WO2023011246A1 - 新型混合基质在maldi-ms细菌鉴定中的应用 - Google Patents

新型混合基质在maldi-ms细菌鉴定中的应用 Download PDF

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WO2023011246A1
WO2023011246A1 PCT/CN2022/107840 CN2022107840W WO2023011246A1 WO 2023011246 A1 WO2023011246 A1 WO 2023011246A1 CN 2022107840 W CN2022107840 W CN 2022107840W WO 2023011246 A1 WO2023011246 A1 WO 2023011246A1
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matrix
chca
bacteria
maldi
mixed matrix
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国新华
赵楠
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吉林大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G01N27/64—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using wave or particle radiation to ionise a gas, e.g. in an ionisation chamber
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention belongs to the technical field of bacterial identification, and in particular relates to the application of a novel mixed matrix in MALDI-MS bacterial identification.
  • Matrix-assisted laser desorption ionization mass spectrometry has been widely used in proteomics, metabolomics and microbial analysis in clinic. It is a fast, sensitive, high-throughput analytical method. Its principle is to absorb laser energy through a special organic small molecule compound as a matrix, carry out protonization transfer, and then ionize the analyte, so as to achieve the purpose of detection.
  • the existing MALDI-MS matrix used to detect bacteria is ⁇ -cyano-4-hydroxycinnamic acid (CHCA), but because CHCA has a good detection effect on hydrophilic proteins, it has certain limitations on the detection of hydrophobic proteins , so the fingerprint spectrum obtained when using traditional matrices to detect bacteria is not comprehensive enough, so it is difficult to distinguish similar bacteria from existing MALDI-MS detection bacteria.
  • CHCA ⁇ -cyano-4-hydroxycinnamic acid
  • the purpose of the embodiments of the present invention is to provide the application of the new mixed matrix in the identification of bacteria by MALDI-MS, aiming to solve the problem that the fingerprint spectrum obtained when the traditional matrix detects bacteria is not comprehensive enough and cannot distinguish similar bacteria.
  • Step 1 Cultivate the bacteria to be tested on a solid medium for 24 hours
  • Step 2 Dissolve CHCA and CHCA-C3 respectively in 50% acetonitrile containing 2.5% trifluoroacetic acid solution to prepare CHCA and CHCA-C3 matrix solutions, and store them in a refrigerator at 4°C for future use;
  • Step 3 Take CHCA matrix solution and CHCA-C3 matrix solution and mix them uniformly at a volume ratio of 1:2;
  • Step 4 Take the bacteria cultured in Step 1 and spread them evenly on the matching target plate of MALDI-MS;
  • Step 5 After the bacteria on the target plate in step 4 are air-dried, spot the mixed matrix solution obtained in step 3 on the dried bacteria;
  • Step 6 The bacteria to which the mixed matrix solution is added dropwise in step 4 are naturally air-dried at room temperature;
  • Step 7 After the solvent in step 6 volatilizes and crystallizes, carry out MALDI analysis directly.
  • the concentration of the substrate solution in the second step is 10-15 mg/ml.
  • the volume of the mixed matrix in step 4 is 0.5-1 ⁇ L.
  • the data in step 7 is collected in the linear positive ion mode to obtain the mass spectrum of the bacteria to be tested.
  • the model of the matrix-assisted laser desorption ionization time-of-flight mass spectrometer used in the step seven is Autoflex speed TOF/TOF, and the laser wavelength is a Nd:YAG laser with a wavelength of 355nm.
  • the acceleration voltage of the matrix-assisted laser desorption ionization time-of-flight mass spectrometer is 20.000kv
  • the delayed extraction voltage is 18.000kv
  • the delayed extraction time is 150ns.
  • the reflector voltage is 20.000kv
  • the lens voltage is 6.000kv
  • the frequency is 500Hz.
  • the present invention also provides a matrix liquid used for bacterial identification, comprising a first matrix liquid and a second matrix liquid; the first matrix liquid is a 50% acetonitrile solution containing CHCA and 2.5% trifluoroacetic acid; the second The matrix solution was 50% acetonitrile solution containing CHCA-C3 and 2.5% trifluoroacetic acid.
  • the volume ratio of the first matrix liquid and the second matrix liquid is 1:2; in the first matrix liquid, the concentration of CHCA is 10-15mg/ml; In the matrix fluid, the concentration of CHCA-C3 is 10-15mg/ml.
  • the present invention also provides the application of the matrix solution described above in the identification of bacteria by MALDI-MS.
  • the bacteria include at least one of Escherichia coli, Enterobacter cloacae, Pseudomonas aeruginosa and Shigella, including the above bacteria but not only limited to this.
  • this novel MALDI mixed matrix can detect hydrophilic protein and hydrophobic protein at the same time, thereby expanding the number of mass spectrum peaks of the analyte, making it It can identify bacteria more accurately, and realize the rapid and highly sensitive identification of bacteria; this mixed matrix can detect the mass spectrum peaks of various hydrophilic proteins and hydrophobic proteins at the same time, thereby improving the accuracy of using MALDI-MS to distinguish bacteria characteristics, allowing it to distinguish between similar bacteria such as Escherichia coli and Shigella. Make clinical drug delivery more accurate.
  • Fig. 1 is the structural formula of CHCA and CHCA-C3;
  • Fig. 2 is that CHCA and CHCA-C3 are respectively used as matrix detection Escherichia coli mass spectrogram;
  • Fig. 3 is the optimal condition MALDI mass spectrogram of optimizing mixed matrix volume ratio
  • Fig. 4 is the mass spectrogram that the mixed matrix detects the bacterium to be tested, and among Fig. 4 A is the spectrogram that Enterobacter cloacae map carries out MALDI detection and obtains, and B among Fig. 4 is that Pseudomonas aeruginosa carries out the spectrogram that MALDI detects and obtains;
  • Figure 5 is the mass spectrograms for the detection of Escherichia coli and Shigella respectively by the mixed matrix.
  • Step 1 Cultivate Escherichia coli on a solid medium for 24 hours;
  • Step 2 Dissolving CHCA in 50% acetonitrile containing 2.5% trifluoroacetic acid solution to prepare a CHCA matrix solution with a concentration of 10-15 mg/ml, and storing it in a refrigerator at 4°C for future use;
  • Step 3 Take the Escherichia coli cultured in step 1 and evenly spread it on the matching target plate of MALDI-MS;
  • Step 4 After the Escherichia coli on the target plate in Step 3 is air-dried, take 0.5-1 ⁇ L of the CHCA matrix solution obtained in Step 2 and spot on the dried Escherichia coli;
  • Step 5 naturally air-dry the Escherichia coli to which the CHCA matrix solution was added dropwise in step 4 at room temperature;
  • Step 6 Perform MALDI analysis directly after the solvent in step 6 volatilizes and crystallizes.
  • Step 1 Cultivate Escherichia coli on a solid medium for 24 hours;
  • Step 2 Dissolving CHCA-C3 in 50% acetonitrile containing 2.5% trifluoroacetic acid solution to prepare a CHCA-C3 matrix solution with a concentration of 10-15 mg/ml, and store it in a refrigerator at 4°C for future use;
  • Step 3 Take the Escherichia coli cultured in step 1 and evenly spread it on the matching target plate of MALDI-MS;
  • Step 4 After the Escherichia coli on the target plate in Step 3 is air-dried, take 0.5-1 ⁇ L of the CHCA-C3 matrix solution obtained in Step 2 and spot it on the dried Escherichia coli;
  • Step 5 naturally air-dry the Escherichia coli to which the CHCA-C3 matrix solution was added dropwise in step 4 at room temperature;
  • Step 6 Perform MALDI analysis directly after the solvent in step 6 volatilizes and crystallizes.
  • the bacteria to be tested are Escherichia coli, and the data is collected in a linear positive ion mode.
  • A is CHCA as a matrix to detect Escherichia coli spectrogram
  • B is CHCA-C3 used as the matrix to detect Escherichia coli mass spectrogram, it shows that when CHCA-C3 is used as the matrix, more mass spectrum peaks are detected, and the fingerprint spectrum obtained when CHCA and CHCA-C3 are used as the matrix alone to detect bacteria The graphs are very different.
  • Step 1 Cultivate Escherichia coli on a solid medium for 24 hours;
  • Step 2 Dissolve CHCA and CHCA-C3 respectively in 50% acetonitrile containing 2.5% trifluoroacetic acid solution to prepare matrix solutions with a concentration of 10-15 mg/ml CHCA and CHCA-C3, and store them in a refrigerator at 4°C for later use;
  • Step 3 Take CHCA matrix solution and CHCA-C3 matrix solution and mix them uniformly at a volume ratio of 2:1;
  • Step 4 Take the Escherichia coli cultured in Step 1 and spread it evenly on the matching target plate of MALDI-MS;
  • Step 5 After the Escherichia coli on the target plate in step 4 is air-dried, take 0.5-1 ⁇ L of the mixed matrix solution obtained in step 3 and spot on the dried Escherichia coli;
  • Step 6 naturally air-dry the Escherichia coli to which the mixed matrix solution was added in step 5 at room temperature;
  • Step 7 After the solvent in step 6 volatilizes and crystallizes, carry out MALDI analysis directly.
  • Step 1 Cultivate Escherichia coli on a solid medium for 24 hours;
  • Step 2 Dissolve CHCA and CHCA-C3 respectively in 50% acetonitrile containing 2.5% trifluoroacetic acid solution to prepare matrix solutions with a concentration of 10-15 mg/ml CHCA and CHCA-C3, and store them in a refrigerator at 4°C for later use;
  • Step 3 Take CHCA matrix solution and CHCA-C3 matrix solution and mix them uniformly at a volume ratio of 1:1;
  • Step 4 Take the Escherichia coli cultured in Step 1 and spread it evenly on the matching target plate of MALDI-MS;
  • Step 5 After the Escherichia coli on the target plate in step 4 is air-dried, take 0.5-1 ⁇ L of the mixed matrix solution obtained in step 3 and spot on the dried Escherichia coli;
  • Step 6 naturally air-dry the Escherichia coli to which the mixed matrix solution was added in step 5 at room temperature;
  • Step 7 After the solvent in step 6 volatilizes and crystallizes, carry out MALDI analysis directly.
  • Step 1 Cultivate Escherichia coli on a solid medium for 24 hours;
  • Step 2 Dissolve CHCA and CHCA-C3 respectively in 50% acetonitrile containing 2.5% trifluoroacetic acid solution to prepare a matrix solution with a concentration of 10-15mg/ml CHCA and CHCA-C3, and store them in a refrigerator at 4°C for later use;
  • Step 3 Take CHCA matrix solution and CHCA-C3 matrix solution and mix them uniformly at a volume ratio of 1:2;
  • Step 4 Take the Escherichia coli cultured in Step 1 and spread it evenly on the matching target plate of MALDI-MS;
  • Step 5 After the Escherichia coli on the target plate in step 4 is air-dried, take 0.5-1 ⁇ L of the mixed matrix solution obtained in step 3 and spot on the dried Escherichia coli;
  • Step 6 naturally air-dry the Escherichia coli to which the mixed matrix solution was added in step 5 at room temperature;
  • Step 7 After the solvent in step 6 volatilizes and crystallizes, carry out MALDI analysis directly.
  • Step 1 Cultivate Escherichia coli on a solid medium for 24 hours;
  • Step 2 Dissolve CHCA and CHCA-C3 respectively in 50% acetonitrile containing 2.5% trifluoroacetic acid solution to prepare matrix solutions with a concentration of 10-15 mg/ml CHCA and CHCA-C3, and store them in a refrigerator at 4°C for later use;
  • Step 3 Take CHCA matrix solution and CHCA-C3 matrix solution and mix them uniformly at a volume ratio of 1:3;
  • Step 4 Take the Escherichia coli cultured in Step 1 and spread it evenly on the matching target plate of MALDI-MS;
  • Step 5 After the Escherichia coli on the target plate in step 4 is air-dried, take 0.5-1 ⁇ L of the mixed matrix solution obtained in step 3 and spot on the dried Escherichia coli;
  • Step 6 naturally air-dry the Escherichia coli to which the mixed matrix solution was added in step 5 at room temperature;
  • Step 7 After the solvent in step 6 volatilizes and crystallizes, carry out MALDI analysis directly.
  • the bacterium to be tested is Escherichia coli, and the data is collected in the linear positive ion mode.
  • the optimal condition for the identification of bacteria in the mixed matrix is that the mixed volume ratio is 1:2.
  • Step 1 respectively culture Enterobacter cloacae and Pseudomonas aeruginosa on solid medium for 24 hours;
  • Step 2 Dissolve CHCA and CHCA-C3 respectively in 50% acetonitrile containing 2.5% trifluoroacetic acid solution to prepare matrix solutions with a concentration of 10-15 mg/ml CHCA and CHCA-C3, and store them in a refrigerator at 4°C for later use;
  • Step 3 Take CHCA matrix solution and CHCA-C3 matrix solution and mix them uniformly at a volume ratio of 1:2;
  • Step 4 Take the Enterobacter cloacae and Pseudomonas aeruginosa cultured in Step 1 and evenly spread them on the MALDI-MS matching target plate;
  • Step 5 After the Enterobacter cloacae and Pseudomonas aeruginosa on the target plate in Step 4 are air-dried, take 0.5-1 ⁇ L of the mixed matrix solution obtained in Step 3 and spot them on the dried Enterobacter cloacae and Pseudomonas aeruginosa respectively ;
  • Step 6 Air-dry the Enterobacter cloacae and Pseudomonas aeruginosa to which the mixed matrix solution was added dropwise in step 5 naturally at room temperature;
  • Step 7 After the solvent in step 6 volatilizes and crystallizes, carry out MALDI analysis directly.
  • Example 1 the data was collected in the linear positive ion mode, and the result is shown in Figure 4 for the mass spectrum of the bacteria to be tested detected by the mixed matrix, which shows that when the mixed matrix is used as the matrix, a better bacterial mass spectrum is detected.
  • Step 1 respectively culturing Escherichia coli and Shigella on solid medium for 24 hours;
  • Step 2 Dissolve CHCA and CHCA-C3 respectively in 50% acetonitrile containing 2.5% trifluoroacetic acid solution to prepare matrix solutions with a concentration of 10-15 mg/ml CHCA and CHCA-C3, and store them in a refrigerator at 4°C for later use;
  • Step 3 Take CHCA matrix solution and CHCA-C3 matrix solution and mix them uniformly at a volume ratio of 1:2;
  • Step 4 Take the Escherichia coli and Shigella cultured in step 1 and evenly spread them on the matching target plate of MALDI-MS;
  • Step 5 After the Escherichia coli and Shigella on the target plate in step 4 are air-dried, take 0.5-1 ⁇ L of the mixed matrix solution obtained in step 3 and spot them on the dried Escherichia coli and Shigella respectively;
  • Step 6 Air-dry the Escherichia coli and Shigella to which the mixed matrix solution was added dropwise in step 5 at room temperature;
  • Step 7 After the solvent in step 6 volatilizes and crystallizes, carry out MALDI analysis respectively.
  • Fig. 5 A is the mixed matrix detection Escherichia coli spectrogram
  • Fig. 5 B is the mixed matrix detection Shigella mass spectrogram, shows When the mixed matrix was used to detect similar bacteria, more differential peaks of the two bacteria were detected. Two types of bacteria can be distinguished.
  • the above-mentioned embodiments of the present invention provide the application of the novel mixed matrix in the identification of bacteria by MALDI-MS.
  • This novel MALDI mixed matrix can simultaneously detect hydrophilic proteins and hydrophobic proteins, thereby expanding the number of mass spectrum peaks of the analytes. It enables more accurate identification of bacteria, and realizes the rapid and highly sensitive identification of bacteria; this mixed matrix can simultaneously detect the mass spectrum peaks of various hydrophilic proteins and hydrophobic proteins, thereby improving the use of MALDI-MS to distinguish bacteria. accuracy, enabling it to differentiate between similar bacteria such as Escherichia coli and Shigella. Make clinical drug delivery more accurate.

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Abstract

一种新型混合基质在MALDI-MS细菌鉴定中的应用，步骤如下：步骤一：在固体培养基上培养待测细菌24h；步骤二：将CHCA和CHCA-C3分别溶于50％乙腈含2.5％三氟乙酸溶液中配制CHCA和CHCA-C3基质液；步骤三：分别取CHCA基质液和CHCA-C3基质液以体积比1:2均匀混合；步骤四：取步骤一培养得到的细菌均匀涂在MALDI-MS靶板上；步骤五：步骤四靶板上的细菌风干后，将步骤三得到的混合基质溶液点在干后的细菌上；步骤六：步骤五中的溶剂挥发结晶后直接进行MALDI分析。这种新型MALDI混合基质可以同时检测到亲水性蛋白质和疏水性蛋白质，从而扩大了分析物的质谱峰数量，使其能够更准确的鉴定细菌，实现了细菌的快速、高灵敏的区分鉴定，从而提高了使用MALDI-MS区分细菌的准确性。

Description

新型混合基质在MALDI-MS细菌鉴定中的应用
本申请要求于2021年8月4日提交中国专利局、申请号为202110890165.0、发明名称为“新型混合基质在MALDI-MS细菌鉴定中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于细菌鉴定技术领域，尤其涉及新型混合基质在MALDI-MS细菌鉴定中的应用。
背景技术
基质辅助激光解析离子化质谱(MALDI-MS)目前已经被广泛应用于蛋白质组学，代谢组学以及临床中的微生物分析。它是一种快速，灵敏，高通量的分析方法。它的原理是通过特殊有机小分子化合物作为基质吸收激光能量，进行质子化传递，进而使分析物离子化，从而达到检测目的。
传统基质检测细菌得到的大多是亲水性蛋白质的指纹谱图，这就导致在传统MALDI检测中，无法区分相似细菌。但是许多相似细菌虽然前期临床表现相似但在疾病后期对感染者造成的后果有较大差异，对其进行准确的区分，在临床治疗上是有十分重要的现实意义的。例如大肠埃希菌和志贺氏菌。大肠埃希菌和志贺氏菌在临床上都是常见的细菌，二者在临床上的早期表现都为腹痛、腹泻和发热，但是与大肠埃希菌相比，志贺氏菌可引起更严重的痢疾疾病，严重者甚至会危及生命。
现有MALDI-MS检测细菌使用的基质是α-氰基-4-羟基肉桂酸(CHCA)，但是由于CHCA对亲水性蛋白质有良好的检测效果，但对于疏水性蛋白质的检测有一定的限制，所以使用传统基质对细菌检测时得到的指纹谱图不够全面，因此现有MALDI-MS检测细菌很难对相似细菌进行区分。
发明内容
本发明实施例的目的在于提供新型混合基质在MALDI-MS细菌鉴定中的应用，旨在解决传统基质对细菌检测时得到的指纹谱图不够全面和无 法区分相似细菌的问题。
本发明实施例是这样实现的，新型混合基质在MALDI-MS细菌鉴定中的应用，步骤如下：
步骤一：在固体培养基上培养待测细菌24h；
步骤二：将CHCA和CHCA-C3分别溶于50％乙腈含2.5％三氟乙酸溶液中配制CHCA和CHCA-C3基质液，保存于4℃的冰箱中以备用；
步骤三：分别取CHCA基质液和CHCA-C3基质液以体积比1:2均匀混合；
步骤四：取步骤一培养得到的细菌均匀涂在MALDI-MS配套的靶板上；
步骤五：步骤四靶板上的细菌风干后，将步骤三得到的混合基质溶液点在干后的细菌上；
步骤六：将步骤四中滴加混合基质溶液的细菌在室温下自然风干；
步骤七：待当步骤六中的溶剂挥发结晶后直接进行MALDI分析。
进一步的技术方案，所述步骤二中所述基质溶液浓度为10-15mg/ml。
进一步的技术方案，所述步骤四中混合基质的体积为0.5-1μL。
进一步的技术方案，所述步骤七数据在线性正离子模式下采集用于获得待测细菌的质谱图。
进一步的技术方案，所述步骤七中使用的基质辅助激光解析离子化飞行时间质谱仪型号为Autoflex speed TOF/TOF，激光波长为355nm的Nd:YAG激光器。
进一步的技术方案，所述基质辅助激光解析离子化飞行时间质谱仪测试时的加速电压为20.000kv，延迟引出电压为18.000kv，延迟引出时间为150ns。
进一步的技术方案，所述基质辅助激光解析离子化飞行时间质谱仪测试时的反射器电压为20.000kv，透镜电压为6.000kv，频率为500Hz。
本发明还提供一种用于细菌鉴定的基质液，包括第一基质液和第二基质液；所述第一基质液为含CHCA和2.5％三氟乙酸的50％乙腈溶液；所述第二基质液为含CHCA-C3和2.5％三氟乙酸的50％乙腈溶液。
在本发明的一些实施方案中，所述第一基质液和第二基质液的体积比为1:2；所述第一基质液中，CHCA的浓度为10-15mg/ml；所述第二基质液中，CHCA-C3的浓度为10-15mg/ml。
本发明还提供了以上所述的基质液在MALDI-MS细菌鉴定中的应用。
本发明所述基质液在MALDI-MS细菌鉴定中的应用中，所述细菌包括大肠埃希菌、阴沟肠杆菌、铜绿假单胞菌和志贺氏菌中的至少一种，包括以上细菌但不仅限于此。
本发明实施例提供的新型混合基质在MALDI-MS细菌鉴定中的应用，这种新型MALDI混合基质可以同时检测到亲水性蛋白质和疏水性蛋白质，从而扩大了分析物的质谱峰数量，使其能够更准确的鉴定细菌，实现了细菌的快速，高灵敏的区分鉴定；这种混合基质可以同时检测到多种亲水蛋白质和疏水蛋白质的质谱峰，从而提高了使用MALDI-MS区分细菌的准确性，使其能够区分大肠埃希菌和志贺氏菌等相似的细菌。使得临床给药更加精准。
附图说明
图1为CHCA和CHCA-C3的结构式；
图2为CHCA和CHCA-C3分别作为基质检测大肠埃希菌质谱图；
图3为优化混合基质体积比的最优条件MALDI质谱图；
图4为混合基质检测待测细菌的质谱图，图4中A为阴沟肠杆菌图进行MALDI检测得到的谱图，图4中B为铜绿假单胞菌进行MALDI检测得到的谱图；
图5为混合基质分别检测大肠埃希菌和志贺氏菌的质谱图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
以下结合具体实施例对本发明的具体实现进行详细描述。
对比例一
步骤一：在固体培养基上培养大肠埃希菌24h；
步骤二：将CHCA溶于50％乙腈含2.5％三氟乙酸溶液中配制浓度为10-15mg/ml的CHCA基质液，保存于4℃的冰箱中以备用；
步骤三：取步骤一中培养得到的大肠埃希菌均匀涂在MALDI-MS配套的靶板上；
步骤四：待步骤三靶板上的大肠埃希菌风干后，将步骤二得到的CHCA基质液取0.5-1μL点在干后的大肠埃希菌上；
步骤五：将步骤四中滴加CHCA基质液的大肠埃希菌在室温下自然风干；
步骤六：待当步骤六中的溶剂挥发结晶后直接进行MALDI分析。
对比例二
步骤一：在固体培养基上培养大肠埃希菌24h；
步骤二：将CHCA-C3溶于50％乙腈含2.5％三氟乙酸溶液中配制浓度为10-15mg/ml的CHCA-C3基质液，保存于4℃的冰箱中以备用；
步骤三：取步骤一中培养得到的大肠埃希菌均匀涂在MALDI-MS配套的靶板上；
步骤四：待步骤三靶板上的大肠埃希菌风干后，将步骤二得到的CHCA-C3基质液取0.5-1μL点在干后的大肠埃希菌上；
步骤五：将步骤四中滴加CHCA-C3基质液的大肠埃希菌在室温下自然风干；
步骤六：待当步骤六中的溶剂挥发结晶后直接进行MALDI分析。
根据对比例1和2所示，所述待测细菌为大肠埃希菌，数据在线性正离子模式下采集，结果如图2中A为CHCA作为基质检测大肠埃希菌谱图和图2中B为CHCA-C3作为基质检测大肠埃希菌质谱图所示，表明用CHCA-C3作基质时，检测到更多的质谱峰，并且CHCA和CHCA-C3单独作为基质检测细菌时得到的指纹谱图有很大不同。
对比例三
步骤一：在固体培养基上培养大肠埃希菌24h；
步骤二：将CHCA和CHCA-C3分别溶于50％乙腈含2.5％三氟乙酸溶液中配制浓度为10-15mg/mlCHCA和CHCA-C3基质液，保存于4℃的冰箱中以备用；
步骤三：分别取CHCA基质液和CHCA-C3基质液以体积比2:1均匀混合；
步骤四：取步骤一培养得到的大肠埃希菌均匀涂在MALDI-MS配套的靶板上；
步骤五：步骤四靶板上的大肠埃希菌风干后，将步骤三得到的混合基质溶液取0.5-1μL点在干后的大肠埃希菌上；
步骤六：将步骤五中滴加混合基质溶液的大肠埃希菌在室温下自然风干；
步骤七：待当步骤六中的溶剂挥发结晶后直接进行MALDI分析。
对比例四
步骤一：在固体培养基上培养大肠埃希菌24h；
步骤二：将CHCA和CHCA-C3分别溶于50％乙腈含2.5％三氟乙酸溶液中配制浓度为10-15mg/mlCHCA和CHCA-C3基质液，保存于4℃的冰箱中以备用；
步骤三：分别取CHCA基质液和CHCA-C3基质液以体积比1:1均匀混合；
步骤四：取步骤一培养得到的大肠埃希菌均匀涂在MALDI-MS配套的靶板上；
步骤五：步骤四靶板上的大肠埃希菌风干后，将步骤三得到的混合基质溶液取0.5-1μL点在干后的大肠埃希菌上；
步骤六：将步骤五中滴加混合基质溶液的大肠埃希菌在室温下自然风干；
步骤七：待当步骤六中的溶剂挥发结晶后直接进行MALDI分析。
对比例五
步骤一：在固体培养基上培养大肠埃希菌24h；
步骤二：将CHCA和CHCA-C3分别溶于50％乙腈含2.5％三氟乙酸 溶液中配制浓度为10-15mg/mlCHCA和CHCA-C3基质液，保存于4℃的冰箱中以备用；
步骤三：分别取CHCA基质液和CHCA-C3基质液以体积比1:2均匀混合；
步骤四：取步骤一培养得到的大肠埃希菌均匀涂在MALDI-MS配套的靶板上；
步骤五：步骤四靶板上的大肠埃希菌风干后，将步骤三得到的混合基质溶液取0.5-1μL点在干后的大肠埃希菌上；
步骤六：将步骤五中滴加混合基质溶液的大肠埃希菌在室温下自然风干；
步骤七：待当步骤六中的溶剂挥发结晶后直接进行MALDI分析。
对比例六
步骤一：在固体培养基上培养大肠埃希菌24h；
步骤二：将CHCA和CHCA-C3分别溶于50％乙腈含2.5％三氟乙酸溶液中配制浓度为10-15mg/mlCHCA和CHCA-C3基质液，保存于4℃的冰箱中以备用；
步骤三：分别取CHCA基质液和CHCA-C3基质液以体积比1:3均匀混合；
步骤四：取步骤一培养得到的大肠埃希菌均匀涂在MALDI-MS配套的靶板上；
步骤五：步骤四靶板上的大肠埃希菌风干后，将步骤三得到的混合基质溶液取0.5-1μL点在干后的大肠埃希菌上；
步骤六：将步骤五中滴加混合基质溶液的大肠埃希菌在室温下自然风干；
步骤七：待当步骤六中的溶剂挥发结晶后直接进行MALDI分析。
根据对比例3-6所示，所述待测细菌为大肠埃希菌，数据在线性正离子模式下采集。综上所述，如图3所示混合基质鉴定细菌最佳条件为混合体积比为1:2。
实施例一
混合基质在MALDI质谱中检测细菌的方法的步骤如下：
步骤一：在固体培养基上分别培养阴沟肠杆菌和铜绿假单胞菌24h；
步骤二：将CHCA和CHCA-C3分别溶于50％乙腈含2.5％三氟乙酸溶液中配制浓度为10-15mg/mlCHCA和CHCA-C3基质液，保存于4℃的冰箱中以备用；
步骤三：分别取CHCA基质液和CHCA-C3基质液以体积比1:2均匀混合；
步骤四：取步骤一培养得到的阴沟肠杆菌和铜绿假单胞菌分别均匀涂在MALDI-MS配套的靶板上；
步骤五：步骤四靶板上的阴沟肠杆菌和铜绿假单胞菌风干后，将步骤三得到的混合基质溶液均取0.5-1μL分别点在干后的阴沟肠杆菌和铜绿假单胞菌上；
步骤六：将步骤五中滴加混合基质溶液的阴沟肠杆菌和铜绿假单胞菌在室温下自然风干；
步骤七：待当步骤六中的溶剂挥发结晶后直接进行MALDI分析。
根据实施例一，数据在线性正离子模式下采集，结果如图4为混合基质检测待测细菌质谱图所示，表明用混合基质作基质时，检测到较好的细菌质谱图。
实施例二
混合基质在MALDI质谱中测定大肠埃希菌和志贺氏菌的步骤如下：
步骤一：在固体培养基上分别培养大肠埃希菌和志贺氏菌24h；
步骤二：将CHCA和CHCA-C3分别溶于50％乙腈含2.5％三氟乙酸溶液中配制浓度为10-15mg/mlCHCA和CHCA-C3基质液，保存于4℃的冰箱中以备用；
步骤三：分别取CHCA基质液和CHCA-C3基质液以体积比1:2均匀混合；
步骤四：取步骤一培养得到的大肠埃希菌和志贺氏菌分别均匀涂在MALDI-MS配套的靶板上；
步骤五：步骤四靶板上的大肠埃希菌和志贺氏菌风干后，将步骤三 得到的混合基质溶液取0.5-1μL分别点在干后的大肠埃希菌和志贺氏菌上；
步骤六：将步骤五中滴加混合基质溶液的大肠埃希菌和志贺氏菌在室温下自然风干；
步骤七：待当步骤六中的溶剂挥发结晶后分别进行MALDI分析。
根据实施例二，数据分别在线性正离子模式下采集，结果如图5中A为混合基质检测大肠埃希菌谱图和图5中B为混合基质检测志贺氏菌质谱图所示，表明用混合基质检测相似细菌时，检测到更多的两种细菌的差异谱峰。可以将两种细菌区分开来。
本发明上述实施例中提供了新型混合基质在MALDI-MS细菌鉴定中的应用，这种新型MALDI混合基质可以同时检测到亲水性蛋白质和疏水性蛋白质，从而扩大了分析物的质谱峰数量，使其能够更准确的鉴定细菌，实现了细菌的快速，高灵敏的区分鉴定；这种混合基质可以同时检测到多种亲水蛋白质和疏水蛋白质的质谱峰，从而提高了使用MALDI-MS区分细菌的准确性，使其能够区分大肠埃希菌和志贺氏菌等相似的细菌。使得临床给药更加精准。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 新型混合基质在MALDI-MS细菌鉴定中的应用，其特征在于，步骤如下：
    步骤一：在固体培养基上培养待测细菌24h；
    步骤二：将CHCA和CHCA-C3分别溶于50％乙腈含2.5％三氟乙酸溶液中配制CHCA和CHCA-C3基质液，保存于4℃的冰箱中以备用；
    步骤三：分别取CHCA基质液和CHCA-C3基质液以体积比1:2均匀混合；
    步骤四：取步骤一培养得到的细菌均匀涂在MALDI-MS配套的靶板上；
    步骤五：步骤四靶板上的细菌风干后，将步骤三得到的混合基质溶液点在干后的细菌上；
    步骤六：将步骤五中滴加混合基质溶液的细菌在室温下自然风干；
    步骤七：待当步骤六中的溶剂挥发结晶后直接进行MALDI分析。
  2. 根据权利要求1所述的新型混合基质在MALDI-MS细菌鉴定中的应用，其特征在于，所述步骤二中所述基质溶液浓度为10-15mg/ml。
  3. 根据权利要求1所述的新型混合基质在MALDI-MS细菌鉴定中的应用，其特征在于，所述步骤四中混合基质的体积为0.5-1μL。
  4. 根据权利要求1-3任一所述的新型混合基质在MALDI-MS细菌鉴定中的应用，其特征在于，所述步骤七数据在线性正离子模式下采集用于获得待测细菌的质谱图。
  5. 根据权利要求1所述的新型混合基质在MALDI-MS细菌鉴定中的应用，其特征在于，所述步骤七中使用的基质辅助激光解析离子化飞行时间质谱仪型号为Autoflex speed TOF/TOF，激光波长为355nm的Nd:YAG激光器。
  6. 根据权利要求5所述的新型混合基质在MALDI-MS细菌鉴定中的应用，其特征在于，所述基质辅助激光解析离子化飞行时间质谱仪测试时的加速电压为20.000kv，延迟引出电压为18.000kv，延迟引出时间为 150ns。
  7. 根据权利要求5所述的新型混合基质在MALDI-MS细菌鉴定中的应用，其特征在于，所述基质辅助激光解析离子化飞行时间质谱仪测试时的反射器电压为20.000kv，透镜电压为6.000kv，频率为500Hz。
  8. 一种用于细菌鉴定的基质液，其特征在于，包括第一基质液和第二基质液；所述第一基质液为含CHCA和2.5％三氟乙酸的50％乙腈溶液；所述第二基质液为含CHCA-C3和2.5％三氟乙酸的50％乙腈溶液。
  9. 根据权利要求1所述的基质液，其特征在于，所述第一基质液和第二基质液的体积比为1:2；所述第一基质液中，CHCA的浓度为10-15mg/ml；所述第二基质液中，CHCA-C3的浓度为10-15mg/ml。
  10. 权利要求8或9所述的基质液在MALDI-MS细菌鉴定中的应用，其特征在于，所述细菌选自大肠埃希菌、阴沟肠杆菌、铜绿假单胞菌和志贺氏菌中的至少一种。
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