Detection of microbial nucleic acids by a one-step sandwich hybridization test

{
  "publication_number": "US4486539A",
  "country": "US",
  "kind": "A",
  "title": "Detection of microbial nucleic acids by a one-step sandwich hybridization test",
  "abstract": "This invention relates to a diagnostic kit based on a one step hybridization procedure and method of using the kit for identifying the nucleic acids of viruses and bacteria contained in a single sample. The procedure requires two nucleic acid reagents for each microbe or group of microbes to be identified.",
  "claims": [
    "1. A method for identifying nucleic acids by a one-step sandwich hybridization test, said method comprising the steps of: (a) rendering the nucleic acids in the sample to be identified single-stranded; (b) allowing said single-stranded nucleic acids of the sample to hybridize simultaneously in a single step with a combination of at least one pair of nucleic acid reagents, said reagents having been purified, the first nucleic acid reagent of said pair comprising a single-stranded fragment of nucleic acid, having a nucleotide sequence of at least 10 bases, and being affixed to a solid carrier, the second nucleic acid reagent of said pair comprising a single-stranded fragment of nucleic acid, having a nucleotide sequence of at least 10 bases, and being labeled with a radioisotope, said first and second nucleic acid reagents being capable of forming hybrid molecules by complementary base pairing with given sequences of the sample nucleic acid to be identified, provided that the second nucleic acid reagent cannot hybridize with the first nucleic acid reagent; (c) washing said solid carrier to substantially remove said label which is not incorporated in said hybrid molecule; and (d) measuring said label on the washed solid carrier, whereby determining whether the sample contains the nucleic acid to be identified.",
    "2. The method of claim 1, wherein the first nucleic acid reagent is a DNA or RNA fragment.",
    "3. The method of claim 1, wherein the second nucleic acid reagent is a DNA or RNA fragment.",
    "4. The method of claim 1, wherein the solid carrier is a nitrocellulose sheet.",
    "5. A kit for the detection of nucleic acids with a one-step sandwich hybridization test, the kit comprising in packaged combination a container of at least one pair of nucleic acid reagents, said reagents having been purified: (a) a first nucleic acid reagent of said pair comprising a single-stranded fragment of nucleic acid, having a nucleotide sequence of at least 10 bases, and being affixed to a solid carrier, said first nucleic acid reagent being capable of forming a double-stranded hybrid molecule by complementary base pairing with a given sequence of the nucleic acid to be identified and (b) a second nucleic acid reagent of said pair comprising a single-stranded fragment of nucleic acid having a nucleotide sequence of at least 10 bases, and being labeled with a radioisotope, said second nucleic acid reagent being capable of forming a double-stranded hybrid molecule by complementary base pairing with a given sequence of the nucleic acid to be identified, provided that the second nucleic acid reagent cannot hybridize with the first nucleic acid reagent.",
    "6. The diagnostic kit of claim 5, wherein the first nucleic acid reagent is a DNA or RNA fragment.",
    "7. The diagnostic kit of claim 5, wherein the second nucleic acid reagent is a DNA or RNA fragment.",
    "8. The diagnostic kit of claim 5, wherein the solid carrier is a nitrocellulose sheet."
  ],
  "description_excerpt": "The present invention relates to a kit for the detection of microbes using a two step hybridization procedure.\n\nIn traditional microbial diagnostics the presence of a microbe in a given sample is demonstrated by isolating the microbe in question. After enrichment cultivations, the microbe is identified either on the basis of its biochemical properties or its immunological properties. Both methods of identification require that the microbe in the sample be viable. Such identification can be laborious and time-consuming. Indeed, the detection of certain viruses, requiring sophisticated biochemical purification or tissue culture techniques, can take as long as 4 to 6 weeks.\n\nThe purpose of this invention is to provide a diagnostic kit for detecting the nucleic acid of a microbe in a sample with the aid of a sensitive and specific nucleic acid hybridization technique. The nucleic acid may be of two types, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).\n\nNucleic acid hybridization is an old and well known method for investigating the identity of nucleic acids. Hybridization is based on complementary base pairing. When single-stranded nucleic acids are incubated in solution, complementary base sequences pair to form double-stranded stable hybrid molecules. The double-stranded hybrid molecules can be separated from the single-stranded molecules by chemical means.\n\nSome methods based on the identification of nucleic acid(s) have already been applied to microbial diagnostics. Enterotoxigenic Escherichia coli has been identified from fecal samples by colony hybridization using the gene for toxin production as a probe.",
  "cpc": [
    "C12Q 1/04",
    "C12Q 1/6813",
    "C12Q 1/6888",
    "C12Q 1/689",
    "C12Q 1/701",
    "Y10S 435/81",
    "Y10S 436/804",
    "Y10S 436/808",
    "Y10S 436/811",
    "Y10S 436/823"
  ],
  "ipc": [
    "C12N 15/00",
    "C12N 15/09",
    "C12Q 1/04",
    "C12Q 1/16",
    "C12Q 1/68",
    "C12Q 1/6813",
    "C12Q 1/6888",
    "C12Q 1/689",
    "C12Q 1/70",
    "G01N 33/50",
    "G01N 33/58"
  ],
  "assignees": [
    "Orion Oyj"
  ],
  "inventors": [
    "Tuula M. Ranki",
    "Soderlund Hans E."
  ],
  "filing_date": "1982-10-14",
  "publication_date": "1984-12-04",
  "grant_date": "1984-12-04",
  "priority_date": "1981-10-16",
  "application_number": "US-43418282-A",
  "family_id": "8514776",
  "cited_by_count": 271,
  "citations": [
    "US4139346A",
    "US4302204A",
    "US4359535A",
    "US4358535A",
    "US4358535B1"
  ]
}