SE455201B - Diagnostic technique for e.g. cell detection - Google Patents

Abstract

Diagnostic procedure uses a DNA hybridisation technique e.g. for specific labelling of tissues, cells, microorganisms or viruses. Species specific fragments of DNA or RNA having a marker are hybridised with a corresponding fragment of the desired material. The marker can be detected by chemilumin-escence. Used as a diagnostic procedure of cells etc. using DNA or 10A. (Provisional Basic previously advised in Week 83-37)

Description

455 201 eukaryotic origin.

Particularly suitable as markers are luminol and isoluminol, which have the formula NHZ OO NH 25-ZZ IE IE I NH loo luminol isoluminol where the NH2 group is intended to be linked to different NH2 groups on the DNA-alternative RNA molecule by means of of, for example, glutaraldehyde or benzoquinone.

After the reaction with released DNA or RNA from the unknown sample and separation of hybridized and non-hybridized DNA or RNA, for example by filtration, the degree of hybridization can be measured by measuring the luminescence strength. Thus, since the labeled DNA or RNA fragment is specific for, for example, a given tissue, cell, microorganism or virus, the method of the invention can be used for both qualitative and quantitative detection of this tissue, cell, etc.

The process according to the invention thus avoids the problems which labeling with radioactive isotopes entails.

However, the labeling procedure of the process of the invention may in some cases adversely affect the hybridization reaction by introducing foreign molecules into the DNA structure and the RNA structure, respectively. However, this disadvantage can be eliminated if the species-specific DNA sequence and the RNA sequence, respectively, are protected during the labeling procedure by hybridizing with the homologous sequence, thereby binding the marker to surrounding, non-specific parts of the DNA or RNA molecule.

The invention will now be further illustrated in the following examples. j! ~. ~: nf-'í5dv. \ 'fw- _ ~: - -..- I-'n-: i 12: 455 201 Example 1 Labeling of DNA with isoluminol Specific DNA (1 pg) is resuspended in 100 μl 0 , 1 M phosphate buffer (pH 6.8), and the solution is heated to 95 ° C for 5 min. After cooling to room temperature, glutaraldehyde (1.25% final conc) is added. After 18 hours at room temperature, DNA-bound glutaraldehyde is not separated by gel filtration, Sephadex G-50 (pasteur pipette) equilibrated with 0.1 M NaCl. Drop fractions (50 μl) are taken out, and fractions 8-15 are combined. To the pooled fractions 8-15 are added 25 pg of isoluminol and 20 μl of 1 M carbonate-bicarbonate buffer (pH 9.5). After 24 hours at 4 ° C, 0.2 M lysine is added.

The DNA-glutaraldehyde-isoluminol complex is separated from other components by an additional gel filtration (as above). Fractions 8-15 contain the isoluminoline-labeled DNA probe, and this material is then used directly in hybridization.

Hybridization on nitrocellulose filters Nitrocellulose filters are moistened in distilled water and placed on a double layer of dry filter paper (Whatman no 3). The bacterial sample is applied as a known volume of a bacterial suspension in PBS buffer (phosphate buffered sodium chloride) or a direct sample from a patient, eg faecal sample (question: presence of enterotoxin-producing E. coli).

Once the samples have dried, each filter is placed on a double layer of filter paper, saturated with 0.5 M NaOH, and incubated for 10 minutes at room temperature.

The filters are then transferred at 1-minute intervals to three successive neutralizations on filter paper saturated with 1 M Tris (trishydroxymethylaminomethane), pH 7. Finally, the filters are placed for 10 minutes on filter paper, which is saturated with 1 M Tris, pH 7 and 1.5 M NaCl and then air dried. The filters are baked for 2 hours at 80 ° C under vacuum. For 15 minutes, the filters are prehybridized in a solution consisting of 50% formamide, 0.75 M NaCl, 0.075 M Na citrate, 0.1% SDS (sodium dodecyl sulfate), 1 mM EDTA (ethylenediaminetetraacetate), 0.02% Ficoll (polymer) (molecular weight = 400,000), 0.02% polyvinylpyrrolidone (molecular weight = 360,000) and 0.02% bovine serum albumin. The prehybridization takes place at 42 ° C.

The filters are placed on double filter papers, which are saturated with hybridization buffer (identical to the prehybridization solution except that heat-denatured calyx thymus DNA 75 pg / ml is added. This DNA has been sonicated to give DNA fragments of co-history 2.5 x 105 daïton). Labeled DNA, so-called probe DNA, is denatured by incubation at 95 ° C for 10 minutes. The probe (50 μl) is then diluted in 42-degree hybridization solution, and about 100 μl of probe solution is applied to each sample on the filter. Hybridization is allowed to proceed for 1 hour at 42 ° C. Finally, the filters are washed twice at 42 ° C (30 min / wash) in a buffer consisting of 0.75 M NaCl, 0.075 M Na-citrate and 0.1% SDS and twice at room temperature (15 min / wash) with 0 , 3 M NaCl; 0.03 M Na-citrate. After the filters have been air-dried, the amount of labeled DNA bound to the DNA of the samples by conventional chemistry analysis is counted. Typical results are shown in Table 11 1.

Example 2 Protection by labeling the active site for hybridization 1 pg - phage M13 DNA containing 200 bp (base pair) specific probe fragment is mixed with 1 pg 200 bp purified probe fragment in 0.1 M phosphate buffer (pH 6.8).

The solution is heated to 9506 for 5 minutes and then allowed to stand at room temperature for 10 hours. The procedure is continued according to Example 1. Typical results can be seen in Table 2.

Tabe11 1 Bound probe Isoiuminoï (mV) cpm (3H) Antaï baked / m1 preliminary values (cf.) Y. pseudotubercuïosis pan 322 108 670 2400 106 30 323 Y. pseudotubercuïosis koneroii 108 25 168 Tamm 2 Enterotoxin-producing e 'C0. 045 i 2163 Ej: Tec e. Em konmm 108 ss 316 455 201 5 Comparison has been made with labeling with radioactive isotopes (3H), and the results obtained are shown in Tables 1 and 2. It is clear that labeling with cheminuminescent substances gives such good results, which shows that the process according to the invention constitutes a good alternative to the radioactive method used hitherto without being encumbered with the disadvantages which the radioactive labeling entails.

Claims (3)

455 201 Patent claims: Method in diagnostics using DNA hybridization techniques, for example for the specific detection of tissues, cells, microorganisms or viruses, characterized in that a species-specific fragment of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) is provided with a marker and is caused to base pair or hybridize with a homogeneous fragment of the unknown substance, using as a marker a substance that can be detected by a chemiluminescence reaction. Method according to claim 1, characterized in that during the labeling process the species-specific DNA sequence or the RNA sequence is protected by hybridization with the homogeneous sequence, after which the marker is copied to surrounding, non-specific parts of DNA or RNA molecules. The DNA fragment is again denatured, for example in an alkaline environment by heating, and combined with the substance to be diagnosed. 3. A method of carrying out the method according to claim 1, characterized in that it consists of a species-specific fragment of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which is provided with a substance which can be detected by a chemiluminescence reaction.