[1]于 博,李博宇,赵 博,等.布鲁氏菌RPA-LFD检测方法的建立[J].中国预防兽医学报,2019,(12):1233-1237.[doi:0.3969/j.issn.1008-0589.201904037]
 YU Bo,LI Bo-yu,ZHAO Bo,et al.Establishment of RPA-LFD detection method for Brucella[J].Chinese journal of preventive veterinary medicine,2019,(12):1233-1237.[doi:0.3969/j.issn.1008-0589.201904037]
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布鲁氏菌RPA-LFD检测方法的建立()
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2019年12
页码:
1233-1237
栏目:
出版日期:
2020-01-25

文章信息/Info

Title:
Establishment of RPA-LFD detection method for Brucella
文章编号:
1008-0589(2019)12-1233-05
作者:
 

于 博1李博宇1赵 博1李 东1李健明2时 坤2曾范利2宗 颖2杜 锐2*

 (1. 吉林农业大学 动物科学技术学院,吉林 长春130118;2. 吉林农业大学 中药材学院,吉林 长春130118)
Author(s):
 

YU Bo1 LI Bo-yu1 ZHAO Bo1 LI Dong1 LI Jian-ming2 SHI Kun2 ZENG Fan-li2 ZONG Ying2 DU Rui2*

 (1. College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China;
2. College of Chinese Medicinal materials, Jilin Agricultural University, Changchun 130118, China)
关键词:
布鲁氏菌重组酶聚合酶扩增技术侧流层析试纸条
分类号:
S852.61
DOI:
0.3969/j.issn.1008-0589.201904037
文献标志码:
A
摘要:
为建立一种快速,便于现场检测布鲁氏菌的方法,本研究基于布鲁氏菌(Brucella) Omp31基因的保守片段设计并合成引物和探针,通过对反应条件优化建立了布鲁氏菌重组酶聚合酶-侧流层析试纸条(RPA-LFD)检测方法,对该方法进行特异性、敏感性、重复性检测。结果显示该检测方法在39 ℃反应20 min以上便可肉眼观察到明显结果。特异性试验结果显示除布鲁氏菌基因组呈阳性外,维氏气单胞菌、大肠杆菌、甲型副伤寒沙门氏菌、肠炎沙门氏菌、乙型溶血性链球菌均为阴性,特异性较强;敏感性试验结果显示,该方法最低可检测到101拷贝/μL的质粒标准品,比常规PCR高出100倍,敏感性较高;重复性试验结果显示,3次批间重复试验无明显差异,稳定性良好。利用该方法检测34份临床样品,其检测结果与普通PCR检测结果符合率为100 %。本研究建立了一种快速检测布鲁氏菌的方法,该方法不需要特殊的仪器,操作简便,灵敏度高,肉眼即可观察到结果,为布鲁氏菌病的现场快速诊断提供了帮助。
  
Abstract:
In order to establish a rapid and convenient diagnostic method for Brucellosis in field, we designed and synthesized primers and probes based on the conserved sequence of Omp31 gene of Brucella, accordingly we established a recombinase polymerase amplification with lateral flow dipstick (RPA-LFD) method to detect Omp31 gene. We also optimized the method and tested for its specificity, sensitivity and reproducibility. Results showed that the reaction can be visualized 20 minutes after incubation at 39℃. Specificity test showed that Aeromonasveronii, Escherichia coli, Paratyphi, Enteritidis, β-Hemolyticstreptoco- ccus were negative except for Brucella. Sensitivity test showed the method could detect as low as 101 copies/μL plasmid standard sample, which was 100 times higher than the ordinary PCR. Repeatability test showed that there was no significant difference among three independent tests. We compared the test results of 34 clinical samples detected by RPA-LFD method and ordinary PCR, we found the coincidence rate of the two methods was 100%. In summary, the Brucella specific RPA-LFD detection methodis rapid, highly sensitive, easy to operate, and the results can be directly visualized without any special instruments, thus the method will be helpful for the rapid diagnosis of Brucellosis in the future.

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(本文编辑:李 爽;英文编辑:温志远)

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更新日期/Last Update: 2020-01-19