牛支原体可视化重组酶聚合酶扩增检测方法的建立

doi:10.3969/j.issn.1004-1524.20230922

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (8): 1811-1819.DOI: 10.3969/j.issn.1004-1524.20230922

牛支原体可视化重组酶聚合酶扩增检测方法的建立

王健霖¹(), 田兴苗¹, 王景松¹^,², 戴莎莎¹, 郭亚男², 何生虎¹, 李继东¹^,^*()

1.宁夏大学动物科技学院,宁夏银川 750021
2.宁夏农林科学院动物科学研究所,宁夏银川 750002

收稿日期:2023-07-28 出版日期:2024-08-25 发布日期:2024-09-06
作者简介:*李继东,E-mail: lijidongi@foxmail.com
王健霖(1997—),女,山西吕梁人,硕士研究生,主要从事兽医微生物学与免疫学研究。E-mail: wangjl1202@163.com
通讯作者: 李继东
基金资助:
宁夏回族自治区重点研发计划项目(2022BBF03025);宁夏回族自治区重点研发计划项目(2022BBF03024);宁夏大学产教融合研究生联合培养示范基地建设项目

Establishment of a visual recombinase polymerase amplification assays for Mycoplasma bovis

WANG Jianlin¹(), TIAN Xingmiao¹, WANG Jingsong¹^,², DAI Shasha¹, GUO Yanan², HE Shenghu¹, LI Jidong¹^,^*()

1. College of Animal Science and Technology, Ningxia University, Yinchuan 750021, China
2. Institute of Animal Science, Ningxia Academy of Agriculture and Forestry Sciences, Yinchuan 750002, China

Received:2023-07-28 Online:2024-08-25 Published:2024-09-06
Contact: LI Jidong

摘要/Abstract

摘要：

为建立一种对牛支原体高效、快捷的可视化重组酶聚合酶扩增(LFD-RPA)临床诊断方法,以牛支原体uvrc基因序列为靶基因,设计特异性引物和探针,通过筛选引物和探针、优化反应条件,并通过灵敏度、特异性、重复性和临床样本检测试验进行验证。结果显示,该试验建立的牛支原体LFD-RPA最佳引物为F2/R2,最佳反应条件为39 ℃、 25 min;检测灵敏度可达2.08 copies·μL^-1,是普通PCR灵敏度的100倍;与滑液囊支原体、沙门氏菌、绵羊肺炎支原体、多杀性巴氏杆菌、金黄色葡萄球菌、无乳链球菌和产气荚膜梭菌之间无交叉反应;重复性稳定;对50份鼻拭子样本进行检测,阳性率为26%,与我国行业标准PCR检测方法的符合率为89.6%。该试验成功建立了牛支原体LFD-RPA检测方法,该方法具有操作简便、快速、高效、敏感等优点,为牛支原体的临床快速诊断提供了技术支撑。

关键词: 牛支原体, 重组酶聚合酶扩增, 可视化, 检测方法

Abstract:

The aim was to establish an efficient and rapid visual recombinase polymerase amplification (LFD-RPA) clinical diagnostic method for Mycoplasma bovis. With the uvrc gene sequence of Mycoplasma bovis as the target gene, specific primers and probes are designed and validated by sensitivity, specificity, repeatability and clinical sample detection test through screening of primers and probes and optimizing the reaction conditions. The results showed that the optimal primers for LFD-RPA of Mycoplasma bovis established in this test were F2/R2, and the optimal reaction condition was 39 ℃ for 25 min; the detection sensitivity was up to 2.08 copies·μL^-1, 100 times of common PCR; there was no cross reaction with Mycoplasma synoviae, Salmonella, Mycoplasma ovipneumoniae, Pasteurella multocida, Staphylococcus aureus, Streptococcus agalactiae and Clostridium perfringens; the repeatability was stable; 50 nasal swab samples were detected, the positive rate was 26%, and the coincidence rate with domestic industry standard PCR detection methods was 89.6%. In this study, the LFD-RPA detection method for Mycoplasma bovis was successfully established, which has the advantages of easy operation, rapidity, high efficiency, and sensitivity, and provides technical support for the rapid clinical diagnosis of Mycoplasma bovis.

Key words: Mycoplasma bovis, recombinase polymerase amplification assays, visualization, test method

中图分类号:

S852.62

王健霖, 田兴苗, 王景松, 戴莎莎, 郭亚男, 何生虎, 李继东. 牛支原体可视化重组酶聚合酶扩增检测方法的建立[J]. 浙江农业学报, 2024, 36(8): 1811-1819.

WANG Jianlin, TIAN Xingmiao, WANG Jingsong, DAI Shasha, GUO Yanan, HE Shenghu, LI Jidong. Establishment of a visual recombinase polymerase amplification assays for Mycoplasma bovis[J]. Acta Agriculturae Zhejiangensis, 2024, 36(8): 1811-1819.

图/表 10

表1 引物和探针序列信息

Table 1 Primer and probe sequences information

引物名称 Primer name	引物序列 Primer sequence (5'-3')	长度 Length/bp
F1	CCTGGTGTTTATCTATGAAAAGATGCTAAA	119
R1	TTAGTTTTATATGAATTAATAGCGCCGTCA
F2	CACAAAACCAAAGCCTTAATTGACCTAGAT	173
R2	CCTTTTATGTTTCTTAGTTTGCCTTCTAGTGA
F3	TTTACTATGGTCCTTTTCCTTCTGGTTATG	207
R3	TGGCTGCTTGATGCATTTTGTTAGTTAGTT
uvrc-LFD-F	CACAAAACCAAAGCCTTAATTGACCTAGAT
uvrc-LFD-R	[5’-biotin]-CCTTTTATGTTTCTTAGTTTGCCTTCTAGTGA
uvrc-LFD-Probe	[5'FAM]-ATGACTTAGAACTATACAACTACTTAGTTCAAAT/idSp/CAAGTTGAAGTTGACC-[3'C3 Spacer]
NY-T-F	TTTTAGCTCTTTTTGAACAAAT	1 900
NY-T-R	GGCTCTCATTAAGAATGTC
uvrc-F	GTGGATGCAATTGAAGCTGAAC	2 098
uvrc-R	AAAGAGCAGAAGAGAGAGAGCT

图1 标准质粒PCR扩增凝胶电泳图 M,DNA 标准 DL 5 000;1,目的片段;C,阴性对照。

Fig.1 Standard plasmid PCR amplification gel electrophoresis M, DNA marker DL 5 000; 1, Target fragment; C, Negative control.

图2 基础RPA引物的筛选 M,DNA 标准 DL 500;1~6,反应时间5、10、15、20、25、30 min;C,阴性对照。下同。

Fig.2 Screening of basic RPA primers M, DNA marker DL500; 1-6, Reaction time was 5, 10, 15, 20, 25, 30 min; C, Negative control. The same as below.

图3 基础RPA反应条件的优化

Fig.3 Optimization of basic RPA reaction conditions

图4 基础RPA灵敏度、特异性与重复性试验

Fig.4 Basic RPA sensitivity, specificity and repeatability test

图5 LFD-RPA反应条件的优化

Fig.5 Optimization of LFD-RPA reaction conditions

图6 LFD-RPA灵敏度试验

Fig.6 Sensitivity test of LFD-RPA

图7 LFD-RPA特异性试验 1,绵羊肺炎支原体;2,滑液囊支原体;3,沙门氏菌;4,多杀性巴氏杆菌;5,金黄色葡萄球菌;6,无乳链球菌;7,产气荚膜梭菌。

Fig.7 LFD-RPA specific assay 1, Mycoplasma ovipneumoniae; 2, Mycoplasma synoviae; 3, Salmonella; 4, Pasteurella multocida; 5, Staphylococcus aureus; 6, Streptococcus agalactiae; 7, Clostridium perfringens.

图8 LFD-RPA重复性试验

Fig.8 LFD-RPA repeatability test

表2 基础RPA与行业标准PCR检测方法结果比较分析

Table 2 The results of basic RPA and industry standard PCR detection methods were compared and analyzed

		行业标准PCR Industry standard PCR		总计 Total
		阳性Positive	阴性Negative	总计 Total
LFD-RPA	阳性Positive	12	1	13	92.31% (PPV)
	阴性Negative	0	36	37	97.29% (NPV)
	总计Total	12	38	50
		92.31% (灵敏度) (Sensitivity)	97.30% (特异度) (Specificity)	0.896(Kappa值) (Kappa value)

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牛支原体可视化重组酶聚合酶扩增检测方法的建立

Establishment of a visual recombinase polymerase amplification assays for Mycoplasma bovis

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