|
|
|
| Establishment and Application of Loop-mediated Isothermal Amplification Assay for Detection of Staphylococcus aureus |
| ZHANG Xiao-Yu, WANG Wen-Wen, MA Chen-Jie, WU Xiao-Ling, DENG Guang-Cun* |
| School of Life Sciences/Key Laboratory of Ministry of Education for Conservation and Utilization of Special Biological Resources in the Western China, Ningxia University, Yinchuan 750021, China |
|
|
|
|
Abstract Bovine mastitis is the most harmful common disease in dairy farms, which seriously affects the healthy development of dairy industry. Bacterial infection is main cause of bovine mastitis. Staphylococcus aureus is one of the most common causative pathogen which causing bovine mastitis. Therefore, the rapid detection method for Staphylococcus aureus is of great significance to realize the early prevention of mastitis in dairy cows. The loop-mediated isothermal amplification (LAMP) technique is a new nucleic acid amplification method that could quickly detect target DNA under isothermal conditions. In this study, a set of 6 specific primers were designed according to the nuc gene (GenBank No. DQ507379.1) of S. aureus, and LAMP technique for S. aureus was established by optimizing the reaction conditions, and then the specificity, sensitivity and clinical detection effect of the method were evaluated. The results showed that the established method could specifically detect S. aureus, the minimum detect limit was 2.7×101 copies/μL, and the sensitivity of LAMP detection was 100 times higher than that of PCR method. The LAMP method which had the advantage of simplicity, rapidity, sensitivity and high specificity, was suitable to rapidly detect S. aureus in dairy farms. This study provides a technical reference for the early diagnosis of dairy cow mastitis caused by other pathogenic bacteria.
|
|
Received: 29 November 2020
|
|
|
|
Corresponding Authors:
* dgc@nxu.edu.cn
|
|
|
|
[1] 杜琳, 冯小慧, 张三粉, 等. 2020. 生鲜乳中金黄色葡萄球菌LAMP检测方法的建立[J]. 动物医学进展, 41(02): 54-57.
(Du L, Feng X H, Zhang S F, et al.2020. Establishment of LAMP assay for detecting Staphylococcus aureus in fresh milk[J]. Advances in Veterinary Medicine, 41(02): 54-57.)
[2] 韩利慧. 2019. 食品中金黄色葡萄球菌检验方法分析[J]. 中国标准化, (24): 245-246.
(Han L H. 2019. Analysis of detection method of Staphylococcus aureus in food[J]. China Standardization, (24): 245-246.)
[3] 侯轩, 付萍, 张海燕, 等. 2012. 牛支原体的环介导等温扩增快速检测技术研究[J]. 农业生物技术学报, 20(02): 218-224.
(Hou X, Fu P, Zhang H Y, et al.2012. Development of loop-mediated isothermal amplification for rapid detection of Mycoplasma bovis[J]. Journal of Agricultural Biotechnology, 20(02): 218-224.)
[4] 刘晓菲, 陈睿元, 郑宇. 2020. 基于环介导等温扩增技术的玫烟色棒束孢可视化检测[J]. 中国生物防治学报, 1-7.
(Liu X F, Chen R Y, Zheng Y.2020. Visual detection of entomogenous fungi Isaria fumosorosea by loop-mediated isothermal amplification technology[J]. Chinese Journal of Biological Control, 1-7.)
[5] 司晓雪. 2020. 金黄色葡萄球菌可视化检测方法的建立与评价[D]. 硕士学位论文, 吉林大学, 导师:李娟, pp. 1-68.
(Si X X.2020. Establishment and evaluation of visual detection method for Staphylococcus aureus[D]. Thesis for M. S., Jilin University, Supervisor: Li J, pp. 1-68.)
[6] 徐美迪. 2019. 基于实时环介导等温扩增技术检测三种病原体的筛查体系研究[D]. 硕士学位论文,内蒙古医科大学, 导师:木兰, pp. 1-91.
(Xu M D.2019. Development of real-time loop mediated isothermal amplification system for detection of three kinds of food borne pathogens[D]. Thesis for M. S., Inner Mongolia Medical University, Supervisor: Mu L, pp. 1-91.)
[7] 于颖. 2015. 实时荧光LAMP技术检测牛乳中金黄色葡萄球菌的研究[D]. 硕士学位论文, 河北农业大学, 导师: 张伟, pp. 1-53.
(Yu Y.2015. Detection of Staphylococcus aureus in milk using real-time fluorescence loop-mediated Iisothermal amplification[D]. Thesis for M. S., Hebei Agricultural University, Supervisor: Zhang W, pp. 1-53.)
[8] Cha E, Hertl J A, Schukken Y H, et al.2013. The effect of repeated episodes of bacteria-specific clinical mastitis on mortality and culling in Holstein dairy cows[J]. Journal of Dairy Science, 96(8): 4993-5007.
[9] Chang T, Wang L, Zhao K, et al.2016. Duplex identification of Staphylococcus aureus by aptamer and gold nanoparticles[J]. Journal of Nanoscience and Nanotechnology, 16(6): 5513-5519.
[10] Hegazy M K, Awad S I, Saleh N E, et al.2020. Loop mediated isothermal amplification (LAMP) of Toxoplasma DNA from dried blood spots[J]. Experimental Parasitology, 211: 107869.
[11] Hoegh S V, Skov M N, Boye K, et al.2014. Variations in the Staphylococcus aureus-specific nuc gene can potentially lead to misidentification of meticillin-susceptible and -resistant S. aureus[J]. Journal of Medical Microbiology, 63(Part 7): 1020-1022.
[12] Kawai K, Inada M, Ito K, et al.2017. Detection of bovine mastitis pathogens by loop-mediated isothermal amplification and an electrochemical DNA chip[J]. Journal of Veterinary Medical Science, 79(12): 1973-1977.
[13] Li T, Lu H, Wang X, et al.2017. Molecular characteristics of Staphylococcus aureus causing bovine mastitis between 2014 and 2015[J]. Frontiers in Cellular and Infection Microbiology, 7: 127.
[14] Liu Y, Zhang J, Yin D J.2016. PCR-based approaches for the detection of clinical methicillin-resistant Staphylococcus aureus[J]. The Open Microbiology Journal. 10: 45-56.
[15] Lu Y, Xininigen Yu J L, et al.2018. Distribution characteristics of pathogenic communities associated with bovine mastitis across different regions of China[J]. Advances in Microbiology, 7(2): 48-57.
[16] Mondal B, Ramlal S, Kingston J.2018. Colorimetric DNAzyme biosensor for convenience detection of enterotoxin B harboring Staphylococcus aureus from food samples[J]. Journal of Agricultural and Food Chemistry, 66(6): 1516-1522.
[17] Notomi T, Okayama H, Masubuchi H, et al.2000. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 28(12): e63-e63.
[18] Schukken Y H, Hertl J, Bar D, et al.2009. Effects of repeated gram-positive and gram-negative clinical mastitis episodes on milk yield loss in Holstein dairy cows[J]. Journal of Dairy Science, 92(7): 3091-3105.
[19] Song H S, Bae Y C, Park S C, et al.2018. Loop-mediated isothermal amplification assay for detection of four immunosuppressive viruses in chicken[J]. Journal of Virological Methods, 256: 6-11.
[20] Sreedeep K S, Sethi S, Yadav R, et al.2020. Loop-mediated isothermal amplification (LAMP) in the respiratory specimens for the diagnosis of pediatric pulmonary tuberculosis: A pilot study[J]. Journal of Infection and Chemotherapy, 26(8): 823-830.
[21] Tomita N, Mori Y, Kanda H, et al.2008. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products[J]. Nature Protocols, 3(5): 877-882.
[22] Xiong J, Huang B, Xu J S, et al.2020. A closed-tube loop-mediated isothermal amplification assay for the visual detection of Staphylococcus aureus[J]. Applied Biochemistry and Biotechnology, 191(1): 201-211.
[23] Yu L Z, Song S Y, Yu C, et al.2020. Rapid detection of Anguina agrostis by loop-mediated isothermal amplification[J]. European Journal of Plant Pathology, 156(3): 819-825. |
|
|
|
