Amplification based detection of bacterial infection

• US 5,654,141 A
• Filed: 11/18/1994
• Issued: 08/05/1997
• Est. Priority Date: 11/18/1994
• Status: Expired due to Fees

• Current Assignee: Thomas Jefferson University (Main Line Health System)
• Original Assignee: Thomas Jefferson University (Main Line Health System)
• Inventors: Tuan, Rocky S., Mariani, Brian D.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US08/342,091
• Time in Patent Office: 991 Days
• Field of Search: 435/6, 435/91.2
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

A method of detecting a bacterial infection in a patient comprising obtaining a patient sample; obtaining a sample of nucleic acids from the patient sample; separating charged cellular components from the nucleic acids using a mixed bed ion exchange resin; amplifying bacterial nucleic acids in the sample; and detecting the presence or absence of amplified nucleic acids wherein the presence of amplified nucleic acids indicates a bacterial infection.

Claims

1. A method of detecting a bacterial infection in a patient comprising:

obtaining a patient sample;

obtaining a sample of nucleic acids from said patient sample;

separating charged cellular contaminants from said nucleic acids with a mixed bed ion exchange resin;

amplifying by the polymerase chain reaction a bacterial nucleic acid sequence suspected of being associated with thereby producing amplified nucleic acids; and

detecting the presence or absence of said amplified nucleic acids wherein the presence of amplified nucleic acids indicates a bacterial infection.

2. The method of claim 1 wherein said patient sample is selected from the group consisting of synovial fluid, cartilage, bone, tendon, ligament, intervertebral disc, synovial membrane, saliva, cerebrospinal fluid, sputum, mucus, bone marrow, serum, blood, urine, lymph, tears, semen, and feces.

3. The method of claim 1 wherein said bacterial infection is selected from the group consisting of Streptococcus, Staphylococcus, and Bacteroides.

4. The method claim 1 wherein amplifyig bacertial nucleic acids comprises using at least one primer selected from the group consisting of SEQ ID NO:

1 and SEQ ID NO;

2.

5. The method of claim 1 wherein amplifying bacterial nucleic acids may be performed by an amplification method selected from the group consisting of polymerase chain reaction, ligase chain reaction, repair chain reaction, cyclic probe reaction, nucleic acid sequence based amplification, strand displacement amplification, and Qβ

replicase.

6. The method of claim 1 wherein amplifying bacterial nucleic acids is performed by the polymerase chain reaction.

7. A method of detecting a bacterial infection in a patient comprising:

obtaining a patient sample;

obtaining a sample of nucleic acids from said patient sample;

separating charged cellular contaminants from said nucleic acids with a mixed bed ion exchange resin;

amplifying by the polymerase chain reaction bacterial nucleic acids present in a patient sample thereby producing amplified nucleic acids wherein said polymerase chain reaction comprises at least one primer selected from the group consisting of SEQ ID NO;

1 and SEQ ID NO;

2; and

detecting the presence or absence of said amplified nucleic acids wherein the presence of amplified nucleic acids indicates a bacterial infection.

8. A method of detecting a bacterial infection in a patient comprising:

obtaining a patient sample;

obtaining a sample of nucleic acids from said patient sample;

separating charged cellular contaminants from said nucleic acids with a mixed bed ion exchange resin;

amplifying by the polymerase chain reaction bacterial nucleic acids present in a patient sample thereby producing amplified nucleic acids wherein said polymerase chain reaction comprises the use of two primers, a first primer comprising the sequence of SEQ ID NO;

1 and a second primer comprising the sequence of SEQ ID NO;

2.

9. A diagnostic kit for the detection of a bacterial infection in synovial fluid comprising a mixed bed ion exchange resin, at least one primer selected from the group consisting of SEQ ID NO:

1 and SEQ ID 2, and extraction buffer;

said kit useful for detecting a bacterial infection.

10. A method of detecting a bacterial infection in a patient comprising:

obtaining a synovial fluid sample from a patient suspected of having a bacterial infection;

obtaining a sample of nucleic acids from said patient sample;

separating charged cellular contaminants from said nucleic acids with a mixed bed ion exchange resin;

amplifying bacterial nucleic acids suspected of being associated with said bacterial infection present in said patient sample thereby producing amplified nucleic acids; and

detecting the presence or absence of said amplified nucleic acids wherein the presence of amplified nucleic acids indicates a bacterial infection.

11. A method of detecting a bacterial infection in a patient comprising:

obtaining a patient sample;

obtaining a sample of nucleic acids from said patient sample;

separating charged cellular contaminants from said nucleic acids with a mixed bed ion exchange resin;

amplifying bacterial nucleic acids specific for 16S rRNA, ial gene sequences, or Enterotoxin A sequences present in a patient sample thereby producing amplified nucleic acids; and

detecting the presence or absence of said amplified nucleic acids wherein the presence of amplified nucleic acids indicates a bacterial infection.

12. A method of detecting a bacterial infection in a patient comprising:

obtaining a synovial fluid sample from a patient suspected of having a bacterial infection;

obtaining a sample of nucleic acids from said patient sample;

separating charged cellular contaminants from said nucleic acids with a mixed bed ion exchange resin;

amplifying a bacterial nucleic acid sequence within a sequence selected from the group consisting of 16S rRNA, ial or Enterotoxin A present in said sample thereby producing amplified nucleic acids; and

detecting the presence or absence of said amplified nucleic acids wherein the presence of amplified nucleic acids indicates a bacterial infection.

13. A method of detecting a bacterial infection in a patient comprising:

obtaining a synovial fluid sample from a patient suspected of having a bacterial infection;

obtaining a sample of nucleic acids from said patient sample;

separating charged cellular contaminants from said nucleic acids with a mixed bed ion exchange resin;

amplifying a bacterial nucleic acid sequence within a sequence suspected of being associated with said bacterial infection present in said sample thereby producing amplified nucleic acids; and

detecting the presence or absence of said amplified nucleic acids wherein the presence of amplified nucleic acids indicates a bacterial infection.

14. A diagnostic kit for the detection of a bacterial infection in synovial fluid comprising a mixed bed ion exchange resin, at least one primer complementary to a bacterial sequence suspected of infecting synovial fluid, and extraction buffer;

said kit useful for detecting a bacterial infection.