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Database establishment method and identification method of BALDI-TOF MS strain

A technique for establishing a method and Bartonella, which is applied in the field of microbial mass spectrometry detection, can solve problems such as impossibility and limitation of technology application and development, and achieve the effects of saving operation steps, shortening identification time, and easy operation

Inactive Publication Date: 2020-06-05
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] There is no domestic research on the identification method of Bartonella mass spectrometry, and there is no reference protein fingerprint of Bartonella in the database of imported and domestic microbial MALDI-TOFMS instruments. General identification, which limits the application and development of this technology in the diagnosis and control of Bartonella-related diseases

Method used

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  • Database establishment method and identification method of BALDI-TOF MS strain
  • Database establishment method and identification method of BALDI-TOF MS strain
  • Database establishment method and identification method of BALDI-TOF MS strain

Examples

Experimental program
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Effect test

Embodiment 1，200、21MALDI-TOF MS ，11ATCC(1 no. 3)。 Embodiment 2

[0078] In Example 1, 200 strains and 21 Bartonella strains were selected for establishing a MALDI-TOF MS identification database, which contained 11 Bartonella ATCC standard bacteria (column 3 in Table 1). In Example 2, 173 strains of Bartonella wild isolates were used to evaluate the identification reliability of the established database (column 4 in Table 1). The experimental strains used were provided by the Vector Biology Control Office of the Institute of Infectious Diseases Control, Chinese Center for Disease Control and Prevention.

[0079] Table 1: Strain information used to establish and evaluate the Bartonella MALDI-TOF-MS database

[0080]

[0081] Note: 1. * is pathogenic Bartonella; 2. "This laboratory" is the vector biological control room of the Chinese Center for Disease Control and Prevention; the Bartonella strains of "this laboratory" can be obtained from public strains It can be obtained from the preservation center or isolated using existing technology...

Embodiment 1

[0083] The present embodiment is the establishment method of the Bartonella database utilizing MALDI-TOF MS, and the method comprises the following steps:

[0084] (1) Cultivation and identification of Bartonella strains

[0085] Among the Bartonella strains (alternative strains) in Table 1, the culture conditions for Bacilliformis (B.bacilliformis) are: at 28°C, 5% CO 2 In the incubator, resuscitate culture on TSA medium of 10% defibrinated sheep blood. The culture conditions for other Bartonella strains are: at 37°C, 5% CO 2 In the incubator, cultivate under the environment of 70% humidity, and revive the culture on the TSA medium of 5% defibrated sheep blood. Harvest the fresh bacteria from the 4th to 6th day of culture according to the growth rate of the colony.

[0086] Using primers BhCS.781p-BhCS.1137n to amplify the specific gene of Bartonella genus citrate synthase gene (gltA) for taxonomic identification [6] . The PCR primers used for identification are as follo...

Embodiment 2

[0107] Embodiment 2: wild strain actual measurement

[0108] This embodiment utilizes the Bartonella standard database of MALDI-TOF MS established in Example 1 to identify whether the strains to be tested (14 species, 173 strains in total) are Bartonella. The method comprises the following steps:

[0109] (1) Cultivation and identification of Bartonella strains:

[0110] Resuscitate and culture the strain to be tested to obtain fresh bacteria; then perform PCR reaction and sequence on the fresh bacteria to determine whether it is Bartonella. The specific operation method and parameters are as described in Example 1.

[0111] (2) Sample pretreatment:

[0112] The identified fresh bacteria were pretreated to obtain a sample protein solution. The specific operation method and parameters are as described in Example 1.

[0113] (3) Target plate preparation:

[0114] Add the sample protein solution dropwise to the target position to prepare a target plate. The specific operati...

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Abstract

The invention belongs to the field of microbial mass spectrometry detection, and particularly relates to a database establishment method and an identification method of a Baltonella strain by utilizing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The establishment method comprises the following steps: preparing a target plate and detecting MALDI-TOFMS to form a Baltonella strain standard database; the identification method comprises the following steps: carrying out MALDI-TOF MS detection on a to-be-detected strain to obtain a mass spectrum of the to-be-detected strain, and analyzing in the database to obtain an identification score. The method is based on an autonomously established Bartonella protein fingerprint spectrum database, and thedatabase comprises 200 and 21 Bartonella reference strains which cover all important pathogenic species; the Bartonella is a causticized bacterium and is difficult to grow, and the database already covers enough Bartonella strains; the MALDI-TOF MS can be used for rapidly and accurately identifying the Bartonella, the identification time is shortened, the operation steps are reduced, and the method has important significance in clinical laboratory inspection and diagnosis of the Bartonella.

Description

technical field [0001] The invention belongs to the field of microbial mass spectrometry detection, and in particular relates to a database establishment method and an identification method for Bartonella strains using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Background technique [0002] Bartonella species are a group of aerobic bacilli with harsh nutritional requirements and Gram-negative strains that parasitize mammals. They have a wide range of hosts and are distributed globally. Some species are pathogenic to humans and animals. Taxonomically they belong to α - Proteobacteria, Rhizobiales, Bartonellaceae, currently 33 species of Bartonella have been officially named, and their related pathogenic bacteria include Brucella that causes human brucellosis and plant pathogenic fungus rhizocarcinoma Agrobacterium. Bartonella invades mammals, replicates and reproduces in the body, and continuously infects red blood cells to f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/62G01N33/68
CPCG01N27/62G01N33/6851
Inventor 栗冬梅刘起勇徐爱玲宋秀平杜小莉崔志刚
Owner ICDC CHINA CDC
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