CN104237367A - Kit applied to small molecular qualitative and quantitative analysis and detection and high-throughout screening of microbial culture solution sample and preparation method of kit - Google Patents
Kit applied to small molecular qualitative and quantitative analysis and detection and high-throughout screening of microbial culture solution sample and preparation method of kit Download PDFInfo
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- CN104237367A CN104237367A CN201410455916.6A CN201410455916A CN104237367A CN 104237367 A CN104237367 A CN 104237367A CN 201410455916 A CN201410455916 A CN 201410455916A CN 104237367 A CN104237367 A CN 104237367A
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Abstract
The invention relates to a preparation method of a kit applied to small molecular qualitative and quantitative analysis and detection and high-throughout screening of a microbial culture solution sample. The preparation method comprises the following steps: placing the microbial culture solution sample at room temperature and fully thawing; adding a mixed solution-basic lysate at the temperature of -20 DEG C and with the volume same as that of the sample, and reacting in a water bath at the temperature of 50 degrees for 5min; centrifugating; and collecting supernatant fluid, wherein the basic lysate is QL2 which comprises the following substances in percentage by volume: 10%-50% of methanol, 50%-90% of acetonitrile and 1%-7% of formic acid. The preparation method of the kit is simple and the cost is relatively low.
Description
Technical field
This technology belongs to analytical chemistry applications field, is specifically related to surface laser and resolves field of mass spectrometry.
Background technology
It is a kind of novel soft ionization biological mass spectrometry that development in recent years is got up that surface laser resolves mass spectrum, wherein with Matrix Assisted Laser Desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) for most important example, the present invention obtained Nobel chemistry Prize.Form primarily of two parts: substance assistant laser desorpted ionized ion gun (MALDI) and time of flight mass analyzer (TOF).The principle of MALDI irradiates sample and substrate formed cocrystallization film with laser, matrix absorbs energy transferring to biomolecule from laser, in ionization process, proton translocation is obtained proton to biomolecule or from biomolecule, and the process that biomolecule is ionized.Therefore it is a kind of Soft ionization techniques, is applicable to the mensuration of potpourri and biomacromolecule.The principle of TOF is that ion accelerates to fly over dirft tube under electric field action, and according to the flight time arriving detecting device is different, the detected mass-to-charge ratio (M/Z) namely measuring ion was directly proportional to the flight time of ion, detects ion.MALDI-TOF-MS have highly sensitive, accuracy is high and resolution high, for the fields such as life science provide a kind of strong analytical test means.
But all mass spectrums of resolving based on surface laser headed by Matrix Assisted Laser Desorption ionization time of flight mass spectrometry all need by sample preparation on special target plate, its operation is that sample dispersion is formed crystal in substrate molecule.When irradiating crystal with laser, matrix absorbs energy from laser, sample desorption, there is Charger transfer between matrix-sample and make ionized sample molecule, the sample of ionization flies over the tof tube of vacuum under electric field action, different and be detected according to the flight time arriving detecting device, to be namely directly proportional to the flight time of ion by the ratio (M/Z) of the quality electric charge of ion and to analyze ion, and record the molecular weight of sample molecule.Focal issue is just that the molecular weight of general substrate molecule is at 1000-3000da, causes excessive background mass spectra peak.This just determines all mass spectrophotometry that this type of is resolved based on surface laser can only be applicable to large Molecular Detection, such as protein, long-chain polypeptide, nucleic acid, macromolecular material etc., and Small molecular detection (molecular weight is less than 1000da) can not be applied to, the advantage that the high sensitivity/high flux etc. of the mass spectrophotometry of resolving based on surface laser is in other words unique cannot be put to good use on small molecule analysis detects. and the Small molecular that molecular weight is less than 1000da is the class chemical products to the most important most using value of the mankind, the Medicine small molecule of such as more than 95%, all amino acid, vitamin etc. relevant chemical substance healthy with human lives.
Currently available technology detects for Small molecular and screening also rests on spectroscopic methodology detection, such as fluorescence radiation detects or derives can the substrate of fluoroscopic examination, but this type of testing requirement target Small molecular molecular structure has photolytic activity group, another drawback is exactly that often photometry sensitivity is lower, can't detect the Small molecular concentration of trace.The starting of liquid chromatography mass spectrometric (LC-MS) and gaseous mass spectrum (GC-MS) is applied in micromolecular detection and high flux screening.But need to consume a large amount of manpowers and solvent consumptive material, and a sample needs quite to grow a process to going out result from being prepared into mass spectrophotometry again, different as requested, analyze a sample and need 30-60 minute from sample preparation to taking result, the high flux screening requirement of modern biotechnology medicine development can not be met far away.
Summary of the invention
Goal of the invention: find simple, the lower-cost sample preparation methods of a kind of preparation method, reduce expenses and the time.
Technical scheme: one is applied to Small molecular qualitative and quantitative analysis and detects and high throughput screening of microorganisms nutrient solution sample reagent box, be made up of the sample of equal volume and basic lysate, basic lysate is QL2, QL2 material composition and volume ratio: 10-50% methyl alcohol, 50-90% second cyanogen, 1-7% formic acid.
Be applied to a preparation for the detection of Small molecular qualitative and quantitative analysis and high throughput screening of microorganisms nutrient solution sample reagent box, microbial culture medium sample placed room temperature, fully thaws; Add the mixed solution-basic lysate of-20 degree equal volume, react 5 minutes in 50 degree of water-baths; Centrifugal; Collect supernatant; Basic lysate is QL2, QL2, QL2 material composition and volume ratio: 10-50% methyl alcohol, 50-90% second cyanogen, 1-7% formic acid.
Preferred version is QL2 material composition and volume ratio: 50% methyl alcohol, 50% second cyanogen, 1% formic acid.
Beneficial effect: 1, preparation method is simple; 2, cost is lower.
Accompanying drawing explanation
Mass spectrogram after the cracking of the high yield fat acid leaven bacterium that Fig. 1 utilizes the present invention to detect.
Embodiment
The basic lysate QL2 of microbial culture medium sample preparation reagents box and sample preparation methods
Microbial culture medium sample (single centrifuge tube, 96 hole porous plates, 384 hole porous plates) is placed room temperature 10 minutes, fully thaws.Add the mixed solution-basic lysate QL2 (QL2 material composition and volume ratio are: 10-50% methyl alcohol, 50-90% second cyanogen, 1-7% formic acid) of-20 degree equal volume, act on 5 minutes, and act on 5 minutes in 50 degree of water-baths.3000rmp, after centrifugal 10 minutes, collects supernatant.
After being illustrated in figure 1 saccharomycetes to make fermentation, the product analysis of cracking bacterium.We can carry out rapid screening high-yield strains from the method.
Claims (3)
1. one kind is applied to the detection of Small molecular qualitative and quantitative analysis and high throughput screening of microorganisms (bacillus, saccharomycete, aspergillus etc.) nutrient solution sample reagent box, it is characterized in that, be made up of the sample of equal volume and basic lysate, basic lysate is QL2, QL2 material composition and volume ratio: 10-50% methyl alcohol, 50-90% second cyanogen, 1-7% formic acid.
2. one kind is applied to the detection of Small molecular qualitative and quantitative analysis and high throughput screening of microorganisms cultivation microorganism (bacillus, saccharomycete, aspergillus etc.) preparation of nutrient solution sample reagent box, it is characterized in that, microbial culture medium sample is placed room temperature, fully thaws; Add the mixed solution-basic lysate of-20 degree equal volume, react 5 minutes in 50 degree of water-baths; Centrifugal; Collect supernatant; Basic lysate is QL2, QL2 material composition and volume ratio: 10-50% methyl alcohol, 50-90% second cyanogen, 1-7% formic acid.
3. the preparation being applied to the detection of Small molecular qualitative and quantitative analysis and high throughput screening of microorganisms nutrient solution sample reagent box according to claim 1, is characterized in that, QL2 material composition and volume ratio are: 50% methyl alcohol, 50% second cyanogen, 1% formic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410455916.6A CN104237367A (en) | 2014-09-09 | 2014-09-09 | Kit applied to small molecular qualitative and quantitative analysis and detection and high-throughout screening of microbial culture solution sample and preparation method of kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410455916.6A CN104237367A (en) | 2014-09-09 | 2014-09-09 | Kit applied to small molecular qualitative and quantitative analysis and detection and high-throughout screening of microbial culture solution sample and preparation method of kit |
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| CN104237367A true CN104237367A (en) | 2014-12-24 |
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| CN201410455916.6A Pending CN104237367A (en) | 2014-09-09 | 2014-09-09 | Kit applied to small molecular qualitative and quantitative analysis and detection and high-throughout screening of microbial culture solution sample and preparation method of kit |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102654490A (en) * | 2011-03-02 | 2012-09-05 | 上海市食品药品检验所 | Method for measuring content of mycotoxins in araliaceae plants by liquid chromatography-tandem mass spectrometry |
| US20140234873A1 (en) * | 2006-11-24 | 2014-08-21 | Agency For Science, Technology And Research | Apparatus for processing a sample in a liquid droplet and method of using the same |
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2014
- 2014-09-09 CN CN201410455916.6A patent/CN104237367A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140234873A1 (en) * | 2006-11-24 | 2014-08-21 | Agency For Science, Technology And Research | Apparatus for processing a sample in a liquid droplet and method of using the same |
| CN102654490A (en) * | 2011-03-02 | 2012-09-05 | 上海市食品药品检验所 | Method for measuring content of mycotoxins in araliaceae plants by liquid chromatography-tandem mass spectrometry |
Non-Patent Citations (5)
| Title |
|---|
| DAVID J. EVASON 等: "Effects of ion mode and matrix additives in the identi®cation of bacteria by intact cell mass spectrometry", 《RAPID COMMUNICATIONS IN MASS SPECTROMETRY》, vol. 14, no. 8, 15 May 2000 (2000-05-15), pages 669 - 672 * |
| JOSHUA D. RABINOWITZ 等: "Acidic Acetonitrile for Cellular Metabolome Extraction from Escherichia coli", 《ANAL. CHEM.》, vol. 79, no. 16, 15 August 2007 (2007-08-15), pages 6167 - 6173 * |
| MASAFUMI TOKUOKA 等: "Simple metabolite extraction method for metabolic profiling of the solid-state fermentation of Aspergillus oryzae", 《J. BIOSCI. BIOENG.》, vol. 110, no. 6, 31 December 2010 (2010-12-31), pages 665 - 669 * |
| 罗方方 等: "HPLC-MS/MS法测定水产品中硫酸粘菌素、杆菌肽及维吉尼霉素M1的残留量", 《南方水产科学》, vol. 9, no. 4, 31 August 2013 (2013-08-31), pages 62 - 68 * |
| 聂存喜 等: "热带假丝酵母固体发酵代谢物提取方法的比较分析", 《动物营养学报》, vol. 24, no. 8, 27 July 2012 (2012-07-27), pages 1590 - 1594 * |
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Application publication date: 20141224 |
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