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

摘要/Abstract

摘要： 旨在采用环介导等温扩增（LAMP）和横向流动试纸条（LFD）相结合的方法，建立一种快速、特异，过程可视化的猪肺炎支原体（Mycoplasma hyopneumoniae，Mhp）检测方法。针对猪肺炎支原体（Mhp）P36基因设计5套特异性引物和1条异硫氰酸荧光素（FITC）标记的探针，进行LAMP扩增反应。将生物素标记的LAMP产物与FITC标记的探针进行特异性杂交，并使用横向流动试纸条完成扩增产物的检测。经优化，LAMP最佳反应条件为65℃，反应15 min，从基因组DNA提取到LFD结果判断只需40 min左右，比常规PCR技术缩短近2 h。LAMP-LFD可特异性地检出猪肺炎支原体（Mhp），对猪鼻支原体（Mhr）及猪圆环病毒（PCV）等常见猪病病原的检测结果为阴性。灵敏性试验表明，LAMP-LFD对Mhp的检测灵敏度为1×10⁰个拷贝DNA，是普通PCR的1 000倍。利用本方法可从采集的88份临床疑似病料样品中检测到64份阳性，国标PCR方法可检测到56份阳性，符合率可达90.9%。综上，本研究建立的LAMP-LFD方法可特异、准确地应用于Mhp的检测，而且灵敏度高、操作简单、仪器设备依赖性低、检测成本低、耗时短，适合基层实验室、应急检测或现场监测等使用，具有较高的推广价值，有望发展成为Mhp快速检测的有效手段。

关键词: 猪肺炎支原体, 环介导等温扩增技术, 横向流动试纸条, 诊断

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

中图分类号:

S852.62

孙盼盼, 时建立, 彭喆, 王硕, 王妍, 吴晓燕, 徐绍建, 李俊. 猪支原体肺炎LAMP-LFD快速检测方法的建立及初步应用[J]. 畜牧兽医学报, 2020, 51(6): 1419-1428.

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.

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