Nucleotide sequences for detection of Bacillus anthracis

• US 6,448,016 B1
• Filed: 06/12/2001
• Issued: 09/10/2002
• Est. Priority Date: 06/12/2001
• Status: Expired due to Fees

• Current Assignee: United States Of America As Represented By The Secretary Of The Army (Government of the United States of America)
• Original Assignee: The United States Of America As Represented By The Secretary Of The Army
• Inventors: Cheng, Tu-Chen, Rastogi, Vipin K.
• Primary Examiner(s): Myers, Carla J.
• Application Number:
  US09/879,027
• Time in Patent Office: 455 Days
• Field of Search: 435/6, 435/91.2, 435/810, 536/23.1, 536/23.7, 536/24.32, 536/24.33
• US Class Current: 435/6.12
• CPC Class Codes: C07K 14/32 from Bacillus (G)
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

The invention provides purified and isolated DNA fragments from Bacillus anthracis chromosomal DNA, primer sets and probes derived therefrom, as well as kits and detection methods for B. anthracis. The methods of the invention provide for specific detection of anthrax over closely related strains of Bacillus, as well as accurate detection of low numbers of B. anthracis in an environmental sample containing large amounts of non-specific DNA. The invention is applicable to food, health care, and military applications.

Claims

1. An isolated and purified DNA fragment from chromosomal DNA of B. Anthracis consisting essentially of the nucleotide sequence of SEQ ID NO:

2.

2. A pair of forward and reverse oligonucleotide primers for use in the amplification-based detection of B. Anthracis, said primer pairs consisting of at least 10 to 30 contiguous nucleotides of SEQ ID NO:

2 and wherein said primer pairs specifically amplify B. Anthracis DNA and do not amplify DNA from related strains of B. cereus, B. thuringienis, and B. subtilis.

3. The primer pairs according to claim 2, wherein said primer pairs have nucleotide sequences represented by SEQ ID NO:

8 and SEQ ID NO;

9.

4. The primer pairs according to claim 2, further including a detectable label.

5. An oligonucleotide probe for use in hybridization-based detection of B. anthracis consisting of a fragment from the nucleotide sequence of SEQ ID NO:

2 and which specifically binds complementary strand DNA from B. anthracis and does not bind DNA from related strains B. cereus, B. thuringiensis, and B. subtilis.

6. The oligonucleotide probe according to claim 5, wherein said probe is bound to a solid support.

7. The probe according to claim 5, wherein said probe further includes a detectable label.

8. A method for detection of B. anthracis in an environmental sample containing non-specific DNA, comprising the steps of:

(a) providing a pair of primers, wherein said primers consist of at least 10 to 30 contiguous nucleotides of SEQ ID NO;

2 and wherein said primer pairs specifically amplify B. Anthracis DNA and do not amplify DNA from related strains of B. cereus, B. thuringienis, and B. subtilis;

(b) mixing said primers with DNA isolated from said environmental sample;

(c) amplifying any DNA to which the primers in step (b) anneal by use of polymerase chain reaction; and

(d) detecting any B. anthracis DNA in said environmental sample based on the amplification products of step (c).

9. The method of claim 8, wherein said primer pairs have nucleotide sequences represented by SEQ ID NO:

8 and SEQ ID NO;

9.

10. The method of claim 8, wherein said primer pairs further include a detectable label.

11. A method for detection of B. anthracis in an environmental sample, comprising the steps of:

(a) providing at least one oligonucleotide probe, wherein said probe consists of a fragment from the nucleotide sequence of SEQ ID NO;

2 and which specifically binds complementary strand DNA from B. anthracis and does not bind DNA from related strains B. cereus, B. thuringiensis, and B. subtilis;

(b) conjugating said probe to a solid support;

(c) contacting said environmental sample with said support-bound probe formed in step (b) under conditions favorable for hybridization; and

(d) detecting any B. anthracis DNA in said sample based on the hybridization products of step (c).

12. The method of claim 11, wherein said probe further includes a detectable label.

13. A kit for the detection of B. anthracis, comprising:

(a) a carrier to receive therein one or more containers; and

(b) at least one of said containers including a pair of oligonucleotide primers wherein said primers consist of at least 10 to 30 contiguous nucleotides of SEQ ID NO;

2 and wherein said primer pairs specifically amplify B. Anthracis DNA and do not amplify DNA from related strains of B. cereus, B. thuringienis, and B. subtilis.

14. The kit of claim 13, wherein said primers further include a detectable label.

15. The kit of claim 13, further comprising a second container containing primers for amplifying the TOX gene and the CAP gene of B. anthracis.

16. A kit for the detection of B. anthracis, comprising:

(a) a carrier to receive therein one or more containers; and

(b) at least one of said containers including an oligonucleotide probe, wherein said probe consists of a fragment from the nucleotide sequence of SEQ ID NO;

2 and which specifically binds complementary strand DNA from B. anthracis and does not bind DNA from related strains B. cereus, B. thuringiensis, and B. subtilis.

17. The kit of claim 16, wherein said probe further includes a detectable label.

18. The kit of claim 16, further comprising a second container containing primers for amplifying the TOX gene and the CAP gene of B. anthracis.