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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa

Definitions

• This invention relates to identification of a specific microorganism in a specimen or sample containing whole microorganisms and pertains more specifically to the diagnosis of diseases characterized by a localized concentration of infectious organisms in tissue, particularly the skin, or in a biological ' fluid, particularly blood; still more particularly it pertains to the rapid diagnosis of such diseases as leishmaniasis, malaria, trypanosomiasis, babesiasis, and herpesvirus diseases.
• leishmaniasis (caused by several different species of Leishmania)
• malaria (caused by several different species of Plasmodium)
• trypanosomiasis (caused by several different species of Trypansoma)
• babesiasis (caused by various species of Babesia)
• the diseases caused by various members of the herpesvirus family are leishmaniasis (caused by several different species of Leishmania)
• malaria caused by several different species of Plasmodium)
• trypanosomiasis (caused by several different species of Trypansoma)
• babesiasis (caused by various species of Babesia)
• the diseases caused by various members of the herpesvirus family The severity and pathogenicity of the disease in each case depends upon the specific identity of the infectious organism. Both the
• Sty iro treatment for the disease and the subsequent medical follow up depend upon rapid, accurate identification of the infecting species.
• Techniques currently widely used for identification of strains of microorganisms and diagnosis and identification of the infecting species in the case of diseases are time-consuming, involving the isolation and cultivation of the microorganism, e.g. the infectious organism and are often unsuccessful because of serious technical difficulties. Delay in identification is particularly serious in the case of those diseases which are endemic to areas where medical services are not readily available.
• Ostrow et al.. Virology, Vol. 108, 21-27 (1981) described extraction and purification of virus from wart tissue, extraction and purification of DNA from the purified virus, and treatment of the purified DNA with restriction enzyme followed by hybridization with labelled DNA from known organisms.
• species of microorganisms can be identified in samples of products or in samples of infected tissue or of biological fluid such as blood from infected mammals by immobilizing DNA from said sample on a solid support, subjecting said immobilized DNA to hybridization with a labeled specimen of species-specific non-cross-hybridizing DNA from a known organism, and determining whether hybridization occurs.
• kinetoplast DNA is a species-specific non-cross-hybridizing DNA which can be used effectively as the labelled probe or hybridizing agent.
• a sample of the product or culture suspected to contain an undesired microorganism, or a sample of infected tissue from a skin lesion or a sample of infected blood, both of which contain whole organisms of the infecting species is simply touched momentarily to any conventional solid support for immobilizing DNA, such as
• OM ⁇ i ⁇ SNATl ⁇ diazobenzyloxymethyl paper a nitrocellulose filter, or a solid DNA support sold under the trade name "Gene Screen", treated with aqueous alkali (preferably at least 0.2 M) to expose the DNA from within the organism, and the alkali removed by washing.
• the sample DNA thus immobilized in a small localized zone of the support and containing all of the DNA of the sample, is then subjected to hybridization with a labelled specimen of species-specific non-cross-hybridizing DNA from an authentic known specimen of an individual species or subspecies of the microorganism.
• OMPI require shorter times, but much higher temperatures tend to destroy the DNA.
• kits can readily be supplied for carrying out the test of the present invention in any clinical laboratory or even in the field.
• a kit contains a supply of supports for the samples and a supply of labelled DNA specimens from known organisms.
• a more complete kit would include a supply of aqueous alkali at least 0.2 M in concentration, and a supply of aqueous buffer.
• One or more standards may also be included in the kit in the form of a separate supply of DNA from a known source, preferably spotted or immobilized at a known location on the supports, as well as supplies of hybridization buffer and washing buffer solutions, and if desired a supply of photographic film for use with radioactivly labelled DNA specimens.
• any known labels can be employed for the DNA specimens; standard radioactive labelling, as with tritium or P, makes it possible to use conventional scintillation counters for rapid and accurate determination of hybridization or to use photographic film for autoradiographic determination. Sensitivity of the test is such that samples containing no more than 1000 organisms and in some cases as few as 300 organisms can readily be identified by the present invention.
• the invention can be used for identification of microorganisms in a sample of any culture or product containing the microorganisms in sufficient concentration so that a sample of convenient size for immobilizing upon a support contains at least the minimum detectable number of microorganisms.
• the invention can also be used for diagnosis or identification of any disease in which the infectious organisms are sufficiently numerous so that a tissue or blood sample of practical size, say 0.1 g, contains at least the minimum detectable number of organisms.
• diseases include leishmaniasis, malaria, trypanosomiasis, babesiasis, and herpesvirus diseases.
• leishmaniasis, malaria, trypanosomiasis, babesiasis, and herpesvirus diseases are intended to illustrate the nature of the invention without acting as limitations upon its scope.
• Example Sample Preparation Animals either Balb/c mice or Golden Syrian hamsters, were infected by subcutaneous infections in the rear hind foot pads of 10 to 10 promastigotes of leishmania.
• Tissue samples for touch preparations were prepared from animals previously infected with either Leishmania mexicana or Leishmania braziliensis promastigotes. The lesion was excised two to three months after infection, the skin was removed and the tissue cut into 2-3 mm pieces. A single tissue piece was used for each touch preparation on nitrocellulose. The tissue was placed on the nitrocellulose filter for a period of 30 seconds to one minute. The nitrocellulose filter was air-dried and placed in a clean envelope for dry storage until the filter could be processed.
• the nitrocellulose filter was treated with aqueous 0.5 M NaOH, 1.5 M NaCl solution for 10 minutes at room temperature to expose the DNA, followed by a 10 minute treatment in 3 M Tris-HCl buffer solution, pH 8, at room temperature to remove the alkali.
• the filter was then air-dried and baked at 80°C for one hour.
• the cells (10 ) in each case were pelleted, washed 2 times in phosphate buffered saline and the kDNA was extracted by resuspending the cells in iysis buffer (0.2 M Nacl, 0.01 M Tris, 0.001 M EDTA, pH 8.0, 10% SDS) , then shearing the chromosomal DNA by passage through a 22 gauge needle; the catenated kDNA was pelleted at 38,000 g for 30 minutes. The pellet was resuspended in a minumum volume of buffer or H 2 0, and cesium chloride was added to bring to a final concentration of 1.7 g/ml. The DNA suspension was then centrifuged for 48 hours at 40,000 rpm.
• iysis buffer 0.2 M Nacl, 0.01 M Tris, 0.001 M EDTA, pH 8.0, 10% SDS
• Fractions (200 ⁇ l) were collected from the bottom of the gradient. The DNA was visualized by mixing 10 1 of each fraction with 10 ⁇ 1 of ethidium bromide (1 ⁇ g/ ⁇ l) and viewed using a source of ultraviolet light. The kDNA fraction was pooled, and dialyzed overnight against 10 mM Tris-Cl pH 8.0, 1 mM EDTA (2 x 4L) .
• Purified kDNA specimens (0.2-0.4 ⁇ g) were labelled by the method of nick translation in a 50 1 reacvtion mixture containing 50 mM NaCl, 10 mM MgCl 2 . 10 mM dithiothreitol, 125 p moles of each deoxynucleotide triphosphate (in most reactions, two radioactive ( 32P) deoxynucleotide triphosphates were used), and 12 units of DNA polymerase I. The reaction mixture was incubated at 15 ⁇ C for two hours. The labelled DNA was separated from unincorporated dATP on a chromatographic gel column run in deionized water.
• the labelled DNA was denatured by boiling for three minutes and chilled on ice immediately before adding to the hybridization mix described below.
• DNA from the sample lesions was immobilized were each soaked for 2 hours at 40°C in hybridization solution (50% formamide, 5 x SSC, 10 x Denhardts, 100 Vig/ml
• OMPI denatured Salmon sperm DNA to which labelled k-DNA from a selected species of organism was added, then incubated for 12 hours at 42°C, washed in 0.1 x SSC with 0.5% SDS three times for 30 min. at 50°C. Each filter paper was air dried and exposed to XAR-5 film to provide an autoradiograph.

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• 1983
  • 1983-07-05 WO PCT/US1983/001029 patent/WO1984001174A1/en unknown
  • 1983-07-05 EP EP83902587A patent/EP0119209A1/de not_active Withdrawn

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