CN102749233A - Pretreatment method for directly detecting infection urine pathogen by MALDI-TOF MS - Google Patents
Pretreatment method for directly detecting infection urine pathogen by MALDI-TOF MS Download PDFInfo
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- CN102749233A CN102749233A CN2012101801239A CN201210180123A CN102749233A CN 102749233 A CN102749233 A CN 102749233A CN 2012101801239 A CN2012101801239 A CN 2012101801239A CN 201210180123 A CN201210180123 A CN 201210180123A CN 102749233 A CN102749233 A CN 102749233A
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Abstract
The invention discloses a pretreatment method for directly detecting infection urine pathogen by MALDI-TOF MS. An urine sample pretreatment part is improved on a methodology, bacteria is washed and collected, formic acid and acetonitrile are used for performing protein digestion on bacteria, and then the detection is carried out, thereby the bacteria identification time is substantially shortened and the identification accuracy is enhanced. The pretreatment method is capable of rapidly and accurately identifying the infection urine sample, and can timely guide the clinical medication with pertinence.
Description
Technical field
The present invention relates to the clinical samples pathogenic microorganism and detect, belong to the microbiology field.
Background technology
Urinary system infection contamination (UTIs) accounts for second in the clinical infection rate; And classic method (urine is cultivated and identified) is long when enduring; The clinician often can not wait for breadboard result, but selects the microbiotic of wide spectrum to treat, and this has just caused the resistant rate of clinical bacteria to rise year by year.
The UTIs Infect And Diagnose is mainly according to clinical symptoms and laboratory result.The patient should clean vulva before specimen taken, and gets midstream urine in sterile chamber.Main laboratory examination comprises: the inspection of urine dry chemical, urine sediment analysis, smear for microscopic examination, gram stain microscopy and urine culture identification.The urine culture identification is considered to the best way now, and urine is inoculated in the double dish, hatches 18-24h, utilizes the single bacterium colony that grows in the double dish to identify.Long when yet classic method is endured, spend morely, and the sample that has a 60-80% is a negative findings.
At present; Ground substance assistant laser based on mass spectrum framework collection of illustrative plates is resolved flight mass spectrum (Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry; MALDI-TOF) progress into clinical; Being applied to microorganism detection, mainly is to cultivate the back bacterium colony but detect sample, and Bacteria Identification time ratio classic method takes early 1 day.Yet MALDI-TOF MS seldom is used for the direct evaluation of type specimen.The present invention wants through the method for MALDI-TOF MS is improved, provide a kind of can be in 30 minutes, directly accurately detect the method for pathogen in the clinical urine specimen, thereby the direct clinical medication.
Summary of the invention
The purpose of this invention is to provide MALDI-TOF MS and identify infection urine sample pre-treating method.
MALDI-TOF MS involved in the present invention identifies that infecting urine sample pre-treating method is behind bacterium washing, collection bacterium, utilizes formic acid and acetonitrile that bacterium is carried out the albumen stripping, and application of sample after the drying, adds matrix, and machine testing is gone up in dry back.Guaranteeing to detect accurately under the prerequisite, shortened minute greatly.
For achieving the above object, technical scheme of the present invention is achieved in that
Get 1ml urine sample 2000 * g, centrifugal 30 seconds, get supernatant.15500rpm centrifugal 5 minutes, gets deposition.Sterilized water washing 1 time, drying, subsequent use.Add 5 μ l formic acid (70%) and 5 μ l acetonitriles, carry out the albumen process in leaching, mixing is got 1 μ l point target, adds 1 μ l matrix (α one cyanic acid one 4 hydroxycinnamic acid saturated solutions include 50% acetonitrile and 2.5% trifluoroacetic acid) after the drying, and machine testing is gone up in dry back.
Detect step and relatively simplify, reduced because the bacterium that complex steps is brought is lost.Minute has been shortened in the simplification of testing process greatly, and antibiotic therapy provides reliable evidence for the clinician implements fast targetedly.
Description of drawings
Fig. 1 is respectively Escherichia coli MALDI-TOF Mass Spectrometer Method mass spectrogram and qualification result in the urine of improving one's methods;
Fig. 2 is respectively enterococcus faecalis MALDI-TOF Mass Spectrometer Method mass spectrogram and qualification result in the urine of improving one's methods.
Embodiment
Embodiment 1MALDI-TOF MS detects the sample pre-treatment
Leave and take doubtful urinary infection patient's 1456 parts of CCMSs clinically, immediately censorship.Get 1ml urine sample 2000 * g, centrifugal 30 seconds, get supernatant.15500rpm centrifugal 5 minutes, gets deposition.Sterilized water washing 1 time, drying, subsequent use.Add 5 μ l formic acid (70%) and 5 μ l acetonitriles, carry out the albumen stripping, mixing is got 1 μ l point target, adds 1 μ l matrix (α one cyanic acid one 4 hydroxycinnamic acid saturated solutions include 50% acetonitrile and 2.5% trifluoroacetic acid) after the drying, and machine testing is gone up in dry back.
Embodiment 2MALDI-TOF MS analyzes
It is 200Hz that the mass spectral acquisition of bacterium needs the laser intensity of autoflex III MALDI-TOF/TOF MS, and sensing range is 2-20kDa.The formation of ion needs the nitrogen laser of 337nm, and parsing time delay is 40ns.The detection of each sample needs 500 laser point to be collected and to analyze (diverse location that 10 * 50 illuminated laser spot derive from the appearance target).Need the potpourri of protein standard 1 and polypeptide standard 2 to calibrate before the mensuration.Sample is measured the back and is formed mass spectrogram, analyzes with BioTyper bacterium database through MALDI BioTyper software 3.0 and compares, and the score value scope is 0-3, measures score value＞1.9, explains to identify kind; 1.7＜score value＜1.9 explain to identify genus; Score value＜1.7, then qualification result is insincere.
Claims (2)
1. a MALDI-TOF MS directly detects and infects urine pathogen pre-treating method, behind bacterium washing, collection bacterium, utilizes formic acid and acetonitrile that bacterium is carried out the albumen stripping, and application of sample after the drying, adds matrix, and machine testing is gone up in dry back.
2. MALDI-TOF MS according to claim 1 identifies pre-treating method, it is characterized in that getting 1ml urine sample 2000 * g, centrifugal 30 seconds, gets supernatant.15500rpm centrifugal 5 minutes, gets deposition.Sterilized water washing 1 time, drying, subsequent use.Add 5 μ l formic acid (70%) and 5 μ l acetonitriles, carry out the albumen stripping, mixing is got 1 μ l point target, adds 1 μ l matrix (α one cyanic acid one 4 hydroxycinnamic acid saturated solutions include 50% acetonitrile and 2.5% trifluoroacetic acid) after the drying, and machine testing is gone up in dry back.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2012101801239A CN102749233A (en) | 2012-06-04 | 2012-06-04 | Pretreatment method for directly detecting infection urine pathogen by MALDI-TOF MS |
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| CN2012101801239A CN102749233A (en) | 2012-06-04 | 2012-06-04 | Pretreatment method for directly detecting infection urine pathogen by MALDI-TOF MS |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107076651A (en) * | 2014-08-18 | 2017-08-18 | 贝克顿·迪金森公司 | Sample preparation methods and its automated system for MALDI |
| CN110455909A (en) * | 2019-04-02 | 2019-11-15 | 天津大学 | A kind of Matrix Assisted Laser Desorption time-of-flight mass spectrometry analyzes the analysis method of condensed tannin in acorn nut |
| CN110628867A (en) * | 2019-10-24 | 2019-12-31 | 南方医科大学南方医院 | Novel urine bacterium detection method |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107076651A (en) * | 2014-08-18 | 2017-08-18 | 贝克顿·迪金森公司 | Sample preparation methods and its automated system for MALDI |
| US10458887B2 (en) | 2014-08-18 | 2019-10-29 | Becton, Dickinson And Company | Method of sample preparation for Maldi and automated system therefor |
| CN107076651B (en) * | 2014-08-18 | 2021-04-06 | 贝克顿·迪金森公司 | Sample preparation method for MALDI and automated system therefor |
| US11385145B2 (en) | 2014-08-18 | 2022-07-12 | Becton, Dickinson And Company | Method of sample preparation for Maldi and automated system therefor |
| US11774332B2 (en) | 2014-08-18 | 2023-10-03 | Becton Dickinson And Company | Method of sample preparation for maldi and automated system therefor |
| CN110455909A (en) * | 2019-04-02 | 2019-11-15 | 天津大学 | A kind of Matrix Assisted Laser Desorption time-of-flight mass spectrometry analyzes the analysis method of condensed tannin in acorn nut |
| CN110628867A (en) * | 2019-10-24 | 2019-12-31 | 南方医科大学南方医院 | Novel urine bacterium detection method |
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Effective date of registration: 20121017 Address after: 1 Department of Microbiology, Tongren Hospital, Tongren East Lane, Beijing, Dongcheng District 100730, China Applicant after: Lu Xinxin Applicant after: Wang Xinhua Address before: 1 Department of Microbiology, Tongren Hospital, Tongren East Lane, Beijing, Dongcheng District 100730, China Applicant before: Wang Xinhua |
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Application publication date: 20121024 |