重荧光定量PCR方法。根据Bb的fla基因，GPS的infB基因，SS的gdh基因保守序列设计特异性引物和探针，经过反应条件优化后，建立一种检测三种病原的TaqMan多重荧光定量PCR方法。结果表明，该方法对胸膜肺炎放线杆菌、多杀性巴氏杆菌、伪狂犬病病毒、猪源肺炎克雷伯菌、猪肺源大肠杆菌等病原核酸检测未出现阳性，具有良好的特异性；对三种标准质粒品的最低检测下限分别为8.06、7.11和7.15 copies·μL^-1，具有较高敏感性；组内和组间变异系数均小于2%，具有良好的稳定性。对2023—2025年收集的513份临床猪肺脏样品进行检测，结果显示，Bb与GPS混合感染的比例为6.63%；Bb与SS混合感染的比例为8.11%；GPS与SS混合感染比例为12.47%；三种病原混合感染的比例为5.46%；所有混合感染的总比例为16.57%。所建立的多重荧光定量PCR方法具有较好的特异性、敏感性和稳定性，可作为临床上三种病原的快速诊断工具。

关键词: 猪支气管败血波氏杆菌, 副猪格拉瑟菌, 猪链球菌, 多重荧光定量 PCR

Abstract:

This study aims to develop a multiplex fluorescent quantitative PCR method for the rapid and simultaneous detection of Bordetella bronchiseptica (Bb), Glaesserella parasuis (GPS) and Streptococcus suis (SS). Specific primers and probes were designed based on the conserved sequences of the fla gene (Bb), infB gene (GPS), and gdh gene (SS). After optimizing the reaction conditions, a TaqMan-based multiplex fluorescent quantitative PCR method for detecting these three pathogens was established. The method demonstrated good specificity, with no positive signals for nucleic acids from Actinobacillus pleuropneumoniae, Pasteurella multocida, Pseudorabies virus, Klebsiella pneumoniae, and Escherichia coli from pigs. It exhibited high sensitivity, with the minimum detection limits of 8.06, 7.11, and 7.15 copies·μL^-1 for the standard plasmid templates of Bb, GPS, and SS, respectively. The method also showed good reproducibility, with intra-assay and inter-assay coefficients of variation both < 2%. Analysis of 513 clinical porcine lung samples collected between 2023 and 2025 revealed that the proportion of mixed infections was 16.57%. The developed multiplex fluorescent quantitative PCR method exhibited superior specificity, sensitivity, and stability, providing a reliable tool for the rapid detection and diagnosis of these three pathogens in clinical settings.

Key words: Bordetela bronchiseptica, Glaesserella parasuis, Streptococcus suis, multiplex TaqMan real-time PCR

中图分类号:

S858.285.1

刘汉元, 王梓浩, 赵梦菲, 黄茜, 王斐, 华琳, 吴斌, 彭忠. 猪支气管败血波氏杆菌、副猪格拉瑟菌和猪链球菌TaqMan三重荧光定量PCR方法的建立与初步应用[J]. 畜牧兽医学报, 2025, 56(11): 5936-5942.

LIU Hanyuan, WANG Zihao, ZHAO Mengfei, HUANG Xi, WANG Fei, HUA Lin, WU Bin, PENG Zhong. Establishment and Preliminary Application of a TaqMan Triple Fluorescence Quantitative PCR Assay for Detection of Bordetella bronchiseptica, Glaesserella parasuis and Streptococcus suis[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5936-5942.

图/表 3

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引物和探针 Primers and probes	序列(5′→3′) Sequences	产物大小/bp Product size
BbP-F	TGCAAGCGAAAGTCCGATGT	75
BbP-R	GGCTCCCAAGAGAGAAAGGC
BbP-P	Cy5-CGGGCAGCTAGGCCGCCCAT-BHQ1
GPSP-F	AGGCGGACGTGATGAAA	103
GPSP-R	ACTGCCTTTCTTCGCGTGTT
GPSP-P	VIC-ACGTCAATCTGACCGTAACCA-BHQ1
SSP-F	TTCGACCTCTTGGTGGACATC	124
SSP-R	CAATATCAGCCTTGCCATCGT
SSP-P	FAM-ACGCCGCGCTCGTTTGACAG-BHQ1
Bb-F	TGCGGGGACAGGCA	267
Bb-R	TTGAGGCTCCCAAGAGAG
GPS-F	TACTTCTAAATATGCGTTAGAAGCAG	336
GPS-R	ATCAAGGTCAGATGGCATGTT
SS-F	CCAATATGCTGTTCAAAAAGCGA	328
SS-R	ATCAAGGTCAGATGGCATGTT

病原 Pathogens	阳性样品 Positive samples	阳性率/% Positive rates	细菌分离样品 Positive samples	分离率/% Positive rates
Bb	87	16.96	11	5.69
GPS	107	20.86	13	6.96
SS	144	28.07	31	13.92
Bb+GPS	34	6.63	3	1.26
Bb+SS	43	8.11	4	2.53
SS+GPS	64	12.47	6	3.16
Bb+GPS+SS	28	5.46	0	0.00

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