CN111366726A - Method for detecting salmonella in food by combining immune enrichment with MALDI-TOF MS and application - Google Patents

Abstract

The invention relates to a method for quickly and accurately detecting salmonella in food by combining an immune enrichment technology and MALDI-TOF MS (matrix-assisted laser Desorption-time of flight mass spectrometry). Compared with the traditional method, the detection method greatly shortens the screening and culturing time of the target bacteria, and the immune enrichment technology related to the detection method has the characteristics of good specificity, high selectivity and the like, and can specifically enrich the target bacteria from a sample to be detected; meanwhile, the MALDI-TOF MS biological mass spectrometer has the characteristics of high sensitivity, quick and accurate detection result and the like, the MALDI-TOF MS biological mass spectrometer and the TOF MS biological mass spectrometer are combined for use, the detection method is used for detecting the salmonella in the food, and has the advantages of good specificity, high selectivity, strong sensitivity and quick and accurate detection result, and when the bacteria concentration in a sample is 3CFU/mL, the MALDI-TOF MS can quickly and accurately identify the bacteria concentration in the sample after enrichment culture for 9 hours.

Description

Translated fromChinese

一种免疫富集联合MALDI-TOF MS检测食品中沙门氏菌的方法及应用A method for detection of Salmonella in food by immune enrichment combined with MALDI-TOF MSand 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检测食品中沙门氏菌的方法及应用。The invention belongs to the field of biotechnology, in particular to a method and application for detecting Salmonella in food by immune enrichment combined with MALDI-TOF MS.

背景技术Background technique

沙门氏菌(Salmonella)是自然界中引起食源性疾病的致病菌之一，在全球范围内常常引起细菌性食物中毒。据2018年世界卫生组织统计，每年约有1/10的人患食源性疾病，约有30000人丧失生命，而沙门氏菌是致病因子之一；同时，据近几年国内外对于食源性疾病的监控显示，沙门氏菌仍然是食源性疾病的主要微生物性致病菌之一，其中，欧盟国家2017年的沙门氏菌致病死亡率高达33.3％，2015年，中国沙门氏菌致病死亡率为0.72％。因此，急需对食品中的沙门氏菌进行有效监测和准确鉴定。Salmonella is one of the pathogenic bacteria that cause food-borne diseases in nature, and it often causes bacterial food poisoning worldwide. According to the statistics of the World Health Organization in 2018, about 1/10 of people suffer from food-borne diseases every year, and about 30,000 people lose their lives, and Salmonella is one of the pathogenic factors. Disease monitoring shows that Salmonella is still one of the main microbial pathogens of foodborne diseases. Among them, the mortality rate of Salmonella in EU countries in 2017 was as high as 33.3%, and in 2015, the mortality rate of Salmonella in China was 0.72%. . Therefore, there is an urgent need for effective monitoring and accurate identification of Salmonella in food.

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，因此可用于沙门氏菌的全细胞蛋白指纹图谱分析，进而对沙门氏菌进行鉴定。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. analysis to identify Salmonella.

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检测食品中沙门氏菌的方法及应用。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 Salmonella 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检测食品中沙门氏菌的方法，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，对食品中沙门氏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、准确鉴定食品中的沙门氏菌。A method for detecting Salmonella 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-assisted laser desorption ionization with high sensitivity and accuracy Time-of-flight mass spectrometry (MALDI-TOF-MS) is used in combination to specifically enrich Salmonella in food and perform whole-cell protein fingerprint analysis, which can efficiently, quickly and accurately identify Salmonella 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：使用抗沙门氏菌抗体制备免疫磁珠，对沙门氏菌进行特异性富集，制得菌体-免疫磁珠复合物；(2) Specific enrichment of target bacteria: use anti-Salmonella antibody to prepare immunomagnetic beads, carry out specific enrichment for Salmonella, and obtain bacterial-immune magnetic bead complexes;

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沙门氏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 bacteria-immune magnetic bead complex in step (2) in the Salmonella selective enrichment medium for enrichment for 12-15 hours, and selectively enrich the bacteria obtained by immune enrichment;

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，显示鉴定结果为沙门氏菌为阳性，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 Salmonella, 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沙门氏菌。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 for the detection of Salmonella 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:

免疫富集沙门氏菌的检测体系为1mL，具体为：The detection system for immune enrichment of Salmonella 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30min；The reaction conditions are: room temperature 30min;

而且，所述步骤⑴中根据《GB 4789.4食品安全国家标准食品微生物学检测沙门氏菌检验》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 4789.4 National Food Safety Standard Food Microbiology Detection Salmonella 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抗沙门氏菌抗体，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-Salmonella antibody to the magnetic beads after discarding the supernatant instep ③, adjust the system to 500 μL with 2-morpholine ethanesulfonic acid, and place it at 37°C for 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沙门氏菌方面中的应用。Application of the method as described above in the detection of Salmonella 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快速检测食品中沙门氏菌，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 Salmonella 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 uses the immune enrichment technology to detect the target bacteria. Selective enrichment after specific enrichment greatly shortens the culture time of bacteria.

2、本发明利用免疫富集技术联合MALDI-TOF MS快速检测食品中沙门氏菌，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 uses immune enrichment technology combined with MALDI-TOF MS to rapidly detect Salmonella 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 the data and display the ratio. to the 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样品中沙门氏菌的浓度为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 Salmonella 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，用于检测食品中沙门氏菌，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 Salmonella in food, and has good specificity, high selectivity and sensitivity. Strong, 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沙门氏菌特异性结果图；其中，A为标准菌株样本，B为沙门-金葡混合菌株样本，C为沙门-大肠混合菌株样本；Fig. 2 is a graph showing the specificity result of Salmonella in the detection of miscellaneous bacteria system in the present invention; wherein, A is a standard strain sample, B is a Salmonella-S. aureus mixed strain sample, and C is a Salmonella-large intestine mixed strain sample;

图3为本发明中检测不同沙门氏菌特异性结果图；其中，A为捕获率结果图，B为样品全细胞蛋白指纹图，其中，S.T为鼠伤寒沙门氏(Salmonella typhimurium)、S.E肠炎沙门氏菌(Salmonella enterica)、S.H海德堡沙门氏菌(Salmonella Heidelberg)；Fig. 3 is a graph showing the specificity results of detecting different Salmonella in the present invention; wherein, A is a result graph of capture rate, B is a sample whole cell protein fingerprint graph, wherein, S.T is Salmonella typhimurium, S.E Salmonella enteritidis (Salmonella typhimurium) enterica), S.H Heidelberg (Salmonella Heidelberg);

图4为本发明检测不同食品样品的细菌全细胞蛋白指纹图结果图；Fig. 4 is the result diagram of bacterial whole cell protein fingerprint of detecting different food samples according to the present invention;

图5为本发明中食品样品中菌浓为3CFU/mL的样本分析结果图；其中，A为增菌6h样本，B为增菌9h样本样本，C为增菌12h样本，D为增菌15h样本；Fig. 5 is a graph showing the analysis result of a sample with a bacterial concentration of 3 CFU/mL in a food sample according to the present invention; wherein, A is a 6-h enrichment sample, B is a 9-h enriched sample, C is a 12-h enriched sample, and D is a 15-h enriched sample sample;

图6为本发明中食品样品中菌浓为48CFU/mL的样本分析结果图；Fig. 6 is the sample analysis result diagram that the bacterial concentration in the food sample of the present invention is 48CFU/mL;

图7为本发明中食品样品中菌浓为1×10²CFU/mL的样本分析结果图；FIG. 7 is a graph showing the analysis result of a sample with a bacterial concentration of 1×10² CFU/mL in a food sample according to the present invention;

图8为本发明中食品样品中菌浓为1×10³CFU/mL的样本分析结果图。FIG. 8 is a graph showing the analysis result of a sample with a bacterial concentration of 1×10³ CFU/mL in a food sample according to the present invention.

表1为本发明中检测不同食品样品的捕获率结果图。Table 1 is the result graph of the capture rate of different food samples detected in the present invention.

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.

一种免疫富集联合MALDI-TOF MS检测食品中沙门氏菌的方法，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，对食品中沙门氏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、准确鉴定食品中的沙门氏菌。A method for detecting Salmonella 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-assisted laser desorption ionization with high sensitivity and accuracy Time-of-flight mass spectrometry (MALDI-TOF-MS) is used in combination to specifically enrich Salmonella in food and perform whole-cell protein fingerprint analysis, which can efficiently, quickly and accurately identify Salmonella in food.

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;

⑵目的菌的特异性富集：使用抗沙门氏菌抗体制备免疫磁珠，对沙门氏菌进行特异性富集，制得菌体-免疫磁珠复合物；(2) Specific enrichment of target bacteria: use anti-Salmonella antibody to prepare immunomagnetic beads, carry out specific enrichment for Salmonella, and obtain bacterial-immune magnetic bead complexes;

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沙门氏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 bacteria-immune magnetic bead complex in step (2) in the Salmonella selective enrichment medium for enrichment for 12-15 hours, and selectively enrich the bacteria obtained by immune enrichment;

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，显示鉴定结果为沙门氏菌为阳性，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 Salmonella, 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.

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沙门氏菌。Preferably, the immuno-enrichment technology with good selectivity and specificity is combined with the MALDI-TOFMS biological mass spectrometer with good accuracy and high sensitivity to detect Salmonella 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:

免疫富集沙门氏菌的检测体系为1mL，具体为：The detection system for immune enrichment of Salmonella 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30min；The reaction conditions are: room temperature 30min;

较优地，所述步骤⑴中根据《GB 4789.4食品安全国家标准食品微生物学检测沙门氏菌检验》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 4789.4 National Food Safety Standard for Food Microbiological Detection Salmonella Inspection".

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:

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抗沙门氏菌抗体，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-Salmonella antibody to the magnetic beads after discarding the supernatant instep ③, adjust the system to 500 μL with 2-morpholine ethanesulfonic acid, and place it at 37°C for 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沙门氏菌方面中的应用。Application of the method as described above in the detection of Salmonella in food.

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沙门氏菌的免疫富集技术联合MALDI-TOF MS方法的操作步骤Example 1: Operation steps of the combined MALDI-TOF MS method for the rapid detection of Salmonella 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抗沙门氏菌抗体，用2-吗啉乙磺酸(MES)调节体系至500μL，置于37℃颠倒混匀反应3h；④Add 31.2 μL of anti-Salmonella antibody to ③, adjust the system to 500 μL with 2-morpholineethanesulfonic acid (MES), and place it at 37°C to invert and mix for 3 hours;

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沙门氏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 bacterial body-immunomagnetic bead complex in (2) in the Salmonella selective enrichment medium for enrichment for 12-15 hours, 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;

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，显示鉴定结果为沙门氏菌为阳性，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 Salmonella, 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。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沙门氏菌的免疫富集技术联合MALDI-TOF MS方法的建立Example 2: Establishment of immuno-enrichment technology combined with MALDI-TOF MS method for rapid detection of Salmonella in food

本发明用于快速检测食品中沙门氏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：600μL 250μg/mL磁分离后的免疫磁珠，1mL待检食品样液；The method for rapidly detecting Salmonella 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: 600μL 250 μg/mL magnetic separation After the immunomagnetic beads, 1mL of the food sample liquid to be tested;

反应条件为：室温30min。The reaction conditions were: room temperature for 30 min.

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

⑴检测杂菌体系中沙门氏菌的特异性⑴Detect the specificity of Salmonella in miscellaneous bacteria system

本发明用于快速检测食品中沙门氏菌的方法的杂菌体系中沙门氏菌检测特异性分析是将金黄色葡萄球菌、大肠杆菌与沙门氏菌分别按上述实验方法制备菌悬液，之后将金黄色葡萄球菌及大肠杆菌与沙门氏菌分别以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上清液涂平板，据公式1计算IMB捕获率并取100μL上清液分别接种于沙门氏菌显色培养基及金黄色葡萄球菌鉴别培养基，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-TOFMS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，大肠杆菌为蓝色；金黄色葡萄球菌在血平板培养基中有溶血现象，其余细菌无溶血现象。The detection specificity analysis of Salmonella in the miscellaneous bacteria system of the method for rapidly detecting Salmonella in food according to the present invention is to prepare bacterial suspensions of Staphylococcus aureus, Escherichia coli and Salmonella respectively according to the above experimental method, and then separate Staphylococcus aureus and Escherichia coli Bacillus and Salmonella were mixed at a ratio of 1:1 and then reacted with immunomagnetic beads under the optimal reaction conditions, and then magnetically separated. Take 100 μL of the supernatant and spread it on the plate. It was inoculated into Salmonella chromogenic medium and Staphylococcus aureus identification medium, and cultured at 37°C for 12-15 hours to observe the phenomenon of the medium. The bacteria-immunomagnetic bead complexes were identified and analyzed by MALDI-TOFMS according to the above experimental method. Among them, Salmonella colonies were lavender in Salmonella chromogenic medium, Escherichia coli was blue; Staphylococcus aureus had hemolysis in blood plate medium, and other bacteria had no hemolysis.

C₀:原菌液菌落数(CFU/mL)；C₀ : the colony count of the original bacterial solution (CFU/mL);

C₁:免疫磁珠捕获后的上清液菌落数(CFU/mL)C₁ : The number of colonies in the supernatant after immunomagnetic bead capture (CFU/mL)

结果如图2所示，不同比例的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，且不同比例杂菌体系的样品指纹数据与数据库比对的鉴定相符率均>81％，结果显示检测样品为沙门氏菌，即免疫富集联合MALDI-TOFMS对于检测杂菌体系中的沙门氏菌具有特异性。The results are shown in Figure 2. The peak intensities of the fingerprints of the samples obtained by different proportions of the mixed bacteria reaction system are different, but the distribution of characteristic peaks is roughly the same, and the fingerprint data of the samples of different proportions of the mixed bacteria system is consistent with the identification of the database comparison. The rates were all >81%, and the results showed that the detected samples were Salmonella, that is, immune enrichment combined with MALDI-TOFMS was specific for detecting Salmonella in the miscellaneous bacteria system.

⑵检测不同沙门氏菌的特异性(2) The specificity of detecting different Salmonella

本发明用于快速检测食品中沙门氏菌的方法的不同沙门氏菌检测特异性是将1mL菌浓为1×10²～1×10⁴CFU/mL的肠炎沙门氏菌(Salmonella enterica,S.E)、鼠伤寒沙门氏菌(Salmonella typhimurium，S.T)及海德堡沙门氏菌(Salmonella Heidelberg,S.H)分别与免疫磁珠在最佳反应条件下反应后据(1)中所述方法计算IMB捕获率，将菌体-免疫磁珠复合物过夜培养后取1mL菌液磁分离，经甲酸-乙醇法处理后进行MALDI-TOF MS鉴定并分析。The different Salmonella detection specificity of the method for rapidly detecting Salmonella in food of the present invention is that 1 mL of bacteria concentration is 1×10² to 1×10⁴ CFU/mL of Salmonella enterica (Salmonella enterica, SE), Salmonella typhimurium (Salmonella enterica) typhimurium, ST) and Salmonella Heidelberg (Salmonella Heidelberg, SH) were reacted with immunomagnetic beads under optimal reaction conditions, respectively, and the IMB capture rate was calculated according to the method described in (1). Then, 1 mL of bacterial liquid was magnetically separated, treated with formic acid-ethanol method, and identified and analyzed by MALDI-TOF MS.

结果如图3(A)所示，IMB对不同沙门氏菌的捕获率均>80％，且由图3(B)可知，3种不同沙门氏菌的指纹图谱峰值强度有所差别，特征峰的分布也有所不同，但是所获取的图谱数据与数据库比对均能获得正确的鉴定结果，与数据库对比结果相符率均>80％，即检测样品为沙门氏菌。所以本研究鉴定方法也可用于S.T、S.E菌株和S.H菌株的特异性富集与鉴定。The results are shown in Figure 3(A), the capture rate of IMB for different Salmonella is >80%, and from Figure 3(B), it can be seen that the peak intensities of the fingerprints of the three different Salmonella are different, and the distribution of characteristic peaks is also different. However, the obtained map data can be compared with the database to obtain correct identification results, and the coincidence rates with the database comparison results are all >80%, that is, the detected sample is Salmonella. Therefore, the identification method in this study can also be used for the specific enrichment and identification of S.T, S.E and S.H strains.

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沙门氏菌的方法的实际样品检测是将牛奶、蛋黄及蛋清进行10倍稀释后分别添加已知浓度分别为1×10²、1×10³CFU/mL的标准菌液，与IMB在最优条件下反应后据实施例3中的方法计算捕获率并进行MALDI-TOF MS鉴定并分析。The actual sample detection of the method for rapid detection of Salmonella in food of the present invention is to dilute milk, egg yolk and egg white by 10 times and then add standard bacterial liquids with known concentrations of 1×10² and 1×10³ CFU/mL respectively. , after reacting with IMB under optimal conditions, the capture rate was calculated according to the method in Example 3 and identified and analyzed by MALDI-TOF MS.

如表1所示，向牛奶、蛋清及蛋黄样品处理液中分别添加已知浓度的菌液，通过加标回收实验得回收率在88％～100％，与实际添加量基本相符；如图4所示，不同实际样品的细菌指纹图谱峰值强度有所差别，但是特征峰的分布基本相同，且指纹图谱数据与数据库比对相符率分别均>85％，即检测样品为沙门氏菌，说明该检测方法可用在牛奶、蛋清和蛋黄样品的检测中。As shown in Table 1, bacterial solutions of known concentrations were added to the milk, egg white and egg yolk sample treatment solutions, respectively, and the recovery rate was 88% to 100% through the standard addition recovery experiment, which was basically consistent with the actual addition amount; Figure 4 As shown, the peak intensity of bacterial fingerprints of different actual samples is different, but the distribution of characteristic peaks is basically the same, and the coincidence rates of fingerprint data and database comparison are all >85%, that is, the detection sample is Salmonella, indicating that this detection method is used. Can be used in the detection of milk, egg white and egg yolk samples.

表1不同实际样品的捕获率Table 1 Capture rates of different real samples

实施例5：检出限及增菌时间探究Example 5: Detection limit and enrichment time exploration

本发明用于快速检测食品中沙门氏菌的方法的检出限及增菌时间探究是用无菌生理盐水将沙门氏菌的浓度调整至低于10CFU/mL，接种于牛奶样品处理液中，与免疫磁珠在最优条件下反应后磁分离，将细菌-免疫磁珠复合物接种于沙门氏菌选择性增菌培养基，37℃震荡培养，设置4个不同增菌时间的实验组，分别为6、9、12、15h。取不同增菌时间的菌液据实施例1中步骤(4)所述方法进行MALDI-TOF MS鉴定并分析。The detection limit and enrichment time of the method for rapid detection of Salmonella in food according to the present invention is to adjust the concentration of Salmonella to less than 10 CFU/mL with sterile saline, inoculate it into the milk sample treatment solution, and combine with immunomagnetic beads Magnetic separation after reaction under optimal conditions, the bacteria-immunomagnetic bead complex was inoculated into Salmonella selective enrichment medium, and cultured with shaking at 37°C. Four experimental groups with different enrichment time were set up, namely 6, 9, and 4. 12, 15h. MALDI-TOF MS identification and analysis were carried out according to the method described in step (4) in Example 1 to take the bacterial liquids with different enrichment time.

结果如图5至图8所示。由图5可知，当牛奶样品中细菌浓度为3CFU/mL时，经免疫富集后选择性增菌9h即可被MALDI-TOF MS准确鉴定，如图6-8所示，随着细菌浓度的增加，目的菌经免疫富集后选择性增菌9h可被MALDI-TOF MS准确鉴定，与《GB 4789.4食品安全国家标准食品微生物学检测沙门氏菌检验》相比，本发明的沙门氏菌检测方法耗时约缩短了35h。The results are shown in Figures 5 to 8. It can be seen from Figure 5 that when the bacterial concentration in the milk sample is 3 CFU/mL, the selective enrichment for 9 hours after immune enrichment can be accurately identified by MALDI-TOF MS, as shown in Figure 6-8, with the increase of bacterial concentration. Increase, the target bacteria can be accurately identified by MALDI-TOF MS after selective enrichment for 9 hours after immune enrichment. Compared with the "GB 4789.4 Food Safety National Standard for Food Microbiological Detection Salmonella Inspection", the Salmonella detection method of the present invention takes about Shortened by 35h.

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.

Claims (7)

Translated fromChinese

1.一种免疫富集联合MALDI-TOF MS检测食品中沙门氏菌的方法，其特征在于：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，对食品中沙门氏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、准确鉴定食品中的沙门氏菌。1. a method for immune enrichment combined with MALDI-TOF MS to detect Salmonella in food, it is characterized in that: described method will selectivity, specificity strong immune enrichment technology and sensitivity high, accurate biological mass spectrometer - The combined use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) can specifically enrich Salmonella in food and perform whole-cell protein fingerprint analysis, which can efficiently, quickly and accurately identify Salmonella in food.2.根据权利要求1所述的免疫富集联合MALDI-TOF MS检测食品中沙门氏菌的方法，其特征在于：步骤如下：2. the method for detection of Salmonella in food by immune enrichment combined with MALDI-TOF MS according to claim 1, is characterized in that: step is 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：使用抗沙门氏菌抗体制备免疫磁珠，对沙门氏菌进行特异性富集，制得菌体-免疫磁珠复合物；(2) Specific enrichment of target bacteria: use anti-Salmonella antibody to prepare immunomagnetic beads, carry out specific enrichment for Salmonella, and obtain bacterial-immune magnetic bead complexes;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沙门氏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 bacteria-immune magnetic bead complex in step (2) in the Salmonella selective enrichment medium for enrichment for 12-15 hours, and selectively enrich the bacteria obtained by immune enrichment;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 by laser 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，显示鉴定结果为沙门氏菌为阳性，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 Salmonella, 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.3.根据权利要求2所述的免疫富集联合MALDI-TOF MS检测食品中沙门氏菌的方法，其特征在于：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沙门氏菌。3. the method for detection of Salmonella in food by immune enrichment combined with MALDI-TOF MS according to claim 2, it is characterized in that: the immune enrichment technique with good selectivity, strong specificity and good accuracy, high sensitivity obtain MALDI- Combined with TOF MS biological mass spectrometry for detection of Salmonella in food.4.根据权利要求2所述的免疫富集联合MALDI-TOF MS检测食品中沙门氏菌的方法，其特征在于：所述步骤⑵中特异性富集的条件为：4. immune enrichment according to claim 2 is combined with MALDI-TOF MS to detect the method for Salmonella in food, it is characterized in that: the condition of specific enrichment in described step (2) is:免疫富集沙门氏菌的检测体系为1mL，具体为：The detection system for immune enrichment of Salmonella 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30min。The reaction conditions were: room temperature for 30 min.5.根据权利要求2所述的免疫富集联合MALDI-TOF MS检测食品中沙门氏菌的方法，其特征在于：所述步骤⑴中根据《GB 4789.4食品安全国家标准食品微生物学检测沙门氏菌检验》中样品的采集与制备方法进行样品采集与处理。5. immune enrichment according to claim 2 is combined with MALDI-TOF MS to detect the method for Salmonella in food, it is characterized in that: in described step (1), according to " GB 4789.4 national food safety standard food microbiology detects Salmonella inspection " in sample collection and preparation methods for sample collection and processing.6.根据权利要求2所述的免疫富集联合MALDI-TOF MS检测食品中沙门氏菌的方法，其特征在于：所述步骤⑵的具体步骤如下：6. immune enrichment according to claim 2 is combined with MALDI-TOF MS to detect the method for Salmonella in food, it is characterized in that: 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抗沙门氏菌抗体，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-Salmonella antibody to the magnetic beads after discarding the supernatant in step ③, adjust the system to 500 μL with 2-morpholine ethanesulfonic acid, and place it at 37°C for 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.7.如权利要求1至6任一项所述的方法在检测食品中沙门氏菌方面中的应用。7. The application of the method according to any one of claims 1 to 6 in detecting Salmonella in food.

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