Method for quickly and accurately identifying microorganism in sample

A technology of microorganisms and samples, applied in the field of microorganism identification, can solve the problem of low positive rate of bacterial culture

Inactive Publication Date: 2017-03-08
浙江和谱生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, this method is still limited by the problem of low bacterial culture positive rate

Method used

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  • Method for quickly and accurately identifying microorganism in sample
  • Method for quickly and accurately identifying microorganism in sample
  • Method for quickly and accurately identifying microorganism in sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Target microorganisms are selectively captured using magnetic beads modified with antibodies against the target microorganisms.

[0046] Take 50 μg of magnetic beads, the diameter of which is about 1 micron, and the surface is modified with recombinant Protein A / G (~50,00 Da). Each set of Protein A / G contains 4 Fc binding sites of Protein A and 2 An Fc-binding site for Protein G. With PBST buffer solution (137mM NaCl, 10mM Na 2 HPO 4 12H 2 O, 2mM NaH 2 PO 4 2H 2 O, 0.05% Tween20) and washed twice (20 μL each time), the magnetic beads were dispersed in 50 μL PBST buffer solution. Add 2 μL of enteropathogenic Escherichia coli antibody to the mixture of the above buffer solution and magnetic beads, and react on a constant temperature mixer for 30 min at room temperature. The reacted magnetic beads were washed three times with blocking solution PBST+1% BSA (20 μL each time), the magnetic beads were separated by a magnet, and dispersed in 50 μL PBST buffer solution. ...

Embodiment 2

[0050] Using human serum immunoglobulin G to achieve broad-spectrum pathogenic microorganism extraction and enrichment.

[0051] Take 100 μL of Escherichia coli ATCC43889 bacterial solution stored at -80°C in a 50 mL centrifuge tube, add 9 mL of liquid tryptone soybean broth (TSB) medium, and mix well. Slightly unscrew the cap of the centrifuge tube and culture in a shaker, set the temperature at 37°C, set the rotational speed at 180rpm, and cultivate for 12 hours. At this time, the bacteria enter the end of the logarithmic phase, the activity is the best, and the bacterial concentration is the largest.

[0052] Take 50 μg of the same magnetic beads as in Example 1, wash twice with 20 μL of PBST buffer solution each time, and then disperse the magnetic beads in 50 μL of PBST buffer solution. Add 2 μL of human serum immunoglobulin G to the mixture of the above buffer solution and magnetic beads, and react on a constant temperature mixer for 30 min at room temperature. The rea...

Embodiment 3

[0057] Using human serum immunoglobulin G to achieve broad-spectrum pathogenic microorganism extraction and enrichment.

[0058] Take 100 μL of Vibrio parahaemolyticus solution stored at -80°C in a 50 mL centrifuge tube, add 9 mL of liquid tryptone soybean broth (TSB) medium, and mix well. Slightly unscrew the cap of the centrifuge tube and culture in a shaker, set the temperature at 37°C, set the rotational speed at 180rpm, and cultivate for 12 hours. At this time, the bacteria enter the end of the logarithmic phase, the activity is the best, and the bacterial concentration is the largest.

[0059] Take 50 μg of the same magnetic beads as in Example 1, wash twice with 20 μL of PBST buffer solution each time, and then disperse the magnetic beads in 50 μL of PBST buffer solution. Add 2 μL of human serum immunoglobulin G to the mixture of the above buffer solution and magnetic beads, and react on a constant temperature mixer for 30 min at room temperature. The reacted magnetic...

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Abstract

The invention provides a method for quickly and accurately identifying microorganism in a sample. The method comprises the following steps: establishing a pure microorganism reference fingerprint mass spectrum library, and using the pure microorganism reference fingerprint mass spectrum library as a standard spectrum; selectively capturing microorganism from the sample containing complicated background matrix by utilizing a functional material; analyzing the microorganism captured by the functional material by using an MALDI (Matrix-Assisted Laser Desorption)-TOF (Time of Flight)-MS (Mass Spectrometry), thus obtaining a microorganism spectrum; comparing the obtained microorganism spectrum with the standard spectrum, and realizing microorganism identification. The method for quickly and accurately identifying the microorganism in the sample, provided by the invention, has a function of extracting and enriching the microorganism in the sample; through an enrichment function, the lowest detection limit of a mass spectrometer is greatly reduced, and the detection sensitivity is increased; microorganism identification is fast, the selectivity is high, the sensitivity is high, and the usage of the sample is less and is just 1 mL.

Description

technical field [0001] The invention belongs to the field of microorganism identification, in particular to a method for rapidly identifying microorganisms by using functional material affinity mass spectrometry. Background technique [0002] Microbial identification is of great significance for clinical diagnosis and food safety. Taking blood bacterial infection as an example, in the identification of bacteria in blood bacterial infection (bacteremia), the diagnosis of infection is difficult due to the atypical clinical manifestations of patients. At present, the gold standard for clinical diagnosis of bacteremia is blood bacterial culture, but the positive rate of blood culture is extremely low, which leads to delay in treatment [P.Phoompoung, M.Chayakulkeeree, Mycopathologia, 2016, 181(5-6), 1 -7; V. Kroumova, E. Gobbato, E. Basso, L. Mucedola, T. Giani, G. Fortina, Rapid Communications in Mass Spectrometry, 2011, 25(15), 2247–2249.]. [0003] Traditional microbial iden...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/64
CPCG01N27/64
Inventor 乔振刚
Owner 浙江和谱生物科技有限公司
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