Probe for DNA and a process for the detection of "shigellae" and entero-invasive strains of Escherichia coli

• US 4,816,389 A
• Filed: 07/12/1985
• Issued: 03/28/1989
• Est. Priority Date: 07/13/1984
• Status: Expired due to Fees

• Current Assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm) (Allianz Global Investors GmbH), Institut Pasteur
• Original Assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm) (Allianz Global Investors GmbH), Institut Pasteur
• Inventors: Sansonetti, Philippe, Boileau, Catherine, D'Hauteville, Helene
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US06/755,051
• Time in Patent Office: 1,355 Days
• Field of Search: 435/6, 435/29, 435/172.3, 435/317, 435/810, 436/501, 436/808, 536/27, 935/29, 935/78
• US Class Current: 435/6.15
• CPC Class Codes: C07K 14/25 Shigella (G)
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/808 Automated or kit

Abstract

The invention concerns a probe for the detection of shigellae and entero-invasive E. coli, containing a nucleic acid sequence originating from the 140 Mdal virulence plasmid of the M 90 T strain of Shigella flexneri; having a maximum size of around 27 kb and inlcuding all or part of the 27 kb Bam HI fragment.

This probe permits the in vitro diagnosis of syndromes of dysentery or diarrhea, of the Shigellosis type.

Claims

1. A kit for the detection of virulent Shigellae and entero-invasive Escherichia coli microorganisms, wherein the kit comprises:

(A) a container means containing a probe comprising a nucleic acid sequence which is present in a 140 Mdal virulence plasmid of a M 90 T strain of Shigellae flexneri, wherein said nucleic acid sequence has a maximum size of about 27 kb and the nucleic acid sequence includes all or part of a 27 kb BamHI fragment of said 140 Mdal plasmid and a 17 kb EcoRI fragment of said 140 Mdal plasmid, wherein said 17 kb fragment has a part in common with said BamHI fragment; and

(B) a container means containing a control preparation of nucleic acid of an avirulent strain of a microorganism or of nucleic acid of a stool from a healthy individual.

2. A process for detecting in vitro the presence of a cell of a virulent Shigellae or entero-invasive Escherichia coli, wherein the process comprises the steps of:

(A) depositing and fixing nucleic acids of the cell on a filter, so as to make the nucleic acids accessible to a probe;

(B) contacting said fixed nucleic acids from step (A) with a labelled probe under conditions permitting hybridization;

(C) washing said filter resulting from step (B), so as to eliminate any non-hybridized probe; and

then

(D) detecting any hybridized probe on said washed filter resulting from step (C);

wherein said labelled probe comprises;

a nucleic acid sequence which is present in a 140 Mdal virulence plasmid of a M 90 T strain of Shigellae flexneri, wherein said nucleic acid sequence has a maximum size of about 27 kb, and the nucleic acid sequence includes all or part of a 27 kb BamHI fragment of said 140 Mdal plasmid and a 17 kb EcoRI fragment of said 140 Mdal plasmid, wherein said 17 kb fragment has a part in common with said 27 kb BamHI fragment; and

wherein the probe has a label selected from the group consisting of radioactive, enzymatic, fluorescent, and luminescent labels.