产气荚膜梭菌四环素耐药基因双重荧光定量PCR检测方法的建立

doi:10.11843/j.issn.0366-6964.2024.10.034

Abstract

Abstract:

The purpose of this study was to establish a rapid and sensitive duplex real-time PCR method for tetA(P) and tetB(P) tetracycline resistance genes detection in Clostridium perfringens, aiming at providing a new strategy for the expedited identification of tetracycline antibiotic resistance within C. perfringens. Primers and probes were designed to target the conserved sequences of tetA(P) and tetB(P), construct recombinant plasmid, and optimize the annealing temperature and system. At the same time, validation of the method was substantiated through disc diffusion test for the determination of strain of tetracycline drugs sensitivity. The results showed that, the optimized reaction program was 95 ℃ 30 s; 95 ℃ 5 s; 56 ℃ 30 s, 39 cycles. And this method exclusively amplified the recombinant plasmid with no amplification curve observed for control strains. Intra-assay and inter-assay coefficients of variation were observed to be less than 1.3% and 1.8%, respectively. The plasmid standard had a minimum detection threshold of 10² copies·μL^-1. Using this method, resistance gene detection was performed on 33 clinical isolates of C. perfringens, complemented by antimicrobial susceptibility testing. The detection method established by the current study yielded a detection rate of 75.8% (25/33) for tetracycline resistance genes, aligning significantly with the antimicrobial susceptibility testing results. In conclusion, this study successfully established a specific, sensitive, and highly reproducible duplex real-time PCR method for the detection of tetA(P) and tetB(P) tetracycline resistance genes in C. perfringens.

Key words: Clostridium perfringens, tetracycline, tetA(P), tetB(P), duplex real-time PCR

CLC Number:

S852.616.3

Lanxin OU, Bijin YE, Mingfei SUN, Nanshan QI, Juan LI, Minna LÜ, Xuhui LIN, Haiming CAI, Junjing HU, Yongle SONG, Xiangjie CHEN, Yibin ZHU, Lijun YIN, Jianfei ZHANG, Shenquan LIAO, Haoji ZHANG. Establishment of a Duplex Real-time PCR Method for Tetracycline Resistance Gene Detection in Clostridium perfringens[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4630-4637.

Figures/Tables 7

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References 21

1	MEHDIZADEH GOHARI I , NAVARRO M A , LI J H , et al. Pathogenicity and virulence of Clostridium perfringens[J]. Virulence, 2021, 12 (1): 723- 753. doi: 10.1080/21505594.2021.1886777
2	NAVARRO M A , MCCLANE B A , UZAL F A . Mechanisms of action and cell death associated with Clostridium perfringens toxins[J]. Toxins (Basel), 2018, 10 (5): 212. doi: 10.3390/toxins10050212
3	ROOD J I , ADAMS V , LACEY J , et al. Expansion of the Clostridium perfringens toxin-based typing scheme[J]. Anaerobe, 2018, 53, 5- 10. doi: 10.1016/j.anaerobe.2018.04.011
4	SILVEIRA SILVA R O , OLIVEIRA JUNIOR C A , CARVALHO GUEDES R M , et al. Clostridium perfringens: a review of the disease in pigs, horses and broiler chickens[J]. Cienc Rural, 2015, 45 (6): 1027- 1034. doi: 10.1590/0103-8478cr20140927
5	OSMAN K M , ELHARIRI M . Antibiotic resistance of Clostridium perfringens isolates from broiler chickens in Egypt[J]. Rev Sci Tech, 2013, 32 (3): 841- 850.
6	LLANCO L A , NAKANO V , AVILA-CAMPOS M J . Sialidase production and genetic diversity in Clostridium perfringens type A isolated from chicken with necrotic enteritis in Brazil[J]. Curr Microbiol, 2015, 70 (3): 330- 337. doi: 10.1007/s00284-014-0722-5
7	NGAMWONGSATIT B , TANOMSRIDACHCHAI W , SUTHIENKUL O , et al. Multidrug resistance in Clostridium perfringens isolated from diarrheal neonatal piglets in Thailand[J]. Anaerobe, 2016, 38, 88- 93. doi: 10.1016/j.anaerobe.2015.12.012
8	WU D , LUO R B , GONG G , et al. Antimicrobial susceptibility and multilocus sequence typing of Clostridium perfringens isolated from yaks in Qinghai-Tibet plateau, China[J]. Front Vet Sci, 2022, 9, 1022215. doi: 10.3389/fvets.2022.1022215
9	LI J Y , ZHOU Y Q , YANG D W , et al. Prevalence and antimicrobial susceptibility of Clostridium perfringens in chickens and pigs from Beijing and Shanxi, China[J]. Vet Microbiol, 2021, 252, 108932. doi: 10.1016/j.vetmic.2020.108932
10	LYRAS D , ADAMS V , BALLARD S A , et al. tISCpe8, an IS1595-Family lincomycin resistance element located on a conjugative plasmid in Clostridium perfringens[J]. J Bacteriol, 2009, 191 (20): 6345- 6351. doi: 10.1128/JB.00668-09
11	NHUNG N T , CHANSIRIPORNCHAI N , CARRIQUE-MAS J J . Antimicrobial resistance in bacterial poultry pathogens: a review[J]. Front Vet Sci, 2017, 4, 126. doi: 10.3389/fvets.2017.00126
12	ZHU Q D , SUN P , ZHANG B K , et al. Progress on gut health maintenance and antibiotic alternatives in broiler chicken production[J]. Front Nutr, 2021, 8, 692839. doi: 10.3389/fnut.2021.692839
13	刘竟雅, 侯晓昱, 吴金燕, 等. 貉源大肠杆菌四环素耐药基因的检测[J]. 野生动物学报, 2020, 41 (2): 500- 504. doi: 10.3969/j.issn.1000-0127.2020.02.033
	LIU J Y , HOU X Y , WU J Y , et al. Detection of tetracycline resistance gene in Escherichia coli derived from Racoon dog (Nyctereutes procyonoides)[J]. Chinese Journal of Wildlife, 2020, 41 (2): 500- 504. doi: 10.3969/j.issn.1000-0127.2020.02.033
14	PARK M , ROONEY A P , HECHT D W , et al. Phenotypic and genotypic characterization of tetracycline and minocycline resistance in Clostridium perfringens[J]. Arch Microbiol, 2010, 192 (10): 803- 810. doi: 10.1007/s00203-010-0605-5
15	SHEYKHSARAN E , BAGHI H B , SOROUSH M H , et al. An overview of tetracyclines and related resistance mechanisms[J]. Rev Med Microbiol, 2019, 30 (1): 69- 75. doi: 10.1097/MRM.0000000000000154
16	KIU R , HALL L J . An update on the human and animal enteric pathogen Clostridium perfringens[J]. Emerg Microbes Infect, 2018, 7 (1): 141.
17	SAKSENA N K , TRUFFAUT N . Cloning of tetracycline-resistance genes from various strains of Clostridium perfringens and expression in Escherichia coli[J]. Can J Microbiol, 1992, 38 (3): 215- 221. doi: 10.1139/m92-036
18	胡付品, 朱德妹. 我国细菌耐药监测工作需要进入2.0时代[J]. 中国感染与化疗杂志, 2018, 18 (2): 129- 131.
	HU F P , ZHU D M . Surveillance of antimicrobial resistance in China will advance into a new era[J]. Chinese Journal of Infection and Chemotherapy, 2018, 18 (2): 129- 131.
19	JOHANESEN P A , LYRAS D , ROOD J I . Induction of pCW3-encoded tetracycline resistance in Clostridium perfringens involves a host-encoded factor[J]. Plasmid, 2001, 46 (3): 229- 232. doi: 10.1006/plas.2001.1543
20	白龙, 邵毅, 黄柳娟, 等. 鸡源细菌中5种四环素耐药基因多重PCR检测方法的建立[J]. 食品与机械, 2018, 34 (10): 55-59, 90.
	BAI L , SHAO Y , HUANG L J , et al. Establishment of a multiplex PCR system for detection of five tetracycline resistance genes in poultry bacteria[J]. Food & Machinery, 2018, 34 (10): 55-59, 90.
21	夏青青, 王红宁, 张安云. 三重PCR方法对猪、鸡源细菌中四环素类药物耐药基因的检测[J]. 四川大学学报(自然科学版), 2013, 50 (1): 171- 176.
	XIA Q Q , WANG H N , ZHANG A Y . Detection of tetracycline resistant genes in bacterial by using a triple-PCR method[J]. Journal of Sichuan University (Natural Science Edition), 2013, 50 (1): 171- 176.

基因 Gene	引物和探针 Primer and probe	序列 Sequence	产物大小/bp Product size
tetA(P)	tetA-F	5′-AGGTGGACCAATTATAGGAATCA-3′	145
	tetA-R	5′-ACCTCCAACTCTATCATCCACC-3′
	tetA-P	HEX-AGCTACAAATATTTCAGTAAG-BHQ1
tetB(P)	tetB-F	5′-AGCAAAGCGGTATGGGATTT-3′	160
	tetB-R	5′-TCACAAACATTCCTCTACCTTCTGT-3′
	tetB-P	FAM-AACTATGAGGGCAACCTTT-BHQ1

质粒 Plasimd	浓度/(copies·μL^-1) Concentration	组内变异试验 Intra-group variation test		组间变异试验 Inter-group variation test
质粒 Plasimd	浓度/(copies·μL^-1) Concentration	平均数 ${\rm{\bar X}}$±SD	变异系数/% CV	平均数 ${\rm{\bar X}}$±SD	变异系数/% CV
tetA	10⁷	19.4±0.26	1.30	19.5±0.36	1.8
	10⁶	22.5±0.26	1.10	22.6±0.20	0.8
	10⁵	22.7±0.29	1.10	25.7±0.39	1.5
tetB	10⁷	20.4±0.15	0.70	20.5±0.32	1.5
	10⁶	23.7±0.18	0.70	23.6±0.30	1.2
	10⁵	26.4±0.09	0.30	26.4±0.18	0.6

菌株毒素型 Toxintype	菌株数 Isolates	耐药菌株数 Resistance strains			双重qPCR检测结果 Duplex real-time PCR test results		普通qPCR检测结果 Normal real-time PCR test results
菌株毒素型 Toxintype	菌株数 Isolates	四环素 Tetracycline	多西环素 Doxycycline	米诺环素 Minocycline	tetA(P)	tetB(P)	tetA(P)	tetB(P)
A	31	26/31	24/31	23/31	24/31	24/31	24/31	24/31
G	2	1/2	1/2	1/2	1/2	1/2	1/2	1/2

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Establishment of a Duplex Real-time PCR Method for Tetracycline Resistance Gene Detection in Clostridium perfringens

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