Kit for rapid mass spectrometric detection of leptospira

A leptospira and spirochete technology, applied in the field of protein fingerprint detection, can solve the problems of no leptospira, unable to identify leptospira, unable to identify pathogenicity, etc., and achieve the effect of eliminating the hazards of personnel and the environment

Active Publication Date: 2013-10-09
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no standard peptide spectrum of Leptospira in these two sets of commercia

Method used

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  • Kit for rapid mass spectrometric detection of leptospira
  • Kit for rapid mass spectrometric detection of leptospira
  • Kit for rapid mass spectrometric detection of leptospira

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Liquid culture Leptospira sample pretreatment optimization

[0053] Due to the complex composition of the liquid medium, there are many factors that affect the quality of the mass spectrometer, so the effective removal of the medium components of the sample before bacterial protein extraction is very critical. Bacteria washing is an effective way to effectively reduce the influence of medium components.

[0054] The formula of the bacteria washing solution A used in this example is 0.2g of potassium dihydrogen phosphate, 2.9g of disodium hydrogen phosphate, 8.0g of sodium chloride, 0.2g of potassium chloride, Tween-200.5mL, add water to 1000mL, use hydrochloric acid Adjust the pH to 7.4 and autoclave.

[0055] The main washing step of the bacteria is completed by the bacteria washing solution A. In this embodiment, the Leptospira are not washed with the washing solution A, and are washed once with the washing solution A for peptide fingerprint analysis. After...

Embodiment 2

[0059] Embodiment 2 Construction of Leptospira mass spectrometry model

[0060] 1. Samples and instruments

[0061] Forty-three strains of pathogenic Leptospira and 22 strains of non-pathogenic Leptospira were used, and 15 strains of ATCC international standard strains were randomly selected for the construction of mass spectrometry models (Table 1). The remaining 35 strains were used for model testing. All strains have been confirmed pathogenicity by PCR using G1 / G2, B64-I / B64-II as primers and 16SrRNA.

[0062] Table 1. List of strains for model construction

[0063]

[0064] 2. Cell pretreatment

[0065] Take 5 mL of Leptospira bacterial suspension cultured to the logarithmic growth phase, centrifuge at 12000g for 5min, discard the upper liquid medium; wash the bacterial cell sediment with cell washing solution A at 12000g, centrifuge for 5min, and discard the supernatant; Add 200 μL bacterial cell washing solution B, mix well, add 600 μL bacterial cell washing solut...

Embodiment 3

[0074] Example 3 Leptospira mass spectrometry model verification

[0075] Sensitivity (sensitivity), also known as true positive rate (true positive rate), is actually the pathogen and is correctly judged as the percentage of the pathogen according to the standard of the detection method. Specificity, also known as true negative rate, is the percentage that is actually not the pathogen but is correctly judged as the pathogen according to the standard of the detection method.

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Abstract

The invention provides a kit for rapid mass spectrometric detection of leptospira pathogenicity (toxicity) and relates to the technical field of protein fingerprinting detection. The kit is characterized in that based on a mass spectrometry technology, a specific polypeptide mass spectrum model for rapid identification of pathogenicity of leptospira is constructed and by the mass spectrometry technology, pathogenicity detection is carried out. The kit can realize identification of pathogenic (toxic) and non-pathogenic (nontoxic) leptospira by a specific fingerprint, has high accuracy and good repeatability, can realize detection of 96 samples in 2h, can solve the problem that pathogenicity detection of mass leptospira consumes labor and time, and has a low cost and a good application prospect.

Description

technical field [0001] The invention relates to the technical field of protein fingerprint detection, in particular to a mass spectrometry model for detecting Leptospira and a kit for pathogenicity detection based on the mass spectrometry model. Background technique [0002] Leptospirosis is a widely distributed zoonotic disease caused by pathogenic Leptospira. Its prevalence is almost all over the world, especially in Southeast Asia, and it can occur all year round. Leptospira belongs to the genus Leptospira in the spirochete family Spirochetaceae and is divided into two species: Leptospira interrogans (pathogenic) and Leptospira hyperbolic (non-pathogenic). Leptospirosis is a systemic infectious disease. In severe cases, symptoms of acute inflammatory liver injury and kidney injury such as jaundice, hemorrhage, uremia, etc. may occur, and meningeal inflammatory symptoms such as mental disturbance and meningeal irritation may also occur; In severe cases, liver and kidney f...

Claims

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Application Information

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IPC IPC(8): G01N27/64
Inventor 肖迪张翠彩张建中蒋秀高张慧芳李秀文
Owner ICDC CHINA CDC
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