Identifying micro-organisms

A microbial and biomarker technology, applied in biological testing, microbial determination/inspection, biological material analysis, etc., can solve the problem of not recommending affinity purification and the use of biomarkers

Inactive Publication Date: 2004-10-06
THE SEC OF STATE FOR DEFENCE IN HER BRITANNIC MAJESTYS GOVERNMENT OF THE UK OF GREAT BRITAIN & NORTHERN IRELAND
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] Direct mass spectrometric analysis of viral proteins has also been reported (WO 99 / 58727), but it does not suggest affinity purification and use of common biomarkers

Method used

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  • Identifying micro-organisms
  • Identifying micro-organisms
  • Identifying micro-organisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Automatic sampling and identification system

[0057] Referring to Figure 1, a vacuum device (not shown) is used to obtain a sample from an aerosol suspected of containing pathogenic bacteria. The aerosol is mixed with the carrier liquid, and the suspension is fed into the system (1) through the sampler (2). From there, the suspension is transported to the ultrasonic sound generator (4), in which the cell wall of any bacteria in the suspension is broken up with ultrasound, thereby releasing the constituent proteins of the bacteria into the lysis solution. Inevitably, the lysate also contains fragments. Therefore, downstream of the ultrasonic sound generator (4) is a filter (5), and the filter (5) prevents unwanted substances from passing through. In some cases, the use of detergents can promote lysis, which should not affect the downstream immunoaffinity step. Suitable mild non-ionic detergents are well known in the art, including polyoxyethylene-based detergents ...

Embodiment 2

[0061] Example 2 Using Hsp60 as a biomarker to identify potential pathogens

[0062] The average molecular weight of Hsp60 obtained from a variety of organisms can be predicted with high accuracy from the known amino acid sequence (corrected based on the presence of isotopic mixtures) or directly measured with a suitable purified recombinant protein. Although Hsp60 is highly conserved in many species (not only bacteria), mass spectrometry can determine the molecular weight with a high degree of accuracy, and can distinguish protein molecules with only three molecular weight units. Comparing these measured values ​​with the known value database can identify the relevant species, as shown in Table 1.

[0063] Hsp60M r

Bacteria

58015.3Da

Chlamydia trachomatis

57301.7Da

Francisella tularensis

57154.8Da

Salmonella typhimurium

56757.3Da

Burkholderia pseudomallei

[0064] Figure 2 shows the molecular weight of Hsp60 in a va...

Embodiment 4

[0148] Example 4 Comparison of Arg-CHsp60 peptides of Brucella of the genus Brucella and Staphylococcus epidermidis

[0149] The endopeptidase Arg-C (Rhizobacter protease), as its name implies, cleaves the carboxy-peptide bond of arginine. Figure 4 is the peptide fingerprint obtained by Arg-C digestion of Brucella and Staphylococcus epidermidis Hsp60 Icon Compare. As shown in Figure 3, the molecular weights of intact Hsp60 proteins from these organisms are similar (57649 and 57529, respectively, including N-terminal methionine). However, the peptide set obtained is very different and characterizes related organisms.

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Abstract

A method of rapidly identifying unknown micro-organisms by means of mass spectrometry of biomarkers that are isolated from lysates of the micro-organisms on the basis of their structural similarity across a number of species. Also disclosed are said biomarkers, in particular Hsp60.

Description

Technical field [0001] The present invention relates to a method for the rapid identification of microorganisms (such as bacteria). Background technique [0002] There are many situations where it is necessary to be able to quickly detect potentially pathogenic organisms, such as bacteria and viruses. Current laboratory methods typically include culturing organisms and applying immunodiagnostic tests, or preparing histological samples and applying special staining and / or immunohistochemical techniques. These techniques require at least a few hours, and if the organism is to be cultured, it will take several days. DNA-based identification, such as PCR, is more sensitive, but it still takes several hours and requires considerable experimental equipment and experts. [0003] It is standard technical practice to identify specific microorganisms by specific polyclonal antiserum and monoclonal antibodies. Immunohistochemical methods can identify organisms present ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/281C12Q1/48G01N27/62G01N30/72G01N30/88G01N33/543G01N33/569G01N33/68
CPCG01N33/6848G01N2333/912G01N33/569G01N33/68
Inventor R·W·蒂特巴尔D·德斯佩罗克斯
Owner THE SEC OF STATE FOR DEFENCE IN HER BRITANNIC MAJESTYS GOVERNMENT OF THE UK OF GREAT BRITAIN & NORTHERN IRELAND
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