Repetitive DNA sequence specific for mycobacterium tuberculosis to be used for the diagnosis of tuberculosis

• US 5,168,039 A
• Filed: 09/28/1990
• Issued: 12/01/1992
• Est. Priority Date: 09/28/1990
• Status: Expired due to Term

• Current Assignee: Board of Trustees of The University of Arkansas The University of Arkansas, The Board of Trustees of the University of Arkansas (University of Arkansas System)
• Original Assignee: The Board of Trustees of the University of Arkansas (University of Arkansas System)
• Inventors: Crawford, Jack T., Eisenach, Kathleen D., Bates, Joseph H., Cave, M. Donald
• Primary Examiner(s): YARBROUGH, AMELIA
• Application Number:
  US07/589,819
• Time in Patent Office: 795 Days
• Field of Search: 536/27, 435/6, 435/91, 436/811, 935/78
• US Class Current: 536/24.32
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: Y10S 436/811 Test for named disease, body condition or organ function

Abstract

A novel composition and/or methods for the diagnosis of tuberculosis wherein the composition comprises a repetitive DNA segment that is specific for members of the Mycobacterium tuberculosis complex. The DNA segment repeats in the chromosome of Mycobacterium tuberculosis complex, and is conserved in all copies of the chromosomes. A method comprises using an entire repetitive DNA sequence, or any part thereof, as a hybridization probe for the direct detection of Mycobacterium tuberculosis complex in clinical material. In another method, a smaller portion of an entire repetitive DNA sequence is amplified using polymerase chain reaction, yielding a 123 base-pair product.

Claims

1. A complete, isolated and purified, repetitive DNA segment for use in detecting Mycobacterium tuberculosis complex in clinical material, said DNA segment being isolated in clones, λ

KE55 and λ

KE58, and wherein said clones each contains 400 base-pair Sal1 fragments and 2 flanking Sal1 fragments.

2. The complete, repetitive DNA segment of claim 1, wherein said 400 base-pair Sal1 fragments in λ

KE55 and λ

KE58 bind to each other in a hybridization assay.

3. The complete, repetitive DNA segment of claim 1, wherein said 2 flanking Sal1 fragments in λ

KE55 and λ

KE58 partially hybridize to each other.

4. The complete, repetitive DNA segment of claim 1, wherein said segment of λ

KE55 and λ

KE58 is subcloned into the Sal1 site of pUC19 to produce subclones;

DC51 (λ

KE55), pDC38 (λ

KE58), pDC22 (λ

KE55), pDC61 (λ

KE58), pDC2 (λ

KE55), pDC26 (λ

KE58), pDC106 (pDC51), pDC73 (pDC22), and pDC92 (λ

KE55).

5. The complete, repetitive DNA segment of claim 4, wherein the subclones, pDC22 and pDC61, each consist of a 400 base-pair Sal1 fragment of the complete, repetitive DNA segment.

6. The complete, repetitive DNA segment of claim 4, wherein the subclones, pDC51, pDC2, pDC38, and pDC26, each possesses a Sal1 fragment of the complete, repetitive DNA segment and nonrepetitive flanking sequences.

7. The complete, repetitive DNA segment of claim 4, wherein mapping of said subclones with restriction endonucleases produces a restriction map that includes restriction sites (5'"'"' to 3'"'"') in the complete, repetitive DNA segment:

Sst11, Smal, Dral, Sst11, Sall₁, BamH1, Sall₂, Xho1.

8. The complete, repetitive DNA segment of claim 7, wherein the restriction sites are conserved in all copies of the repetitive DNA segment int he chromosomes of M. tuberculosis complex.

9. The complete, repetitive DNA segment of claim 1, wherein the DNA segment is specific for detecting strains of the M. tuberculosis complex and not nontuberculous mycobacteria in a hybridization assay.

10. A hybridization probe, subclone pDC92, for use in the direct detection of isolates of M. tuberculosis complex in clinical material, comprising a complete, isolated and purified, repetitive DNA segment in the chromosomes of M. tuberculosis complex.