畜禽源3种革兰氏阴性杆菌多重PCR检测方法的建立与应用

doi:10.16431/j.cnki.1671-7236.2016.03.003

Abstract

Abstract: To establish a rapid and accurate multiplex PCR method for the clinical detection of three main Gram-negative bacilli from livestock and poultry in Guizhou province,three pairs of specific primers were designed and synthetized according to 23S rRNA gene of Escherichia coli(E.coli),KMT gene of Pasteurella and invA gene of Salmonella.In this study,A mutiplex PCR reaction system was established for detecting the three pathogens at the same time,and the reaction system was optimized and it's performance was evaluated.The results showed that the best concentration of primers was 1.0,1.5and 1.0 μmol/L,respectively,and the best annealing temperature was 56 ℃.The results of performance evaluation showed that the method had good specificity and sensitivity,the sensitivity of Salmonella could reach to 1.44 pg/μL,was the highest.70 Gram-negative clinical isolated strains were detected by the multiplex PCR and it was proofed that the method could identify the three pathogens rapidly and accurately,and it could provide effective technical for the rapid clinical diagnosis and epidemiological survey.

Key words: multiplex PCR; E.coli; Pasteurella; Salmonella

CLC Number:

S852.61

MA Guang-qiang, LIU Li-juan, LONG Dan-dan, YUE Jun, LI Yi, WEN Ming, CHENG Zhen-tao, ZHOU Bi-jun. Establishment and Application of Multiplex PCR Detection Method for Three Main Gram-negative Bacilli from Livestock and Poultry[J]. China Animal Husbandry & Veterinary Medicine, 2016, 43(3): 577-584.

References

[1] 刁有祥,李久芹,陈庆普.山东省鸡大肠杆菌的分离鉴定[J].中国预防兽医学报,2002,24(1):23-25.
[2] 严玉霖,雷华,杨尚斌,等.鹅大肠杆菌的分离鉴定及药敏试验[J].中国畜牧兽医,2011,38(10):60-162.
[3] 吴信明,连洪梅,王艳,等.38株鸭源大肠杆菌的分离鉴定与药敏试验[J].中国畜牧兽医,2010,37(10):78-180.
[4] 李浩,刘阳,李长安.多杀性巴氏杆菌病研究进展[J].畜牧兽医杂志,2011,30(2):31-33.
[5] 张萍,冯芳.沙门氏菌的检测技术和方法的研究进展[J].食品安全质量检测学报,2015,6(5):1834-1841.
[6] 张江英.沙门氏菌检测方法的研究进展[J].家禽科学,2012,10:47-50.
[7] 游勇来.沙门氏菌的分离鉴定及PCR快速分型的研究[D].广州:华南理工大学,2013.
[8] 张曼.牛乳中金黄色葡萄球菌、大肠杆菌、产气荚膜梭菌多重PCR检测方法的建立及应用[D].杨凌:西北农林科技大学,2010.
[9] 白文彬,于康震.动物传染病诊断学[M].北京:中国农业出版社,2002.
[10] 徐海花,牛钟相,秦爱建,等.细菌耐药性研究进展[J].山东农业大学学报(自然科学版),2010,41(1):156-160.
[11] 刘晓丹.六种牛源致病性细菌多重PCR检测方法的建立[D].哈尔滨:东北农业大学,2013.
[12] 毕艳妮,宋宇,马淑青,等.多重PCR技术在病原微生物检测中的应用进展[J].中国微生态学杂志,2014,26(12):1476-1478.
[13] 王红宁.畜禽规模化养殖主要病原菌耐药性检测及控制技术研究进展[J].畜牧兽医科技信息,2002,9:12-15.
[14] 杨丽花.链球菌、金黄色葡萄球菌、沙门氏菌和大肠杆菌多重PCR诊断方法的建立[D].杨凌:西北农林科技大学,2011.
[15] 许一平,成炜,邵彦春,等.沙门菌、大肠杆菌和金黄色葡萄球菌的多重PCR检测[J].微生物学通报,2006,33(6):89-94.
[16] Townsend K M,Boyce J D,Frost A J,et al,Development of PCR assays for species and type-specific identification of Pasteurella multocida isolates[J].J Clin Microbiol,1998,36(4):1096-1100.
[17] Rhan K,De Grandis S A,Clarke R C.Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella[J].Mol Cell Probes,1992,6(4):271-279.

Establishment and Application of Multiplex PCR Detection Method for Three Main Gram-negative Bacilli from Livestock and Poultry

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	YU Kun, ZHAO Jie, MA Qin, SHI Yanhong, ZHANG Xiao, LIU Zihan, ZHANG Xinting, WANG Jianhua, LI Yufeng. Isolation,Identification,Drug Resistance and Pathogenicity Analysis of Salmonella Enteritidis from Commercial Meat Ducks [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(5): 2353-2363.
[2]	GUO Qing, WANG Hongli, FENG Yuxuan, ZHANG Yumeng, MA Qiyin, CHENG Jia, LIU Mingchao. Isolation and Identification of Chicken Probiotics and Analysis of Their Probiotic Properties [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(4): 1852-1861.
[3]	MA Yonghui, CAO Qing, FAN Ziqiu, ZHAO Xuehui, ZHI Ji, MA Jinrui, HE Zengwen, ZHANG Haohao, DENG Jing, CHONG Qian, ZHANG Kunzhong, SONG Weili, GOU Huitian, XUE Huiwen. Isolation,Identification and Biological Characterization Analysis of Salmonella from Livestock and Poultry Slaughtering and Marketing in Gansu Province and Neighboring Areas [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(8): 3566-3576.
[4]	YUAN Liang, HAN Xiaoxi, GONG Yingchao, FAN Xianan, HAO Beili, HAO Shuangshuang, LI Changwen, CHANG Yicong, LI Rui, LIU Fangping. Bioinformatics Analysis and Prokaryotic Expression of SipD Protein of Salmonella Enteritidis in Chickens [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(7): 2998-3007.
[5]	SU Hui, WANG Yunfei, ZHAO Xinling, LIU Mimi, ZOU Mingxiao, DONG Wenlong, LI Guojiang, YIN Baishuang. Physicochemical Properties Research of Lactiplantibacillus plantarum MU-6 Bacteriocin and Its Coding Gene Mining [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(7): 3166-3175.
[6]	HUANG Xinmiao, YAO Min, HE Hao, XIAO Huiping, CHEN Jingzhuo, FENG Hao, ZHANG Li, ZHENG Sunli, ZHANG Mengdie, HUANG Tinghua. Preparation and Application of Polyclonal Antibodies Against the Virulence Protein SptP in Salmonella Typhimurium [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(6): 2597-2604.
[7]	LIU Chunhong, LYU Zhihang, ZHANG Yuqian, LIAN Chunyang, ZHENG Xucan, YAN Changyan, ZHANG Xuelian. Isolation and Identification of Chicken-derived Salmonella Senftenberg and Detection of Drug Resistance and Virulence Genes [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(4): 1773-1783.
[8]	ZHOU Di, WEI Ke, DUAN Qianxi, CAO Weisheng. Isolation,Identification and Detection of Drug Resistance and Virulence Genes of Salmonella Pullorum from a Breeding Chicken Farm of Guangdong [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(11): 5033-5042.
[9]	WANG Qiaoyu, ZHEN Mingzhe, ZHANG Nianjie, YAN Dongxue, WANG Xin. Inhibitory Effect of Quercetin on Biofilm Formation of Salmonella [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(11): 5043-5050.
[10]	ZHANG Xinguo. Identification of Exosomes from Supernatant of IPEC-J2 Cells Before and After Infected with Salmonella,and Expression and Transcriptome Analysis of m⁶A Related Proteins [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(11): 5086-5096.
[11]	TIAN Guangming, MA Li, PU Yuejin, YOU Lei, WANG Haojie, ZHANG Yinghu, FANG Chun, LIANG Xiongyan, WEN Zhenyu, TAN Lei, ZHENG Minghao. Effect of Bacillus coagulans on Growth Performance，Antioxidant Capacity and Immune Function of Laying Chicks Infected with Salmonella Pullorum [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(10): 4352-4363.
[12]	CHEN Liyin, PENG Yating, YAN Hongya, CHEN Peifu, XIN Aiguo, LI Ke. Evaluation of the Prevention and Therapeutic Effect of Lytic Bacteriophage KM104 on Different Serotypes of Salmonella spp.from Chickens [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(1): 278-291.
[13]	TAN Ju, WANG Yongjuan, GUO Guangfu, ZHAO Changjing, XIA Aihong, LI Juyin, WU Minqiu, WANG Yuhang, QIN Qiuping. Construction of a Multi-epitope Vaccine Against Salmonella Pullorum by Immunoinformatics Approach [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(1): 312-322.
[14]	ZHANG Lei, CHEN Liang, JIN Zhenhua, FENG Wanyu, MIAO Yan, LAN Shijie, QIN Pingwei, SHEN Sisi, LI Dan, ZHANG Bei, DONG Jiaqiang, HUANG Xuankai, LI Xuye, JIANG Botao, WANG Hongbao, SHI Tongrui. Isolation, Identification and Pathogenic Characteristics of One Pasteurella multocida Isolate from Sheep [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(1): 347-356.
[15]	WANG Yuyan, ZHANG Linji, CHI Lan, CHEN Yongliang, REN Shifei, SUN Peng. Analysis of Salmonella Resistance and PFGE Typing in Chicken Production Chain [J]. China Animal Husbandry & Veterinary Medicine, 2023, 50(8): 3345-3353.