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Pretreatment method for directly detecting infection urine pathogen by MALDI-TOF MS

A technology for pathogenic bacteria and urine, applied in the field of microbiology, can solve problems such as direct identification with less original specimens, and achieve the effects of shortening the measurement time, simplifying the detection steps, and simplifying the detection process.

Inactive Publication Date: 2012-10-24
鲁辛辛 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, MALDI-TOF MS is rarely used for direct identification of primary specimens

Method used

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  • Pretreatment method for directly detecting infection urine pathogen by MALDI-TOF MS
  • Pretreatment method for directly detecting infection urine pathogen by MALDI-TOF MS
  • Pretreatment method for directly detecting infection urine pathogen by MALDI-TOF MS

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1 MALDI-TOF MS detection sample pretreatment

[0013] Clinically, 1456 samples of clean midstream urine from patients with suspected urinary tract infection were collected and submitted for examination immediately. Take 1ml of urine sample at 2000×g, centrifuge for 30 seconds, and take the supernatant. Centrifuge at 15500rpm for 5 minutes to collect the precipitate. Wash once with sterile water, dry, and set aside. Add 5 μl formic acid (70%) and 5 μl acetonitrile for protein dissolution, mix well, take 1 μl spot target, add 1 μl matrix (α-cyano-4 hydroxycinnamic acid saturated solution, containing 50% acetonitrile and 2.5% trifluoroacetic acid), and tested on the machine after drying.

Embodiment 2

[0014] Embodiment 2 MALDI-TOF MS analysis

[0015] The acquisition of bacterial mass spectrometry requires autoflex III MALDI-TOF / TOF MS with a laser intensity of 200Hz and a detection range of 2-20kDa. Ion formation requires a 337nm nitrogen laser with a resolution delay time of 40ns. The detection of each sample requires 500 laser points to be collected and analyzed (10×50 laser irradiation points originate from different positions of the loading target). A mixture of protein standard 1 and peptide standard 2 is required for calibration before determination. The mass spectrogram is formed after the sample is measured, and analyzed by MALDI BioTyper software 3.0 and compared with the BioTyper bacterial database. The score range is 0-3, and the measurement score > 1.9 indicates that the species is identified; 1.7 < score < 1.9 indicates the identification to the genus; if the score is <1.7, the identification result is not credible.

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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 [0001] The invention relates to the detection of pathogenic microorganisms in clinical specimens and belongs to the field of microbiology. Background technique [0002] Urinary tract infections (UTIs) occupy the second place in the clinical infection rate, and the traditional method (urine culture and identification) takes a long time, and clinicians often do not wait for the results of the laboratory, but choose broad-spectrum antibiotics for treatment. This has caused the drug resistance rate of clinical bacteria to increase year by year. [0003] The diagnosis of UTIs infection is mainly based on clinical symptoms and laboratory results. The patient should wash the vulva before taking the specimen, and take the midstream urine in a sterile container. The main laboratory tests include: urine dry chemical examination, urine sediment analysis, smear microscopy, Gram stain microscopy, and urine culture identification. Urine culture identification is now con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28
Inventor 王新华
Owner 鲁辛辛
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