Assay for nucleic acid sequences in an unpurified sample

• US 5,348,855 A
• Filed: 10/04/1991
• Issued: 09/20/1994
• Est. Priority Date: 03/05/1986
• Status: Expired due to Fees

• Current Assignee: Miles, Inc. (Bayer AG)
• Original Assignee: Miles, Inc. (Bayer AG)
• Inventors: Dattagupta, Nanibhushan, Rae, Peter M. M., Rabin, Daniel U., Huguenel, Edward D.
• Primary Examiner(s): Parr, Margaret
• Assistant Examiner(s): Horlick, Kenneth R.
• Application Number:
  US07/772,625
• Time in Patent Office: 1,082 Days
• Field of Search: 435/6, 435/91.1, 435/29, 435/34, 435/35, 435/36, 536/24.3, 935/78, 436/63, 436/501
• US Class Current: 435/6.14
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• 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 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

A method for detecting (i) one or more microorganisms or (ii) nucleic acid sequences from a prokaryotic source or an eukaroytic source in an unpurified nucleic acid-containing test sample comprising

(a) labeling the nucleic acids in the test sample,

(b) contacting, under hybridization conditions, the labeled hybridizable nucleic acid and one or more immobilized hybridizable nucleic acid probes comprising (i) one or more known microorganisms or (ii) sequences from eukaroytic or prokaryotic sources, to form hybridized labeled nucleic acids, and

(d) assaying for the hybridized nucleic acids by detecting the label. The method can be used to detect genetic disorders, e.g., sickle-cell anemia.

Claims

1. A method for detecting at least one polynucleotide sequence from a microorganism or eucaryotic source in a biological test sample, comprising the steps of:

(a) without prior centrifugation or filtration treating said biological test sample to lyse cells and to release nucleic acids therefrom,

(b) without purification or isolation of specific nucleic acid sequences, photochemically labeling released sample nucleic acids directly in the lysate produced in step (a),

(c) contacting the resulting labeled nucleic acids, under hybridization conditions, with at least one immobilized oligonucleotide or nucleic acid probe hybridizable with the sequence or sequence to be detected, thereby to form hybridized labeled nucleic acids, and

(d) assaying for the hybridized nucleic acids by detecting the label.

2. The method according to claim 1, wherein the biological test sample is selected from the group consisting of urine, blood, cerebrospinal fluid, pus, amniotic fluid, tears, sputum, saliva, lung aspirate, vaginal discharge, stool, a solid tissue sample, a skin swab sample, a throat swab sample and a genital swab sample.

3. The method according to claim 1, wherein said labeling is effected by photochemically reacting a labeled form of a nucleic acid binding ligand with the sample nucleic acids.

4. The method according to claim 1, wherein the nucleic acids released from the sample are in double-standard form and, prior to step (c), the labeled nucleic acids are denatured to provide labeled single-stranded nucleic acids.

5. The method according to claim 1, wherein the biological sample is a eucaryotic source selected from the group consisting of algae, protozoa, fungi, slime molds and mammalian cells.

6. The method according to claim 1, wherein the biological sample is a microorganism selected from the group consisting of Escherichia, Proteus, Klebsiella, Staphylococcus, Streptococcus, Pseudomonas and Lactobacillus.

7. The method according to claim 1, wherein lysis in (a) is effected by treatment with alkali.

8. The method according to claim 1, wherein the label in (b) is selected from the group consisting of a protein binding ligand, haptens, antigen, fluorescent compound, dye, radioactive isotope and enzyme.

9. The method according to claim 1, wherein in (c) the probe is immobilized by covalent coupling or adsorption to a solid surface.

10. The method according to claim 9, wherein the probe is immobilized in the form of at least one dot on a solid support strip.

11. The method according to claim 1, wherein in (c) the hybridization is accelerated by the addition of polyethylene glycol.