[1]郝亚南,王越,岳晓彤,等.金黄色葡萄球菌TSST-1毒素基因荧光定量PCR检测方法的建立及应用[J].江苏农业学报,2026,42(04):835-841.[doi:doi:10.3969/j.issn.1000-4440.2026.04.020]
　HAO Yanan,WANG Yue,YUE Xiaotong,et al.Establishment and application of real-time PCR for detection of Staphylococcus aureus TSST-1 toxin gene[J].,2026,42(04):835-841.[doi:doi:10.3969/j.issn.1000-4440.2026.04.020]
点击复制

金黄色葡萄球菌TSST-1毒素基因荧光定量PCR检测方法的建立及应用()

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
42
期数:
2026年04期
页码:
835-841
栏目:
加工贮藏·质量安全
出版日期:
2026-04-30

文章信息/Info

Title:
Establishment and application of real-time PCR for detection of Staphylococcus aureus TSST-1 toxin gene
作者:
郝亚南1; 2; 王越2; 岳晓彤2; 李雪婷2; 王羽2
(1.吉林省生物基础实验教学示范中心,吉林四平136000；2.吉林师范大学生命科学学院,吉林四平136000)
Author(s):
HAO Yanan1; 2; WANG Yue2; YUE Xiaotong2; LI Xueting2; WANG Yu2
(1.Jilin Province Demonstration Center for Basic Experiment Biology Eduction, Siping 136000, China;2.College of Life Sciences, Jilin Normal University, Siping 136000, China)
关键词:
金黄色葡萄球菌; TSST-1毒素基因（tst）; 实时荧光定量PCR
Keywords:
Staphylococcus aureus; TSST-1 toxin gene (tst); real-time PCR
分类号:
R378.1+1
DOI:
doi:10.3969/j.issn.1000-4440.2026.04.020
文献标志码:
A
摘要:
中毒性休克综合征(TSS)是由金黄色葡萄球菌(Staphylococcus aureus)tst基因编码的TSST-1引发的一种死亡率较高的全身性感染疾病。为了快速检测含有TSST-1毒素基因的金黄色葡萄球菌菌株,本研究基于GenBank中金黄色葡萄球菌TSST-1毒素基因tst设计特异性引物,通过PCR扩增将目标片段插入pMD-18T载体,转化到大肠杆菌(Escherichia coli)DH5α感受态细胞中构建重组质粒。对引物浓度、退火温度进行优化,建立快速检测金黄色葡萄球菌tst基因的实时荧光定量PCR方法,并验证其灵敏度、特异性和重复性。结果表明,以重组质粒为标准品获得的标准曲线方程为Y=-3.494x+28.800,决定系数（R2）=0.998,具有良好的线性关系。确定荧光定量PCR方法的最佳引物浓度为0.1 μmol/L,最佳退火温度为53 ℃,且特异性强,与金黄色葡萄球菌RN4220菌株、大肠杆菌菌株、枯草芽孢杆菌菌株、猪豕链球菌菌株、铜绿假单胞菌菌株的DNA均无交叉反应。此外,该方法具有较高的灵敏性,可检测的最低拷贝数为1 μL 1.29×102 个,其敏感性比普通PCR高100倍。组内和组间的变异系数均小于2.00%,重复性较好。综上,金黄色葡萄球菌TSST-1毒素基因tst的实时荧光定量PCR检测方法灵敏度高、重复性好且特异性强,本研究结果为金黄色葡萄球菌TSST-1毒素基因的检测提供了可靠的技术手段。
Abstract:
Toxic shock syndrome (TSS) is a systemic infectious disease with a high mortality rate, caused by TSST-1, which is encoded by the tst gene of Staphylococcus aureus. To rapidly detect Staphylococcus aureus strains containing the TSST-1 toxin gene, in this study, specific primers were designed based on the TSST-1 toxin gene tst of Staphylococcus aureus in GenBank. The target fragment was inserted into the pMD-18T vector by PCR amplification, and transformed into Escherichia coli DH5α competent cells to construct a recombinant plasmid. The primer concentration and annealing temperature were optimized to establish a real-time PCR method for rapid detection of the tst gene of Staphylococcus aureus, and its sensitivity, specificity and repeatability were verified. The results showed that the standard curve equation obtained using the recombinant plasmid as the standard was Y=-3.494x+28.800, with a coefficient of determination (R2) of 0.998, indicating a good linear relationship. The optimal primer concentration for the real-time PCR method was determined to be 0.1 μmol/L, and the optimal annealing temperature was 53 ℃, with strong specificity and no cross-reaction with the DNA of Staphylococcus aureus strain RN4220, Escherichia coli strain, Bacillus subtilis strain, Streptococcus suis strain, and Pseudomonas aeruginosa strain. In addition, this method exhibited high sensitivity, with the minimum detectable copy number being 1.29×102 copies per microliter, and its sensitivity was 100 times higher than that of ordinary PCR. The intra-group and inter-group coefficients of variation were both less than 2.00%, showing good repeatability. In conclusion, the real-time PCR detection method for the TSST-1 toxin gene tst of Staphylococcus aureus has high sensitivity, good repeatability and strong specificity. The results of this study provide a reliable technical means for the detection of the TSST-1 toxin gene of Staphylococcus aureus.

参考文献/References:

[1]AHMAD-MANSOUR N, LOUBET P, POUGET C, et al. Staphylococcus aureus toxins:an update on their pathogenic properties and potential treatments[J]. Toxins,2021,13(10):677.
[2]HAN J, SHI W L, XU X G, et al. Conditions and mutations affecting Staphylococcus aureus L-form formation[J]. Microbiology,2015,161:57-66.
[3]PENG L C, JIANG J L, CHEN T T, et al. Toxic shock syndrome toxin 1 induces immune response via the activation of NLRP3 inflammasome[J]. Toxins,2021,13(1):68.
[4]TUFFS S W, GONCHEVA M I, XU S X, et al. Superantigens promote Staphylococcus aureus bloodstream infection by eliciting pathogenic interferon-gamma production[J]. Proceedings of the National Academy of Sciences of the United States of America,2022,119(8):2115987119.
[5]HU D L, LI S W, FANG R D, et al. Update on molecular diversity and multipathogenicity of staphylococcal superantigen toxins[J]. Animal Diseases,2021,1(1):7.
[6]HANSEN N S, LETH S, NIELSEN L T. Toxic shock syndrome[J]. Ugeskrift for Laeger,2020,182(20):11190673.
[7]GRUMANN D, RUOTSALAINEN E, KOLATA J, et al. Characterization of infecting strains and superantigen-neutralizing antibodies in Staphylococcus aureus bacteremia[J]. Clinical and Vaccine Immunology,2011,18(3):487-493.
[8]ABDURRAHMAN G, SCHMIEDEKE F, BACHERT C, et al. Allergy-a new role for T cell superantigens of Staphylococcus aureus?[J]. Toxins,2020,12(3):176.
[9]DEURENBERG R H, NIEUWENHUIS R F, DRIESSEN C, et al. The prevalence of the Staphylococcus aureus tst gene among community- and hospital-acquired strains and isolates from Wegener’s Granulomatosis patients[J]. FEMS Microbiology Letters,2005,245(1):185-189.
[10]ZHENG Y, QIN C H, ZHANG X F, et al. The tst gene associated Staphylococcus aureus pathogenicity island facilitates its pathogenesis by promoting the secretion of inflammatory cytokines and inducing immune suppression[J]. Microbial Pathogenesis,2020,138:103797.
[11]ZAREI KOOSHA R, MAHMOODZADEH HOSSEINI H, MEHDIZADEH AGHDAM E, et al. Distribution of tsst-1 and MecA genes in Staphylococcus aureus isolated from clinical specimens[J]. Jundishapur Journal of Microbiology,2016,9(3):29057.
[12]褚敏君. tst基因在儿童金黄色葡萄球菌肺炎中的临床应用价值研究[D]. 昆明:昆明医科大学,2022.
[13]MOHAMMAD S M, HEGAZY L A, ABDEL HADY R S, et al. Real-time PCR versus traditional and Nano-based ELISA in early detection of murine trichinellosis[J]. Journal of Helminthology,2023,97:67.
[14]KRISHNANNAIR GEETHA D, SIVARAMAN B, RAMMOHAN R, et al. A SYBR Green based multiplex Real-Time PCR assay for rapid detection and differentiation of ocular bacterial pathogens[J]. Journal of Microbiological Methods,2020,171:105875.
[15]DUFRESNE K, DIMAGGIO D A, MADUTA C S, et al. Discovery of an antivirulence compound that targets the Staphylococcus aureus SaeRS two-component system to inhibit toxic shock syndrome toxin-1 production[J]. Journal of Biological Chemistry,2024,300(7):107455.
[16]FERNANDES S M, LUNA A, HALL T, et al. Toxic shock syndrome after orthopaedic surgery[J]. Cureus,2023,15(7):42609.
[17]MEHROTRA M, WANG G, JOHNSON W M. Multiplex PCR for detection of genes for Staphylococcus aureus enterotoxins,exfoliative toxins,toxic shock syndrome toxin 1,and methicillin resistance[J]. Journal of Clinical Microbiology,2000,38(3):1032-1035.
[18]NAVARRO E, SERRANO-HERAS G, CASTAO M J, et al. Real-time PCR detection chemistry[J]. Clinica Chimica Acta,2015,439:231-250.
[19]MNSTER P, PPPEL L, ANTAKLI A, et al. The detection of Salmonella enteritidis on German layer farms after cleaning and disinfection[J]. Animals,2023,13(16):2588.
[20]LIU A X, PHILLIPS K, JIA J Y, et al. Development of a qPCR detection approach for pathogenic Burkholderia cenocepacia associated with fresh vegetables[J]. Food Microbiology,2023,115:104333.
[21]COSTA-RIBEIRO A, AZINHEIRO S, ROUMANI F, et al. Multiplex real-time PCR for the detection of shiga toxin-producing Escherichia coli in foods[J]. Methods in Molecular Biology,2023,2967:63-73.
[22]CAO N, TANG Z H, ZHANG X Y, et al. Development and application of a triplex TaqMan quantitative real-time PCR assay for simultaneous detection of feline calicivirus,feline parvovirus,and feline herpesvirus 1[J]. Frontiers in Veterinary Science,2022,8:792322.
[23]WANG J H, GUO K, LI S J, et al. Development and application of real-time PCR assay for detection of Salmonella abortusequi[J]. Journal of Clinical Microbiology,2023,61(3):0137522.

相似文献/References:

[1]李晓霞,贾淼,金君华,等.原料乳中金黄色葡萄球菌环介导等温扩增（LAMP）检测方法的建立及应用[J].江苏农业学报,2017,(05):1171.[doi:doi:10.3969/j.issn.1000-4440.2017.05.032]
　LI Xiao-xia,JIA Miao,JIN Jun-hua,et al.Establishment and application of loop-mediated isothermal amplification(LAMP) of Staphylococcus aureus in raw milk[J].,2017,(04):1171.[doi:doi:10.3969/j.issn.1000-4440.2017.05.032]
[2]张娇娇,吴海虹,孙晋跃,等.超声波联合乳酸对金黄色葡萄球菌的杀菌效果[J].江苏农业学报,2022,38(06):1686.[doi:doi:10.3969/j.issn.1000-4440.2022.06.027]
　ZHANG Jiao-jiao,WU Hai-hong,SUN Jin-yue,et al.Bactericidal effect of ultrasound combined with lactic acid against Staphylococcus aureus[J].,2022,38(04):1686.[doi:doi:10.3969/j.issn.1000-4440.2022.06.027]
[3]陶鸿伟,孟苓凤,张建岭,等.复配植物精油的协同抗菌活性及作用机制[J].江苏农业学报,2025,(07):1429.[doi:doi:10.3969/j.issn.1000-4440.2025.07.019]
　TAO Hongwei,MENG Lingfeng,ZHANG Jianling,et al.Synergistic antimicrobial activity and mechanism of action of composite plant essential oils[J].,2025,(04):1429.[doi:doi:10.3969/j.issn.1000-4440.2025.07.019]

备注/Memo

备注/Memo:
收稿日期：2025-04-06基金项目：吉林省教育厅科学技术研究规划项目(JJKH20230519)；吉林师范大学研究生科研创新计划项目(202038)作者简介：郝亚南(1980-),女,吉林大安人,硕士,副研究员,主要从事微生物学教学与科研管理。(E-mail)jl3292019@163.com通讯作者：王羽,(E-mail)wang2311472521@163.com
更新日期/Last Update: 2026-05-11