猪支原体肺炎LAMP-LFD快速检测方法的建立及初步应用

doi:10.11843/j.issn.0366-6964.2020.025

Abstract

Abstract: The purpose of this study was to establish a rapid, specific and visual assay for the detection of Mycoplasma hyopneumoniae (Mhp) by the combination of the loop-mediated isothermal amplification (LAMP) method and lateral flow dipstick (LFD) system. Five sets of specific primers and one FITC labeled probe targeting to P36 gene of Mhp were designed for LAMP. Biotinylated LAMP amplicons were hybridized exclusively with the FITC-labeled probe and the amplification product was detected by LFD assay. After optimization, the optimal reaction condition of LAMP is 65℃, reaction time is 15 minutes. It only takes about 40 minutes from the extraction of genomic DNA to the visualization of the amplicons by LFD, which is nearly 2 hours shorter than that of conventional PCR. Mhp could be accurately detected by LAMP-LFD. LAMP-LFD can specifically detect Mhp, and the detection results of common pathogens of swine diseases such as Mycoplasma hyorhinis and porcine circovirus are negative. Sensitivity test showed that the sensitivity of LAMP-LFD to Mhp was approximately 1×10⁰ copies of DNA, which was 1 000 times higher than that of standard PCR. Using this method, 64 Mhp positive samples were detected from 88 clinical suspected samples, 56 positive samples were detected by parallel detection of national standard PCR method, and the coincidence rate was 90.9%. The results showed that the LAMP-LFD assay can be used in the detection of Mhp specifically and accurately, and it has advantages of high sensitivity, simple operation, low dependence of instruments and equipment, low detection cost and short time-consuming. It is suitable for the use of basic laboratory, emergency detection or on-site monitoring. It has high promotion value and is expected to develop into a rapid, easy-to-operate tool for detection of Mhp.

Key words: Mycoplasma hyopneumoniae, loop-mediated isothermal amplification combined, lateral flow dipstick, diagnosis

CLC Number:

S852.62

SUN Panpan, SHI Jianli, PENG Zhe, WANG Shuo, WANG Yan, WU Xiaoyan, XU Shaojian, LI Jun. Rapid and Sensitive Diagnosis of Mycoplasma hyopneumoniae by Loop-mediated Isothermal Amplification Combined with a Lateral Flow Dipstick[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(6): 1419-1428.

References 35

[1]	SIBILA M, NOFRARIAS M, LÓPEZ-SORIA S, et al. Chronological study of Mycoplasma hyopneumoniae infection, seroconversion and associated lung lesions in vaccinated and non-vaccinated pigs[J]. Vet Microbiol, 2007, 122(1-2):97-107.
[2]	武昱孜, 邢宪平, 韦艳娜, 等. 不同猪支原体肺炎活疫苗菌株P46和P97基因序列差异性分析[J]. 畜牧兽医学报, 2017, 48(12):2365-2373.WU Y Z, XING X P, WEI Y N, et al. Comparison of nucleotide and deduced amino acid sequences of P46 and P97 for the different live vaccine strains of Mycoplasmal pneumonia of swine[J]. Acta Veterinaria et Zootechnica Sinica, 2017, 48(12):2365-2373. (in Chinese)
[3]	MAES D, SIBILA M, KUHNERT P, et al. Update on Mycoplasma hyopneumoniae infections in pigs:knowledge gaps for improved disease control[J]. Transbound Emerg Dis, 2018, 65(S1):110-124.
[4]	MAES D, SEGALES J, MEYNS T, et al. Control of Mycoplasma hyopneumoniae infections in pigs[J]. Vet Microbiol, 2008, 126(4):297-309.
[5]	MARE C J, SWITZER W P. New species:Mycoplasma hyopneumoniae:a causative agent of virus pig pneumonia[J]. Vet Med Small Anim Clin. 1965, 60:841-846.
[6]	NIELSEN G B, HAUGEGAARD J, JOLIE R. Field evaluation of a ready-to-use combined Porcine circovirus type 2 and Mycoplasma hyopneumoniae vaccine in Denmark- a historical comparison of productivity parameters in 20 nursery and 23 finishing herds[J]. Porcine Health Manag, 2018, 4:29.
[7]	刘建民. 猪支原体肺炎诊断及防治[J]. 畜牧兽医科学:电子版, 2019(11):104-105.LIU J M. The diagnosis and prevention of Mycoplasmal pneumonia of swine[J]. Graziery Veterinary Sciences:Electronic Version, 2019(11):104-105. (in Chinese)
[8]	许立阳. 一起猪支原体肺炎的诊断与防治[J]. 当代畜禽养殖业, 2019(7):47-48.XU L Y. A diagnosis and prevention of Mycoplasmal pneumonia of swine[J]. Modern Animal Husbandry, 2019(7):47-48. (in Chinese)
[9]	王贵平, 李君佩, 罗广, 等. 猪肺炎支原体对猪肺泡巨噬细胞外源性抗原递呈加工功能相关因子mRNA表达量的影响[J]. 中国兽医学报, 2017, 37(11):2101-2107.WANG G P, LI J P, LUO G, et al. Effect of Mycoplasma hyopneumoniae on mRNA expression of the some factors' related to presentation and processing of exogenous antigen in porcine alveolar macrophage[J]. Chinese Journal of Veterinary Science, 2017, 37(11):2101-2107. (in Chinese)
[10]	TSAI S M, LIU H J, SHIEN J H, et al. Rapid and sensitive detection of infectious bursal disease virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick[J]. J Virol Methods, 2012, 181(1):117-124.
[11]	杨若松, 冯志新, 华利忠, 等. 猪支原体肺炎发病模型的建立[J]. 河北农业大学学报, 2015, 38(6):79-82, 87.YANG R S, FENG Z X, HUA L Z, et al. The establishment of a swine model with high virulence organization of Mycoplasma pneumonia[J]. Journal of Agricultural University of Hebei, 2015, 38(6):79-82, 87. (in Chinese)
[12]	武昱孜, 熊祺琰, 刘蓓蓓, 等. 猪肺炎支原体和猪鼻支原体双重荧光定量PCR检测方法的建立[J]. 畜牧兽医学报, 2017, 48(8):1491-1498.WU Y Z, XIONG Q Y, LIU B B, et al. Duplex real-time PCR method for detection of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis[J]. Acta Veterinaria et Zootechnica Sinica, 2017, 48(8):1491-1498. (in Chinese)
[13]	NOTOMI T, OKAYAMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J/OL]. Nucleic Acids Res, 2000, 28(12):e63.[2020-01-01]https://doi.org/10.1093/nar/28.12.e63.
[14]	李秀梅, 石瑜, 朱雅宁, 等. 环介导等温扩增技术与横向流动试纸条法快速检测鸡贫血病毒的研究[J]. 中国动物传染病学报, 2018, 26(1):25-31.LI X M, SHI Y, ZHU Y N, et al. Rapid and sensitive detection of chicken anemia virus by loop-mediated isothermal amplification combined with lateral-flow dipstick[J]. Chinese Journal of Veterinary Parasitology, 2018, 26(1):25-31. (in Chinese)
[15]	李秀梅, 梁智选, 李颖, 等. 环介导等温扩增技术与横向流动试纸条法快速检测布鲁氏杆菌[J]. 食品与生物技术学报, 2017, 36(12):1276-1282.LI X M, LIANG Z X, LI Y, et al. Rapid and sensitive detection of Brucella spp. by loop-mediated isothermal amplification combined with a lateral-flow dipstick[J]. Journal of Food Science and Biotechnology, 2017, 36(12):1276-1282. (in Chinese)
[16]	乔艳, 周前进, 李孝军, 等. 环介导等温扩增联合横向流动试纸条检测简单异尖线虫/派氏异尖线虫方法的建立[J]. 海洋与湖沼, 2019, 50(2):324-335.QIAO Y, ZHOU Q J, LI X J, et al. A loop-mediated isothermal amplification technique combined with a lateral flow dipstick for the detection of Anisakis simplex sensu stricto/Anisakis pegreffii in commercial fish[J]. Oceanologia et Limnologia Sinica, 2019, 50(2):324-335. (in Chinese)
[17]	ZHANG B, NAN T G, XIN J. et al. Development of a colloidal gold-based lateral flow dipstick immunoassay for rapid detection of chlorogenic acid and luteoloside in Flos Lonicerae Japonicae[J]. J Pharm Biomed Anal, 2019, 170:83-88.
[18]	柴方超, 周前进, 陈炯. 环介导等温扩增联合横向流动试纸条可视化检测迟缓爱德华菌[J]. 中国兽医学报, 2017, 37(6):1103-1110.CHAI F C, ZHOU Q J, CHEN J. Visual detection of Edwardsiella tarda based on loop-mediated isothermal amplification combined with a lateral flow dipstick[J]. Chinese Journal of Veterinary Science, 2017, 37(6):1103-1110. (in Chinese)
[19]	中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会.猪肺炎支原体PCR检测方法:GB/T 35909-2018[S]. 北京:中国标准出版社,2018.General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China, Standardization Administration of the P.R.C. Detection of Mycoplasma hyopneumoniae using PCR method:GB/T 35909-2018[S]. Beijing:China Standard Press, 2018(in Chinese)
[20]	徐引弟, 张青娴, 王治方, 等. 快速直接PCR检测猪肺炎支原体方法的建立及其应用[J]. 养猪, 2019(6);100-102.XU Y D, ZHANG Q X, WANG Z F, et al. Establishment and application of rapid direct PCR for the detection of Mycoplasma pneumoniae[J]. Swine Production, 2019(6):100-102. (in Chinese)
[21]	赵国胜. 猪支原体肺炎临床诊断及综合防治[J]. 畜牧兽医科学:电子版, 2019(16):110-111.ZHAO G S. Clinical diagnosis and comprehensive prevention and treatment of Mycoplasma hyopneumoniae[J]. Graziery Veterinary Sciences:Electronic Version, 2019(16):110-111. (in Chinese)
[22]	李石, 李劼, 周霞. 猪肺炎支原体生长培养基的筛选[J]. 黑龙江畜牧兽医, 2014(18):61-63.LI S, LI J, ZHOU X. The screen of growth medium by Mycoplasma pneumonia[J]. Heilongjiang Animal Science and Veterinary Medicine, 2014(18):61-63. (in Chinese)
[23]	刘金媛. 猪支原体肺炎诊断与防治措施[J]. 畜牧兽医科学:电子版, 2019(7):100-101.LIU J Y. Diagnosis and prevention of Mycoplasma pneumonia in pigs[J]. Graziery Veterinary Sciences:Electronic Version, 2019(7):100-101. (in Chinese)
[24]	师丽刚, 周飞. 猪肺炎支原体PCR检测方法的建立与应用[J]. 现代畜牧兽医, 2016(9):11-15.SHI L G, ZHOU F. Establishment and application of PCR detection method of mycoplasma pneumonia in pigs[J]. Modern Journal of Animal Husbandry and Veterinary Medicine, 2016(9):11-15. (in Chinese)
[25]	宋建领, 高华峰, 信爱国, 等. 猪肺炎支原体实时荧光定量PCR检测方法的建立与应用[J]. 畜牧与兽医, 2013, 45(5):71-74.SONG J L, GAO H F, XIN A G, et al. Establishment and application of real-time fluorescence quantitative PCR for Mycoplasma pneumoniae[J]. Animal Husbandry & Veterinary Medicine, 2013, 45(5):71-74. (in Chinese)
[26]	KIMARU W K, BAI F F, WU Y Z, et al. Comparative analysis for the detection and monitoring of Mycoplasma hyopneumoniae infection by nested PCR (n-PCR) and real time PCR (q-PCR) from field swine herds[J]. Agric Sci Technol, 2014, 15(6):918-921.
[27]	LI J H, MINION C F, PETERSEN A C, et al. Loop-mediated isothermal amplification for rapid and convenient detection of Mycoplasma hyopneumoniae[J].World J Microbiol Biotechnol, 2013, 29(4):607-616.
[28]	武昱孜, 刘茂军, 杜改梅, 等. 快速敏感的环介导恒温扩增技术检测猪肺炎支原体P36基因[C]//中国畜牧兽医学会动物传染病学分会第十六次学术研讨会论文集. 济南:中国畜牧兽医学会动物传染病学分会, 解放军军事医学科学院军事兽医研究所, 2015:169.WU Y Z, LIU M J, DU G M, et al. The detection for the P36 gene of mycoplasma pneumonia by rapid sensitive loop-mediated thermostatic amplification[C]//Proceedings of the 16th Symposium of Zoology Branch of Chinese Society of Animal Husbandry and Veterinary Medicine. Ji'nan:Animal Infectious Diseases Branch of Chinese Society of Animal Husbandry and Veterinary Medicine, Institute of Military Veterinary Medicine, PLA Academy of Military Medical Sciences, 2015:169. (in Chinese)
[29]	张莉, 王利丽, 魏财文, 等. 环介导等温扩增技术结合横向流动试纸条检测猪链球菌2型的应用研究[J]. 中国人兽共患病学报, 2016, 32(12):1077-1082, 1090.ZHANG L, WANG L L, WEI C W, et al. Rapid detection of Streptococcus suis serotype 2 with loop mediated isothermal amplification combined with a lateral flow dipstick[J]. Chinese Journal of Zoonoses, 2016, 32(12):1077-1082, 1090. (in Chinese)
[30]	LALLE M, POSSENTI A, DUBEY J P, et al. Loop-mediated isothermal amplification-lateral-flow dipstick (LAMP-LFD) to detect Toxoplasma gondii oocyst in ready-to-eat salad[J]. Food Microbiol, 2018, 70:137-142.
[31]	WU Y Y, TIAN K Y, ZHANG Y H, et al. Rapid and visual detection of Lawsonia intracellularis with an improved recombinase polymerase amplification assay combined with a lateral flow dipstick[J]. BMC Vet Res, 2019, 15(1):97.
[32]	张洁, 曹军军, 祝明松, 等. 绵羊肺炎支原体环介导等温扩增联合横向流动试纸条可视化检测方法的建立[J]. 动物医学进展, 2019, 40(3):1-8.ZHANG J, CAO J J, ZHU M S, et al. Establishment of loop-mediated isothermal amplification-lateral-flow dipstick for detecting Mycoplasma ovipneumoniae[J]. Progress in Veterinary Medicine, 2019, 40(3):1-8. (in Chinese)
[33]	DENG J R, PEI J J, GOU H C, et al. Rapid and simple detection of Japanese encephalitis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick[J]. J Virol Methods, 2015, 213:98-105.
[34]	朱俊灵, 叶佐东, 邓洁汝, 等. 猪瘟病毒RT-LAMP-LFD检测方法的建立与应用[J]. 华南农业大学学报, 2016, 37(1):1-7.ZHU J L, YE Z D, DENG J R, et al. Rapid detection of classical swine fever virus by loop-mediatedisothermal amplification combined with lateral flow dipstick method[J]. Journal of South China Agricultural University, 2016, 37(1):1-7. (in Chinese)
[35]	程蝶, 柴方超, 蔡怡, 等. 环介导等温扩增联合横向流动试纸条可视化检测哈维氏弧菌的研究[J]. 生物技术通报, 2016, 32(6):60-68.CHENG D, CHAI F C, CAI Y, et al. Visual detection of Vibrio harveyi based on loop-mediated isothermal amplification combined with a lateral flow dipstick[J]. Biotechnology Bulletin, 2016, 32(6):60-68. (in Chinese)

Rapid and Sensitive Diagnosis of Mycoplasma hyopneumoniae by Loop-mediated Isothermal Amplification Combined with a Lateral Flow Dipstick

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