# US 5,352,580 A · Selective detection of mycobacteria by nucleicacid probes derived from Mycobacterium kansasii

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- Number: US 5,352,580 A
- Document ID: US5352580A
- Status: Granted
- Kind: A
- Application number: 07/889,651
- Filed: 1993-05-26
- Granted: 1994-10-04
- Earliest claimed: 1993-05-26
- Assignees: Becton, Dickinson and Company
- Inventors: Patricia A. Spears; Daryl D. Shank
- Web page: https://patentgrep.com/patent/US5352580A

## CPC

- C12Q 1/689: for bacteria
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Oligonucleotide probes derived from Mycobacteria Kansasii capable of selectively hybridizing to Mycobacteria nucleic acid are disclosed. The oligonucleotide probe is selected from the group consisting of: (a) an oligonucleotide probe consisting essentially of the DNA sequence given herein as SEQ ID NO:1 (MK14); (b) oligonucleotide probes comprising fragments of MK14 which retain the capability of MK14 of selectively hybridizing to Mycobacteria nucleic acid; (c) oligonucleotide probes which hybridize to MK14 and are capable of selectively hybridizing to Mycobacteria nucleic acid; and (d) oligonucleotide probes which are complementary to any of the foregoing and are capable of selectively hybridizing to Mycobacteria nucleic acid. The probes are useful in nucleic acid amplification and hybridization assays for genus-specific detection of the mycobacteria, specific detection of M. kansasii and specific detection of the slow-growing mycobacteria. Methods of using the probes to detect and amplify Mycobacteria nucleic acid and kits containing the same are also disclosed.

## Claims

### Claim 1

An oligonucleotide probe capable of selectively hybridizing to Mycobacteria nucleic acid, said oligonucleotide probe selected from the group consisting of:
(a) an oligonucleotide probe consisting of a DNA sequence selected from the group consisting of DNA sequences given herein as SEQ ID NO: 1, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:25, and;
(b) an oligonucleotide probe consisting of a DNA sequence selected from the group consisting of the complementary DNA sequence to any of the probes of (a) and are capable of selectively hybridizing to Mycobacteria nucleic acid.

### Claim 2 (depends on 1)

An oligonucleotide probe according to claim 1 selected from the group consisting of probes consisting of the sequences given herein as SEQ ID NO:1 (MK14), SEQ ID NO:14 (MK14-C) and SEQ ID NO:21 (MK14-C2) which is capable of specifically hybridizing to nucleic acid of the genus Mycobacteria.

### Claim 3 (depends on 1)

An oligonucleotide probe according to claim 1 selected from the group consisting of probes consisting of the sequences given herein as SEQ ID NO:20 (MK14-C1) and SEQ ID NO:25 which is capable of specifically hybridizing to nucleic acid of slow growing Mycobacteria.

### Claim 4

An oligonucleotide probe selected from the group consisting of an oligonucleotide probe consisting of the DNA sequence given herein as SEQ ID NO:22 (M. Kansasii fragment BC), an oligonucleotide probe consisting of the DNA sequence given herein as SEQ ID NO:23 (M. tuberculosis fragment BC), and an oligonucleotide probe consisting of the sequence given herein as SEQ ID NO:24 (M. avium fragment BC).

### Claim 5 (depends on 1)

An oligonucleotide probe according to claim 1 in which guanine plus cytosine comprise less than 70% of the total nucleotide composition of said probe.

### Claim 6

A strand displacement amplification oligonucleotide probe which selectively hybridizes to Mycobacteria nucleic acid, said oligonucleotide probe consisting of:
(a) a DNA sequence selected from the group consisting of DNA sequences given herein as SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:25 and the complementary DNA sequence of SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:25,
(b) a restriction enzyme recognition site positioned 5' of the DNA sequence of (a), and
(c) a flanking DNA sequence positioned 5' of the restriction enzyme recognition site of (b) which facilitates binding of a restriction enzyme specific for the restriction enzyme recognition site.

### Claim 7 (depends on 1)

An oligonucleotide probe according to claim 1 labelled with a detectable marker.

### Claim 8

An oligonucleotide probe capable of selectively hybridizing to Mycobacteria kansasii nucleic acid, said oligonucleotide probe selected from the group consisting of:
(a) an oligonucleotide probe selected from the group consisting of probes consisting of the DNA sequences given herein as SEQ ID NO:15 (MK14-D) and SEQ ID NO:13, and;
(b) an oligonucleotide probe consisting of a DNA sequence selected from the group consisting of the complementary DNA sequence to any of the foregoing oligonucleotide probes and arc capable of selectively hybridizing to Mycobacteria kansasii nucleic acid.

### Claim 9 (depends on 8)

An oligonucleotide probe according to claim 8 consisting of the DNA sequence given herein as SEQ ID NO:15 (MK14-D).

### Claim 10 (depends on 8)

An oligonucleotide probe according to claim 8 in which guanine plus cytosine comprise less than 70% of the total nucleotide composition of said probe.

### Claim 11 (depends on 8)

An oligonucleotide probe according to claim 8 labelled with a detectable marker.

### Claim 12

A method of detecting Mycobacteria nucleic acid in a nucleic acid sample, comprising:
(a) contacting an oligonucleotide probe to the nucleic acid sample under conditions permitting the hybridization of said oligonucleotide probe to Mycobacteria nucleic acid, wherein said oligonucleotide probe is selected from the group consisting of
(i) an oligonucleotide probe selected from the group consisting of probes consisting of the DNA sequences given herein as SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:25, and;
(ii) an oligonucleotide probe consisting of a DNA to any of the probes in (i) and which is capable of selectively hybridizing to Mycobacteria nucleic acid; and then
(b) detecting whether or not said oligonucleotide probe hybridizes to said nucleic acid sample, the hybridization of the oligonucleotide probe to the nucleic acid sample indicating that the nucleic acid sample contains Mycobacteria nucleic acid.

### Claim 13 (depends on 12)

A method according to claim 12, wherein said nucleic acid sample is DNA.

### Claim 14 (depends on 12)

A method according to claim 12, wherein said nucleic acid sample is RNA.

### Claim 15 (depends on 12)

A method according to claim 12, wherein said oligonucleotide probe is labelled with a detectable marker, and wherein said detecting step is carried out by detecting said detectable marker.

### Claim 16 (depends on 12)

A method according to claim 12, further comprising a strand displacement amplification step preceeding said detecting step.

### Claim 17 (depends on 12)

A method according to claim 12, further comprising a polymerase chain reaction amplification step preceeding said detecting step.

### Claim 18 (depends on 12)

A method according to claim 12, further comprising a ligase chain reaction amplification step preceeding said detecting step.

### Claim 19

A method of selectively amplifying a target sequence present in slow growing Mycobacteria in a nucleic acid sample, comprising:
(a) contacting at least one pair of oligonucleotide probes to the nucleic acid sample under conditions permitting the hybridization of each of said oligonucleotide probes to the Mycobacteria target sequence, wherein said pair of probes together comprise an amplification pair,
said pair of oligonucleotide probes consisting of the DNA sequences given herein as SEQ ID NO:2 and SEQ ID NO:9; and
(b) amplifying said Mycobacteria target sequence by cyclically reacting said at least one pair of oligonucleotide probes with said nucleic acid sample to produce an amplification product.

### Claim 20 (depends on 19)

A method according to claim 19, wherein said amplifying step is carried out by strand displacement amplification.

### Claim 21 (depends on 19)

A method according to claim 19, wherein said amplifying step is carried out by polymerase chain reaction.

### Claim 22

A kit for detecting Mycobacteria nucleic acid in a nucleic acid sample, comprising a hybridization solution together with an oligonucleotide probe selected from the group consisting of
(i) an oligonucleotide probe selected from the group consisting of probes consisting of the DNA sequences given herein as SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:25, and;
(ii) an oligonucleotide probe consisting of a DNA sequence selected from the group consisting of the complementary DNA sequence to any of the probes of (i) and which is capable of selectively hybridizing to Mycobacteria nucleic acid.

### Claim 23 (depends on 22)

A kit according to claim 22, wherein said oligonucleotide probe is lyophylized.

### Claim 24 (depends on 22)

A kit according to claim 22, wherein said oligonucleotide probe is carried in said hybridization solution.

### Claim 25 (depends on 22)

A kit according to claim 22, said oligonucleotide probe selected from the group consisting of probes consisting of the sequences given herein as SEQ ID NO:1, SEQ ID NO:14 and SEQ ID NO:21 and capable of selectively detecting nucleic acid of the genus Mycobacteria.

### Claim 26 (depends on 22)

A kit according to claim 22, said oligonucleotide probe selected from the group consisting of oligonucleotide probes consisting of the sequences given herein as SEQ ID NO: 15 and SEQ ID NO:13 and capable of selectively detecting nucleic acid of Mycobacterium kansasii.

### Claim 27 (depends on 22)

A kit according to claim 22, said oligonucleotide probe selected from the group consisting of an oligonucleotide probe consisting of the DNA sequence given herein as SEQ ID NO:22 (M. Kansasii fragment BC), an oligonucleotide probe consisting of the DNA sequence given herein as SEQ ID NO:23 (M. tuberculosis fragment BC), and an oligonucleotide probe consisting of the sequence given herein as SEQ ID NO:24 (M. avium fragment SC).

### Claim 28 (depends on 22)

A kit according to claim 22, said oligonucleotide probe selected from the group consisting of probes consisting of the sequences given herein as SEQ ID NO:20 and SEQ ID NO:25 and capable of selectively hybridizing to nucleic acid of slow growing Mycobacteria.

### Claim 29 (depends on 12)

A method according to claim 12 wherein the nucleic acid sample is contacted with an oligonucleotide probe selected from the group consisting of probes consisting of the sequences given herein as SEQ ID NO:1, SEQ ID NO:14 and SEQ ID NO:21 and nucleic acid of the genus Mycobacteria is detected.

### Claim 30 (depends on 12)

A method according to claim 12 wherein the nucleic acid sample is contacted with an oligonucleotide probe selected from the group consisting of probes consisting of the sequences given herein as SEQ ID NO:15 and SEQ ID NO:13 and hybridization to Mycobacteria kansasii is detected.

### Claim 31 (depends on 12)

A method according to claim 12 wherein the nucleic acid sample is contacted with an oligonucleotide probe consisting of the sequence given herein as SEQ ID NO:25 and hybridization to nucleic acid of slow growing Mycobacteria is detected.