Nucleotide sequences for detection of Bacillus anthracis
AssigneeThe United States of America as represented by the Secretary of the Army
InventorsVipin K. Rastogi, Tu-Chen Cheng
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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.
Claims16
An isolated and purified DNA fragment from chromosomal DNA of B. anthracis consisting essentially of the nucleotide sequence of SEQ ID NO:5.
An isolated pair of forward and reverse oligonucleotide primers for use in the amplification-based detection of B. anthracis, each of said forward and reverse primers consisting of at least 20 to 30 contiguous nucleotides of SEQ ID NO:5 and optionally containing a detectable label, and wherein said forward and reverse primers specifically amplify B. anthracis DNA and do not amplify DNA from related strains of B. cereus, B. thuringienis, and B. subtilis.
An isolated oligonucleotide probe for use in hybridization-based detection of B. anthracis, said probe consisting of a fragment of at least 30 nucleotides from the nucleotide sequence of SEQ ID NO:5 which specifically binds complementary strand DNA from B. anthracis and does not bind DNA from related strains B. cereus, B. thuringiensis, and B. subtilis, and wherein said probe optionally includes a detectable label.
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 each of said primers consist of at least 20 to 30 contiguous nucleotides of SEQ ID NO:5 and wherein said pair of primers specifically amplify B. anthracis DNA and do not amplify DNA from related strains of B. cereus, B. thuringienis, and B. subtilis;
- and wherein said primers optionally include a detectable label;
- (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).
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:5 and which specifically binds complementary stand DNA from B. anthracis and does not bind DNA from related strains B. cereus, B. thuringiensis, and B. subtilis, said probe optionally including a detectable label;
- (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).
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 each of said primers consist of at least 20 to 30 contiguous nucleotides of SEQ ID NO:5 and wherein said pair of primers specifically amplify B. anthracis DNA and do not amplify DNA from related strains of B. cereus, B. thuringienis, and B. subtilis said pair of primers optionally including a detectable label.
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 of at least 30 nucleotides from the nucleotide sequence of SEQ ID NO:5 and which specifically binds complementary stand DNA from B. anthracis and does not bind DNA from related strains B. cereus, B. thuringiensis, and B. subtilis, said probe optionally including a detectable label.
Dependencies are inferred from the claim text.