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

Abstract

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

CLC Number:

S858.285.1

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.

Figures/Tables 3

Table 1

Fig. 1

Table 2

References 28

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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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Establishment and Preliminary Application of a TaqMan Triple Fluorescence Quantitative PCR Assay for Detection of Bordetella bronchiseptica, Glaesserella parasuis and Streptococcus suis

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