METHOD FOR DETECTING A BACTERIAL PATHOGEN

• US 20090081656A1
• Filed: 03/27/2008
• Published: 03/26/2009
• Est. Priority Date: 03/30/2007
• Status: Active Grant

• Current Assignee: Case Western Reserve University
• Original Assignee: Case Western Reserve University
• Inventors: Ikegami, Akihiko, Han, Yiping W.
• Granted Patent: US 9,181,592 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

A method is provided for detecting a bacterial pathogen in a sample. One step of the method includes obtaining a sample and then subjecting the sample to nested PCR. The nested PCR is conducted in the presence of at least two outer oligonucleotide primers complementary to a target nucleotide sequence of the bacterial pathogen so that a first amplified product is produced. The target nucleotide sequence includes at least a portion of a 16S-23S ribosomal RNA sequence. The first amplified product is subjected to the nested PCR in the presence of at least two inner oligonucleotide primers complementary to the nucleotide sequence of the first amplified product so that a second amplified product is obtained. Detection of the second amplified product indicates the presence of the bacterial pathogen in the sample.

Claims

1. A method for detecting a bacterial pathogen in a sample, the method comprising the steps of:

obtaining the sample;

subjecting the sample to nested PCR;

wherein the nested PCR is conducted in the presence of at least two outer oligonucleotide primers complementary to a target nucleotide sequence of the bacterial pathogen so that a first amplified product is produced, the target nucleotide sequence comprising at least a portion of a 16S-23S ribosomal RNA sequence;

wherein the first amplified product is subject to the nested PCR in the presence of at least two inner oligonucleotide primers complementary to the nucleotide sequence of the first amplified product so that a second amplified product is obtained; and

detecting the second amplified product, wherein the second amplified product indicates the presence of the bacterial pathogen in the sample.

2. The method of claim 1, the at least two outer oligonucleotide primers being selected from the group consisting of 5′

-GGATTAGATACCCTGGTAGTC-3′

(SEQ ID NO;

1) and 5′

-GGAGTATTTAGCCTT-3′

(SEQ ID NO;

2).

3. The method of claim 1, the at least two inner oligonucleotide primers being selected from the group consisting of 5′

-GGATTAGATACCCTGGTAGTC-3′

(SEQ ID NO;

1), 5′

-GTTTGATCCTGGCTCAG-3′

(SEQ ID NO;

3),5′

-GGTACTTAGATGTTTCAGTTC-3′

(SEQ ID NO;

4), and 5′

-(G/T)TTCGCTCGCC(A/G)CTAC-3′

(SEQ ID NO;

5).

4. The method of claim 1 further comprising the step of identifying the bacterial pathogen corresponding to the second amplified product.

5. The method of claim 4, the step of identifying the bacterial pathogen including a PCR conducted in the presence of at least two detection oligonucleotide primers selected from the group consisting of 5′

-GTAAAACGACGGCCAGT-3′

(SEQ ID NO;

6) and 5′

-CAGGAAACAGCTATGAC-3′

(SEQ ID NO;

7).

6. The method of claim 1, the target nucleotide sequence comprising at least a portion of a 16S ribosomal RNA sequence.

8. The method of claim 1, the at least two outer oligonucleotide primers being selected from the group consisting of 5′

-GGATTAGATACCCTGGTAGTC-3′

(SEQ ID NO;

1) and 5′

-GGAGTATTTAGCCTT-3′

(SEQ ID NO;

2).

7. A method for detecting a bacterial pathogen in amniotic fluid, the method comprising the steps of:

obtaining a sample of the amniotic fluid;

subjecting the sample to nested PCR;

wherein the nested PCR is conducted in the presence of at least two outer oligonucleotide primers complementary to a target nucleotide sequence of the bacterial pathogen so that a first amplified product is produced, the target nucleotide sequence comprising at least a portion of a 16S-23S ribosomal RNA sequence;

wherein the first amplified product is subject to the nested PCR in the presence of at least two inner oligonucleotide primers complementary to the nucleotide sequence of the first amplified product so that a second amplified product is obtained; and

detecting the second amplified product, wherein the second amplified product indicates the presence of the bacterial pathogen in the sample.

9. The method of claim 7, the at least two inner oligonucleotide primers being selected from the group consisting of 5′

-GGATTAGATACCCTGGTAGTC-3′

(SEQ ID NO;

     1),5′

-GTTTGATCCTGGCTCAG-3′

(SEQ ID NO;

2),5′

-GGTACTTAGATGTTTCAGTTC-3′

(SEQ ID NO;

4), and 5′

-(G/T)TTCGCTCGCC(A/G)CTAC-3′

(SEQ ID NO;

5).

10. The method of claim 7 further comprising the step of identifying the bacterial pathogen corresponding to the second amplified product.

11. The method of claim 10, the step of identifying the bacterial pathogen including a PCR conducted in the presence of at least two detection oligonucleotide primers selected from the group consisting of 5′

-GTAAAACGACGGCCAGT-3′

(SEQ ID NO;

6) and 5′

-CAGGAAACAGCTATGAC-3′

(SEQ ID NO;

7).

12. The method of claim 7, the target nucleotide sequence comprising at least a portion of a 16S ribosomal RNA sequence.

13. A method for detecting a bacterial pathogen in amniotic fluid, the method comprising the steps of:

obtaining a sample of the amniotic fluid;

subjecting the sample to nested PCR;

wherein the nested PCR is conducted in the presence of at least two outer oligonucleotide primers complementary to a target nucleotide sequence of the bacterial pathogen so that a first amplified product is produced, the at least two outer oligonucleotide primers being selected from the group consisting of 5′

-GGATTAGATACCCTGGTAGTC-3′

(SEQ ID NO;

1) and 5′

-GGAGTATTTAGCCTT-3′

(SEQ ID NO;

2);

wherein the first amplified product is subject to the nested PCR in the presence of at least two inner oligonucleotide primers complementary to the nucleotide sequence of the first amplified product so that a second amplified product is obtained, the at least two inner oligonucleotide primers being selected from the group consisting of 5′

-GGATTAGATACCCTGGTAGTC-3′

(SEQ ID NO;

1), 5′

-GTTTGATCCTGGCTCAG-3′

(SEQ ID NO;

3),5′

-GGTACTTAGATGTTTCAGTTC-3′

(SEQ ID NO;

4), and 5′

-(G/T)TTCGCTCGCC(A/G)CTAC-3′

(SEQ ID NO;

5), and

detecting the second amplified product, wherein the second amplified product indicates the presence of the bacterial pathogen in the sample.

14. The method of claim 13 further comprising the step of identifying the bacterial pathogen corresponding to the second amplified product.

15. The method of claim 14, the step of identifying the bacterial pathogen including a PCR conducted in the presence of at least two detection oligonucleotide primers selected from the group consisting of 5′

-GTAAAACGACGGCCAGT-3′

(SEQ ID NO;

6) and 5′

-CAGGAAACAGCTATGAC-3′

(SEQ ID No;

7).

16. The method of claim 13, the target nucleotide sequence comprising at least a portion of a 16S-23S ribosomal RNA sequence.

17. The method of claim 16, the target nucleotide sequence comprising at least a portion of a 16S ribosomal RNA sequence.

18. A kit for detecting a bacterial pathogen in a sample comprising:

at least two outer oligonucleotide primers complementary to a target nucleotide sequence comprising at least a portion of a 16S-23S ribosomal RNA sequence of the bacterial pathogen, wherein the at least two outer oligonucleotide primers are used to obtain a first amplified product in a nested PCR; and

at least two inner oligonucleotide primers complementary to the nucleic acid sequence of the first amplified product, wherein the at least two inner oligonucleotide primers are used to obtain a second amplified product in a nested PCR.

19. The kit of claim 18 further including at least two detection oligonucleotide primers for identifying the bacterial pathogen in a PCR.

20. The kit of claim 18, the at least two outer oligonucleotide primers being selected from the group consisting of 5′

-GGATTAGATACCCTGGTAGTC-3′

(SEQ ID NO;

1) and 5′

-GGAGTATTTAGCCTT-3′

(SEQ ID NO;

2).

21. The kit of claim 18, the at least two inner oligonucleotide primers being selected from the group consisting of 5′

-GGATTAGATACCCTGGTAGTC-3′

(SEQ ID NO;

1), 5′

-GTTTGATCCTGGCTCAG-3′

(SEQ ID NO;

3),5′

-GGTACTTAGATGTTTCAGTTC-3′

(SEQ ID NO;

4), and 5′

-(G/T)TTCGCTCGCC(A/G)CTAC-3′

(SEQ ID NO;

5).

22. The kit of claim 19, the at least two detection oligonucleotide primers being selected from the group consisting of 5′

-GTAAAACGACGGCCAGT-3′

(SEQ ID NO;

6) and 5′

-CAGGAAACAGCTATGAC-3′

(SEQ ID NO;

7).

23. The kit of claim 18, the target nucleotide sequence comprising at least a portion of a 16S ribosomal RNA sequence.

24. The kit of claim 18 further comprising at least one temperature resistant, DNA-dependent DNA polymerase and deoxynucleotide triphosphates.