Insertion sequence from a virulent isolate of Burkholderia cepacia, and diagnostic and identification procedures based thereon

• US 6,171,789 B1
• Filed: 04/24/1998
• Issued: 01/09/2001
• Est. Priority Date: 08/17/1995
• Status: Expired due to Fees

• Current Assignee: Her Majesty The Queen In Right of Canada As Represented By The Minister of National Defence
• Original Assignee: Her Majesty The Queen In Right of Canada As Represented By The Minister of Industry (The Republic of Canada)
• Inventors: Johnson, Wendy M., Tyler, Shaun D., Rozee, Kenneth R.
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US09/011,197
• Time in Patent Office: 991 Days
• Field of Search: 536/23.1, 536/24.3, 435/6, 435/91.2, 935/76, 935/77, 935/78
• US Class Current: 435/6.15
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

A novel IS (insertion sequence) element characteristic of a virulent isolate (ET12/cblA isolate) of Burkholderia cepacia. The IS element is a hybrid of two other IS elements commonly found in isolates of B. cepacia, namely IS402 [SEQ ID NO:2] and IS1356 [SEQ ID NO:3]. The IS hybrid IS element has the sequence shown in the Figure [SEQ ID NO:1]. The fact that the hybrid IS element is characteristic of the virulent isolate means that it can be used as an indicator of that isolate. Thus, the invention includes a method of testing for an ET12/cblA isolate of B. cepacia by testing a sample for the presence of the hybrid IS element, eg. by amplification of the IS element by PCR and identification of the amplified sequence.

Claims

1. An isolated and purified insertion element characteristic of the ET12 strain of Burkholderia cepacia which insertion element consists of SEQ ID NO:

1 or a minor variant thereof not rendering the sequence biologically or genetically unrecognizable from SEQ ID NO;

1.

2. A method of testing for a virulent isolate of Berkholderia cepacia in a sample which comprises:

a) testing said sample for the presence or absence of an insertion element which insertion element consists of SEQ ID NO;

1 or a minor variant thereof not rendering the sequence biologically or genetically unrecognizable from SEQ ID NO;

1;

b) providing an indication of the presence or absence of said insertion element thereby indicating the presence or absence of a virulent isolate of Berkholderia cepacia in said sample.

3. The method of claim 2, wherein said sequence is tested for by a method based on polymerase chain reaction or ELISA.

4. The method of claim 3, comprising amplifying said insertion element sequence by polymerase chain reaction to form an amplified sequence, and indicating the presence of said sequence.

5. The method of claim 4, wherein the presence of said amplified sequence is indicated by identifying the size (DNA length) of the amplified sequence, by gel elecrophoresis, by means of labeled primers used for said polymerase chain reaction, which primers are incorporated into the amplified sequences, or by means of a labeled DNA probe for annealing with a unique part of the amplified sequence followed by Southern blot analysis.

6. A diagnostic test kit for detecting a virulent isolate of Berkholderia cepacia in a sample which comprises:

a) reagents required to carry out PCR;

b) primers capable of amplifying by PCR a hybrid insertion sequence consisting of SEQ ID NO;

1 or a unique portion thereof;

c) a positive control nucleic acid sequence which sequence comprises an isolated and purified insertion element characteristic of the ET12 strain of Burkholderia cepacia which insertion element consists of SEQ ID NO;

1 or a minor variant thereof not rendering the sequence biologically or genetically unrecognizable from SEQ ID NO;

1.