CN111337686A

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Info

Publication number: CN111337686A
Application number: CN202010168896.XA
Authority: CN
Inventors: 赵宏; 杨柳; 赵良娟; 董志珍; 张宏伟; 师三媛; 王洪彬; 张滨; 张霞; 赵化冰
Original assignee: Tianjin Customs Animal Plant And Food Inspection Center; Tianjin University of Science and Technology
Current assignee: Tianjin Customs Animal Plant And Food Inspection Center; Tianjin University of Science and Technology
Priority date: 2020-03-12
Filing date: 2020-03-12
Publication date: 2020-06-26

Abstract

Translated fromChinese

本发明涉及一种利用免疫富集技术与MALDI‑TOF MS生物质谱联用快速，准确的检测食品中金黄色葡萄球菌的方法。本检测方法与传统方法相比大大缩短了目标菌的筛选培养时间，检测方法所涉及的免疫富集技术具有特异性好，选择性高等特点，可将目的菌从待检测样品中特异性富集；同时所使用的MALDI‑TOF MS生物质谱仪具有灵敏度高，检测结果快速准确等特点，本发明将二者联合使用，用于检测食品中金黄色葡萄球菌，具有特异性好，选择性高，灵敏度强，检测结果快速准确的特点，可用于牛奶等食品样品中的金黄色葡萄球菌快速准确鉴定。

The invention relates to a rapid and accurate method for detecting Staphylococcus aureus in food by using immune enrichment technology and MALDI-TOF MS biological mass spectrometry. Compared with the traditional method, the detection method greatly shortens the screening and culture time of the target bacteria. The immune enrichment technology involved in the detection method has the characteristics of good specificity and high selectivity, and can specifically enrich the target bacteria from the samples to be detected. At the same time, the used MALDI-TOF MS biological mass spectrometer has the characteristics of high sensitivity, fast and accurate detection results, etc. The invention uses the two in combination to detect Staphylococcus aureus in food, and has good specificity and high selectivity. With strong sensitivity and fast and accurate detection results, it can be used for rapid and accurate identification of Staphylococcus aureus in food samples such as milk.

Description

Translated fromChinese

免疫富集联合MALDI-TOF MS检测食品中金黄色葡萄球菌的方法及应用Method for detection of Staphylococcus aureus in food by immune enrichment combined with MALDI-TOF MSlaw and application

技术领域technical field

本发明属于生物技术领域，尤其是一种免疫富集联合MALDI-TOF MS检测食品中金黄色葡萄球菌的方法及应用。The invention belongs to the field of biotechnology, in particular to a method and application of immune enrichment combined with MALDI-TOF MS for detecting Staphylococcus aureus in food.

背景技术Background technique

金黄色葡萄球菌(Staphyloccocus aureus)是引起食源性疾病的一种重要的细菌，其在自然界中广泛存在，因而引起食品污染的几率也很高。金黄色葡萄球菌可以分泌20多种毒性蛋白，引起多种严重的食物中毒。几年来，由金黄色葡萄球菌等食源性致病菌引起的食物中毒事件频发，因此急需对金黄色葡萄球菌进行快速有效的检测和控制。Staphyloccocus aureus is an important bacterium that causes food-borne diseases, and it exists widely in nature, so it has a high probability of causing food contamination. Staphylococcus aureus can secrete more than 20 kinds of toxic proteins, causing a variety of serious food poisoning. In recent years, food poisoning incidents caused by food-borne pathogens such as Staphylococcus aureus have occurred frequently, so there is an urgent need for rapid and effective detection and control of Staphylococcus aureus.

免疫磁珠(Immunomagnetic beads，IMB)富集技术是利用抗原抗体的特异性结合，在磁场的作用下将目标抗原在较短的时间内分离富集起来的一项技术。具有选择性好，特异性强的特点，可以将目的菌从复杂体系中快速分离富集，因而被广泛应用于分离富集特定微生物、蛋白质及微量有毒有害物质等。Immunomagnetic beads (IMB) enrichment technology is a technology that uses the specific binding of antigen and antibody to separate and enrich the target antigen in a short period of time under the action of a magnetic field. It has the characteristics of good selectivity and strong specificity, and can quickly separate and enrich the target bacteria from complex systems, so it is widely used in the separation and enrichment of specific microorganisms, proteins and trace toxic and harmful substances.

基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)，是一种通过离子的质荷比(M/Z)与离子的飞行时间成正比来分析离子，测得样品分子的分子量并分析混合生物大分子的软电离技术，其可通过测得的蛋白质肽段指纹图谱在数据库中查询识别而鉴定蛋白质，是目前研究蛋白质组学中最主要的鉴定方法，因此可用于金黄色葡萄球菌的全细胞蛋白指纹图谱分析，进而对金黄色葡萄球菌进行鉴定。Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a method to analyze ions by the mass-to-charge ratio (M/Z) of ions proportional to the time-of-flight of ions, measure the molecular weight of sample molecules and analyze mixed biological Soft ionization technology of macromolecules, which can identify proteins by querying and identifying proteins in the database through the measured protein peptide fingerprints. Protein fingerprint analysis was used to identify Staphylococcus aureus.

通过检索，尚未发现与本发明专利申请相关的专利公开文献。Through searching, no patent publications related to the patent application of the present invention have been found.

发明内容SUMMARY OF THE INVENTION

本发明目的在于克服现有技术的不足之处，提供一种免疫富集联合MALDI-TOF MS检测食品中金黄色葡萄球菌的方法及应用。The purpose of the present invention is to overcome the deficiencies of the prior art, and to provide a method and application for the detection of Staphylococcus aureus in food by immune enrichment combined with MALDI-TOF MS.

本发明解决其技术问题所采用的技术方案是：The technical scheme adopted by the present invention to solve its technical problems is:

一种免疫富集联合MALDI-TOF MS检测食品中金黄色葡萄球菌的方法，所述方法将选择性好、特异性强的免疫富集技术与灵敏度高、准确性好的生物质谱仪-基质辅助激光解吸电离飞行时间质谱即MALDI-TOF-MS联合使用，对食品中金黄色葡萄球菌进行特异性富集并进行全细胞蛋白质指纹图谱分析，能够高效、快速、准确鉴定食品中的金黄色葡萄球菌。A method for the detection of Staphylococcus aureus in food by immune enrichment combined with MALDI-TOF MS, the method combines immune enrichment technology with good selectivity and specificity with biological mass spectrometer-matrix aided by high sensitivity and good accuracy Laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is used in combination to specifically enrich Staphylococcus aureus in food and perform whole-cell protein fingerprint analysis, which can efficiently, quickly and accurately identify Staphylococcus aureus in food. .

而且，步骤如下：And, the steps are as follows:

⑴样品的采集及处理；⑴ sample collection and processing;

⑵目的菌的特异性富集：使用抗金黄色葡萄球菌抗体制备免疫磁珠，对金黄色葡萄球菌进行特异性富集，制得菌体-免疫磁珠复合物；(2) Specific enrichment of target bacteria: use anti-Staphylococcus aureus antibody to prepare immunomagnetic beads, and carry out specific enrichment of Staphylococcus aureus to obtain a bacterial-immune magnetic bead complex;

⑶目的菌选择性增菌：将步骤⑵中的菌体-免疫磁珠复合物接种于金黄色葡萄球菌选择性增菌培养基增菌12-15h，对免疫富集所得的细菌进行选择性增菌；(3) Selective enrichment of the target bacteria: inoculate the bacterial body-immunomagnetic bead complex in step (2) in the selective enrichment medium of Staphylococcus aureus for 12-15 hours, and selectively enrich the bacteria obtained by immune enrichment. bacteria;

⑷MALDI-TOF MS数据采集与分析：⑷MALDI-TOF MS data acquisition and analysis:

①点样①Spotting

将增菌样品经甲酸/乙醇提取法处理后取1μL上清液加至点样靶，每种处理方法得到的样品平行点4个孔，待样液滴晾干后，覆盖1μL饱和基质CHCA，干燥后进行MALDI-TOF MS鉴定；After the enriched samples were treated with formic acid/ethanol extraction, 1 μL of the supernatant was added to the spotting target, and the samples obtained by each treatment method were placed in 4 wells in parallel. MALDI-TOF MS identification after drying;

②MALDI-TOF MS数据采集与分析②MALDI-TOF MS data acquisition and analysis

用MALDI-TOF MS对样品进行数据采集；Data acquisition of samples by MALDI-TOF MS;

分析采用线性模式；激光能量：60～90Hz；收集质荷比范围m/z：2000～20000；每个样品激光轰击100次；采集到的数据导入数据库进行分析；每次试验前在采集数据的质量范围内使用大肠杆菌ATCC 8739标准品进行校准；The analysis adopts linear mode; laser energy: 60~90Hz; collection mass-to-charge ratio range m/z: 2000~20000; each sample is bombarded bylaser 100 times; the collected data is imported into the database for analysis; Calibration using Escherichia coli ATCC 8739 standard within the mass range;

⑸结果判断：与数据库对比，显示鉴定结果为金黄色葡萄球菌为阳性，无鉴定结果为阴性，记录每个样品里阳性结果的比例，最后分析数据，并以多种方式显示检测结果；⑸Result judgment: Compared with the database, it shows that the identification result is positive for Staphylococcus aureus, and no identification result is negative. Record the proportion of positive results in each sample, and finally analyze the data and display the test results in various ways;

其中，每次检测均设立阴性对照，阴性对照为未与待检样品反应的免疫磁珠。Among them, a negative control is set up for each test, and the negative control is the immunomagnetic beads that have not reacted with the sample to be tested.

而且，将选择性好、特异强的免疫富集技术与准确性好、灵敏度高得MALDI-TOF MS生物质谱联合用于检测食品中金黄色葡萄球菌。Moreover, the immuno-enrichment technology with good selectivity and specificity is combined with the MALDI-TOF MS biological mass spectrometry with good accuracy and high sensitivity to detect Staphylococcus aureus in food.

而且，所述步骤⑵中特异性富集的条件为：Moreover, the conditions for specific enrichment in the step (2) are:

免疫富集金黄色葡萄球菌的检测体系为1mL，具体为：The detection system for immune enrichment of Staphylococcus aureus is 1 mL, specifically:

600μL 250μg/mL磁分离后的免疫磁珠，1mL待检食品样液；600μL of 250μg/mL magnetically separated immunomagnetic beads, 1mL of food sample liquid to be tested;

反应条件为：室温颠倒混匀反应30min；The reaction conditions are: inversion and mixing at room temperature for 30 min;

而且，所述步骤⑴中根据《GB 4789.10食品安全国家标准食品微生物学检测金黄色葡萄球菌检验》中样品的采集与制备方法进行样品采集与处理。Moreover, in the described step (1), sample collection and processing are carried out according to the collection and preparation method of the sample in "GB 4789.10 National Food Safety Standard Food Microbiology Detection Staphylococcus aureus Inspection".

而且，所述步骤⑵的具体步骤如下：And, the concrete steps of described step (2) are as follows:

每1mg磁珠的处理如下：The processing of each 1 mg of magnetic beads is as follows:

①将磁珠混匀后取1mg置于1.5ml离心管，用500μL 2-吗啉乙磺酸-Tween清洗2次；① After mixing the magnetic beads, take 1 mg and place it in a 1.5 ml centrifuge tube, and wash it twice with 500 μL of 2-morpholinoethanesulfonic acid-Tween;

2-吗啉乙磺酸-Tween的配方为：2-吗啉乙磺酸10mM，pH 6.0，体积百分数0.05％的Tween-20；The formula of 2-morpholineethanesulfonic acid-Tween is: 2-morpholineethanesulfonic acid 10mM, pH 6.0, Tween-20 of 0.05% by volume;

②分别加入200μL新配制的浓度为5mg/mL的N-(3-二甲氨基丙基)-N'-乙基碳二亚胺盐酸盐水溶液和浓度为5mg/mL的N-羟基丁二酰亚胺水溶液，混匀，于37℃反应30min，活化磁性微球表面的羟基基团，磁分离去除未反应的N-(3-二甲氨基丙基)-N'-乙基碳二亚胺盐酸盐和N-羟基丁二酰亚胺；②Add 200μL of newly prepared N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride aqueous solution with a concentration of 5mg/mL and N-hydroxybutanediol with a concentration of 5mg/mL, respectively. Imide aqueous solution, mix well, react at 37°C for 30min, activate the hydroxyl groups on the surface of magnetic microspheres, remove unreacted N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide by magnetic separation Amine hydrochloride and N-hydroxysuccinimide;

③加入500μL 2-吗啉乙磺酸-Tween悬浮磁珠后转移至新的离心管，用500μL 2-吗啉乙磺酸清洗3次，磁分离后弃上清液；③ Add 500 μL of 2-morpholineethanesulfonic acid-Tween suspended magnetic beads and transfer to a new centrifuge tube, wash three times with 500 μL of 2-morpholineethanesulfonic acid, and discard the supernatant after magnetic separation;

④向步骤③中弃上清液后的磁珠中加入31.2μL抗金黄色葡萄球菌抗体，用2-吗啉乙磺酸调节体系至500μL，置于37℃颠倒混匀反应3h；④ Add 31.2 μL of anti-Staphylococcus aureus antibody to the magnetic beads after discarding the supernatant in step ③, adjust the system to 500 μL with 2-morpholineethanesulfonic acid, and place it at 37°C by inversion and mixing for 3 hours;

⑤磁分离去除未偶联的抗体，然后加入1ml的pH 7.4、含质量浓度为1％BSA的PBST缓冲液，重悬，于37℃颠倒混匀反应30min，封闭磁珠表面的自由基团；⑤ Remove unconjugated antibodies by magnetic separation, then add 1 ml of pH 7.4 PBST buffer containing 1% BSA by mass, resuspend, invert and mix at 37°C for 30 min to block the free radicals on the surface of the magnetic beads;

⑥加入500μL的pH 7.4、含质量浓度为1％BSA的PBST清洗4次，最后加入500μL的pH7.4、含质量浓度为0.02％的NaN₃及质量浓度为0.5％BSA的PBST缓冲液悬浮磁珠，即得菌体-免疫磁珠复合物，于4℃冰箱中保存备用。⑥ Add 500 μL of pH 7.4 and PBST containing 1% BSA to wash 4 times, and finally add 500 μL of pH 7.4, 0.02% NaN₃ and 0.5% BSA PBST buffer for magnetic suspension. Beads, that is, the bacteria-immunomagnetic bead complexes, which are stored in a refrigerator at 4°C for later use.

如上所述的方法在检测食品中金黄色葡萄球菌方面中的应用。The application of the method as described above in the detection of Staphylococcus aureus in food.

本发明取得的优点和积极效果为：The advantages and positive effects obtained by the present invention are:

1、本发明利用免疫富集技术联合MALDI-TOF MS快速检测食品中金黄色葡萄球菌，检测过程无需对待测样品进行复杂处理及目的菌的长时间增菌培养，而是先利用免疫富集技术对目的菌进行特异性富集后选择性增菌，大大缩短了细菌的培养时间。1. The present invention utilizes immune enrichment technology combined with MALDI-TOF MS to rapidly detect Staphylococcus aureus in food. The detection process does not require complex processing of the sample to be tested and long-term enrichment culture of the target bacteria, but first utilizes immune enrichment technology. Selective enrichment after specific enrichment of the target bacteria greatly shortens the bacterial culture time.

2、本发明利用免疫富集技术联合MALDI-TOF MS快速检测食品中金黄色葡萄球菌，检测过程所使用的MALDI-TOF MS生物质谱仪具有灵敏度好，结果准确等特点，相关数据库可自动分析数据并显示比对结果。利用MALDI-TOF MS对选择性增菌后的目的菌进行鉴定，鉴定过程快速，鉴定结果直观、准确，特异性好，检出限低，最低可检测至3CFU/mL。2. The present invention utilizes immune enrichment technology combined with MALDI-TOF MS to rapidly detect Staphylococcus aureus in food. The MALDI-TOF MS biological mass spectrometer used in the detection process has the characteristics of good sensitivity and accurate results, and the relevant database can automatically analyze data and display the comparison results. Using MALDI-TOF MS to identify the target bacteria after selective enrichment, the identification process is fast, the identification results are intuitive and accurate, the specificity is good, the detection limit is low, and the minimum detection limit is 3 CFU/mL.

3、本发明方法具有较高的特异性和准确性，检测方法的检测过程简单，快速，鉴定结果准确。据研究结果显示，当1mL样品中金黄色葡萄球菌的浓度为3CFU/mL时通过免疫富集后选择性增菌9h即可被MALDI-TOF MS快速准确鉴定。与传统鉴定方法相比大大缩短了鉴定所需时间，且该方法结果判读直观准确、精确度高、灵敏度高，重复性好，检出限低，最低可检测至3CFU/mL。3. The method of the present invention has high specificity and accuracy, the detection process of the detection method is simple and fast, and the identification result is accurate. According to the research results, when the concentration of Staphylococcus aureus in 1mL sample is 3CFU/mL, it can be quickly and accurately identified by MALDI-TOF MS after selective enrichment for 9h after immune enrichment. Compared with the traditional identification method, the time required for identification is greatly shortened, and the results of this method are intuitive and accurate, high precision, high sensitivity, good repeatability, low detection limit, and the lowest detection is 3CFU/mL.

4、本检测方法与传统方法相比大大缩短了目标菌的筛选培养时间，检测方法所涉及的免疫富集技术具有特异性好，选择性高等特点，可将目的菌从待检测样品中特异性富集；同时所使用的MALDI-TOF MS生物质谱仪具有灵敏度高，检测结果快速准确等特点，本发明将二者联合使用，用于检测食品中金黄色葡萄球菌，具有特异性好，选择性高，灵敏度强，检测结果快速准确。4. Compared with the traditional method, this detection method greatly shortens the screening and cultivation time of the target bacteria. The immune enrichment technology involved in the detection method has the characteristics of good specificity and high selectivity, which can specifically separate the target bacteria from the samples to be detected. At the same time, the MALDI-TOF MS biological mass spectrometer used has the characteristics of high sensitivity, fast and accurate detection results, etc. The invention uses the two in combination to detect Staphylococcus aureus in food, and has good specificity and selectivity. High sensitivity, fast and accurate detection results.

附图说明Description of drawings

图1为本发明中阴性对照的样本分析结果图；Fig. 1 is the sample analysis result diagram of negative control in the present invention;

图2为本发明中检测杂菌体系中金黄色葡萄球菌特异性结果图；其中，A为金黄色葡萄球菌标准菌株样本，B为沙门-金葡混合菌株样本；2 is a graph showing the specificity results of Staphylococcus aureus in the detection of miscellaneous bacteria system in the present invention; wherein, A is a standard strain sample of Staphylococcus aureus, and B is a Salmonella-Staphylococcus aureus mixed strain sample;

图3为本发明中牛奶样品中菌浓为61CFU/mL时细菌全细胞蛋白指纹结果图；其中，A为金黄色葡萄球菌标准菌株样本，B为牛奶样品中菌浓为61CFU/mL样本。3 is a graph showing the result of bacterial whole cell protein fingerprinting when the bacterial concentration in the milk sample is 61 CFU/mL in the present invention; wherein, A is a standard strain sample of Staphylococcus aureus, and B is a sample with a bacterial concentration of 61 CFU/mL in the milk sample.

具体实施方式Detailed ways

下面详细叙述本发明的实施例，需要说明的是，本实施例是叙述性的，不是限定性的，不能以此限定本发明的保护范围。The embodiments of the present invention will be described in detail below. It should be noted that the embodiments are descriptive, not restrictive, and cannot limit the protection scope of the present invention.

本发明中所使用的原料，如无特殊说明，均为常规的市售产品；本发明中所使用的方法，如无特殊说明，均为本领域的常规方法。The raw materials used in the present invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional methods in the art unless otherwise specified.

较优地，步骤如下：Preferably, the steps are as follows:

⑴样品的采集及处理；⑴ sample collection and processing;

①点样①Spotting

较优地，将选择性好、特异强的免疫富集技术与准确性好、灵敏度高得MALDI-TOFMS生物质谱联合用于检测食品中金黄色葡萄球菌。Preferably, the immuno-enrichment technology with good selectivity and specificity is combined with the MALDI-TOFMS biological mass spectrometry with good accuracy and high sensitivity to detect Staphylococcus aureus in food.

较优地，所述步骤⑵中特异性富集的条件为：Preferably, the conditions for specific enrichment in the step (2) are:

反应条件为：室温颠倒混匀30min；The reaction conditions are: inversion and mixing at room temperature for 30 min;

较优地，所述步骤⑴中根据《GB 4789.10食品安全国家标准食品微生物学检测金黄色葡萄球菌检验》中样品的采集与制备方法进行样品采集与处理。Preferably, in the step (1), sample collection and processing are carried out according to the sample collection and preparation method in "GB 4789.10 National Food Safety Standard for Food Microbiology Detection of Staphylococcus aureus".

较优地，所述步骤⑵的具体步骤如下：Preferably, the concrete steps of described step (2) are as follows:

具体地，相关制备、实施例及检测可以如下：Specifically, relevant preparation, embodiment and detection can be as follows:

实施例1：用于快速检测食品中金黄色葡萄球菌的免疫富集技术联合MALDI-TOFMS方法的操作步骤Example 1: Operation steps of the combined MALDI-TOFMS method for the rapid detection of Staphylococcus aureus in food

⑴磁珠活化及免疫磁珠制备(1) Activation of magnetic beads and preparation of immunomagnetic beads

①将磁珠混匀后取1mg置于1.5ml离心管，用500μL 2-吗啉乙磺酸-Tween(MEST10mM，pH 6.0，0.05％Tween-20)清洗2次；① After mixing the magnetic beads, take 1 mg and place it in a 1.5 ml centrifuge tube, and wash it twice with 500 μL of 2-morpholinoethanesulfonic acid-Tween (MEST 10 mM, pH 6.0, 0.05% Tween-20);

②分别加入200μL新配制的N-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)(5mg/mL)和羟基丁二酰亚胺(NHS)(5mg/mL)混匀，于37℃反应30min活化磁性微球表面的羟基基团，磁分离去除未反应的EDC和NHS；②Add 200 μL of freshly prepared N-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (5 mg/mL) and hydroxysuccinimide (NHS) (5 mg respectively) /mL), mix well, react at 37 °C for 30 min to activate the hydroxyl groups on the surface of the magnetic microspheres, and remove unreacted EDC and NHS by magnetic separation;

③加入500μL MEST悬浮磁珠后转移至新的离心管，用500μL MEST清洗3次，磁分离后弃上清液；③ Add 500 μL MEST suspended magnetic beads and transfer to a new centrifuge tube, wash 3 times with 500 μL MEST, and discard the supernatant after magnetic separation;

④向③中加入31.2μL抗金黄色葡萄球菌抗体，用2-吗啉乙磺酸(MES)调节体系至500μL，置于37℃颠倒混匀反应3h；④ Add 31.2 μL of anti-Staphylococcus aureus antibody to ③, adjust the system to 500 μL with 2-morpholineethanesulfonic acid (MES), and place it at 37°C for 3 hours by inverting and mixing;

⑤磁分离去除未偶联的抗体，然后加入1ml PBST缓冲液(pH 7.4，含1％BSA)重悬，于37℃颠倒混匀反应30min，封闭磁珠表面的自由基团；⑤ Remove unconjugated antibodies by magnetic separation, then add 1 ml of PBST buffer (pH 7.4, containing 1% BSA) to resuspend, invert and mix at 37°C for 30 minutes to block the free radicals on the surface of the magnetic beads;

⑥加入500μL PBST清洗4次，最后加入500μL PBST缓冲液(pH 7.4，含0.02％NaN₃及0.5％BSA)悬浮磁珠，4℃冰箱中保存备用。⑥ Add 500 μL PBST to wash 4 times, and finally add 500 μL PBST buffer (pH 7.4, containing 0.02% NaN₃ and 0.5% BSA) to suspend the magnetic beads, and store in a refrigerator at 4°C for later use.

⑵目的菌富集(2) Enrichment of target bacteria

取600μL制备好的免疫磁珠磁分离后与1mL样液于室温反应30min后磁分离，弃上清。Take 600 μL of prepared immunomagnetic beads for magnetic separation and react with 1 mL of sample solution at room temperature for 30 min, then magnetically separate, and discard the supernatant.

⑶目的菌选择性增菌(3) Selective enrichment of target bacteria

将(2)中的菌体-免疫磁珠复合物接种于金黄色葡萄球菌选择性增菌培养基增菌12-15h,对免疫富集所得的细菌进行选择性增菌；Inoculate the thalline-immunomagnetic bead complex in (2) in a Staphylococcus aureus selective enrichment medium for enrichment for 12-15h, and selectively enrich the bacteria obtained by immune enrichment;

⑷MALDI-TOF MS检测⑷MALDI-TOF MS detection

①点样①Spotting

将(3)中增菌样品经甲酸/乙醇提取法处理后取1μL上清液加至点样靶，每种处理方法得到的样品平行点4个孔，待样液滴晾干后，覆盖1μL饱和基质CHCA，干燥后进行MALDI-TOF MS鉴定；After the enrichment samples in (3) were treated with formic acid/ethanol extraction, 1 μL of the supernatant was added to the spotting target. The samples obtained by each treatment method were placed in 4 wells in parallel. After the sample droplets were dried, cover with 1 μL. Saturated matrix CHCA, dried and identified by MALDI-TOF MS;

用MALDI-TOF MS对样品进行数据采集；Data collection of samples was performed with MALDI-TOF MS;

其中，每次检测均设立阴性对照，阴性对照为未与待检样品反应的免疫磁珠。如图1所示，未与待检样品反应的免疫磁珠样本无检测结果。Among them, a negative control is set up for each test, and the negative control is the immunomagnetic beads that have not reacted with the sample to be tested. As shown in Figure 1, the immunomagnetic bead sample that did not react with the sample to be tested has no detection result.

实施例2：用于快速检测食品中金黄色葡萄球菌的免疫富集技术联合MALDI-TOFMS方法的建立Example 2: Establishment of an immunoenrichment technique combined with MALDI-TOFMS method for rapid detection of Staphylococcus aureus in food

本发明用于快速检测食品中金黄色葡萄球菌的方法是一种基于免疫富集技术和MALDI-TOF MS生物质谱分析的方法，其中免疫富集的反应体系总体积为1mL，包括：600μL250μg/mL磁分离后的免疫磁珠，1mL待检食品样液；The method for rapidly detecting Staphylococcus aureus in food is a method based on immune enrichment technology and MALDI-TOF MS biological mass spectrometry analysis, wherein the total volume of the immune enrichment reaction system is 1 mL, including: 600 μL, 250 μg/mL Magnetically separated immunomagnetic beads, 1mL of food sample liquid to be tested;

反应条件为：室温颠倒混匀30min。The reaction conditions were as follows: inversion and mixing at room temperature for 30 min.

实施例3：特异性分析Example 3: Specificity Analysis

⑴检测杂菌体系中金黄色葡萄球菌的特异性(1) The specificity of detecting Staphylococcus aureus in the miscellaneous bacteria system

本发明用于快速检测食品中金黄色葡萄球菌的方法的杂菌体系中金黄色葡萄球菌检测特异性分析是将金黄色葡萄球菌与沙门氏菌分别制备菌悬液，之后将金黄色葡萄球菌及沙门氏菌以1:1的比例混匀后在最佳反应条件下与免疫磁珠反应后磁分离，取100μL上清液分别接种于金黄色葡萄球菌鉴别培养基及沙门氏菌显示培养基，37℃恒温培养12-15h后观察培养基现象。并将菌体-免疫磁珠复合物据上诉实验方法进行MALDI-TOF MS鉴定并分析。其中，沙门氏菌的菌落在沙门氏显色培养基中为淡紫色，金黄色葡萄球菌不显色；金黄色葡萄球菌在血平板培养基中有溶血现象，沙门氏菌无溶血现象。The method for rapidly detecting Staphylococcus aureus in food according to the present invention is used for the detection specificity analysis of Staphylococcus aureus in the mixed bacteria system, which is to prepare bacterial suspensions of Staphylococcus aureus and Salmonella respectively, and then separate Staphylococcus aureus and Salmonella as After mixing at a ratio of 1:1, it was reacted with immunomagnetic beads under the optimal reaction conditions and then magnetically separated. 100 μL of the supernatant was inoculated into Staphylococcus aureus identification medium and Salmonella display medium, respectively, and incubated at 37°C for 12- The phenomenon of culture medium was observed after 15h. The bacteria-immunomagnetic bead complexes were identified and analyzed by MALDI-TOF MS according to the above experimental method. Among them, the colony of Salmonella was lavender in Salmonella chromogenic medium, and Staphylococcus aureus did not develop color; Staphylococcus aureus had hemolysis in blood plate medium, but Salmonella had no hemolysis.

结果如图2所示，杂菌反应体系所得的样品指纹图谱峰值强度有所差别，但特征峰的分布大致相同，且杂菌体系的样品指纹数据与数据库比对的鉴定相符率均>90％，结果显示检测样品为金黄色葡萄球菌，即免疫富集联合MALDI-TOF MS对于检测杂菌体系中的金黄色葡萄球菌具有特异性。The results are shown in Figure 2. The peak intensities of the fingerprints of the samples obtained from the mixed bacteria reaction system are different, but the distribution of characteristic peaks is roughly the same, and the identification coincidence rates of the sample fingerprint data of the mixed bacteria system and the database are all >90%. , the results show that the detected sample is Staphylococcus aureus, that is, immune enrichment combined with MALDI-TOF MS is specific for detecting Staphylococcus aureus in the miscellaneous bacteria system.

实施例4：实际样品检测Example 4: Actual sample detection

本发明用于快速检测食品中金黄色葡萄球菌的方法的实际样品检测，是向1mL牛奶样品中添加已知浓度为61CFU/mL的标准菌液，与IMB在最优条件下反应后据上述方法进行MALDI-TOF MS鉴定并分析。The actual sample detection of the method for rapid detection of Staphylococcus aureus in food according to the present invention is to add a standard bacterial solution with a known concentration of 61 CFU/mL to 1 mL of milk sample, react with IMB under optimal conditions, and follow the method described above. MALDI-TOF MS identification and analysis were performed.

如图3所示，当牛奶样品中细菌浓度为61CFU/mL时，经免疫富集后选择性增菌12-15h即可被MALDI-TOF MS准确鉴定，且样品指纹数据与数据库比对的鉴定相符率>85％即本发明免疫富集联合MALDI-TOF MS可用于牛奶样品中金黄色葡萄球菌的检测。As shown in Figure 3, when the bacterial concentration in the milk sample is 61 CFU/mL, the selective enrichment after immune enrichment can be accurately identified by MALDI-TOF MS for 12-15 hours, and the identification of the sample fingerprint data is compared with the database. If the coincidence rate is greater than 85%, the immune enrichment combined with MALDI-TOF MS of the present invention can be used for the detection of Staphylococcus aureus in milk samples.

本发明是一种基于免疫富集技术和MALDI-TOF MS生物质谱鉴定的方法，其特异性强，灵敏度高，重复性好，结果准确，同时具有高效性。The invention is a method based on immune enrichment technology and MALDI-TOF MS biological mass spectrometry identification, which has strong specificity, high sensitivity, good repeatability, accurate results and high efficiency.

尽管为说明目的公开了本发明的实施例，但是本领域的技术人员可以理解：在不脱离本发明及所附权利要求的精神和范围内，各种替换、变化和修改都是可能的，因此，本发明的范围不局限于实施例和附图所公开的内容。Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, therefore , the scope of the present invention is not limited to the contents disclosed in the embodiments and drawings.