GB2228485A - DNA probes for tuberculosis - Google Patents

Abstract

DNA probes having potential applications to the diagnosis of tuberculosis can be derived from deoxyribonucleotide sequences capable of hybridizing with those present in a naturally occuring plasmid known to exist in Mycobacterium fortuitum. The hybridizing sequence is preferably one occurring in a DNA fragment of approximately 5000 base pairs which is present in DNA of M. tuberculosis cut with restriction endonuclease EcoRI and which detects banding patterns on Southern blotting distinguishing different strains of M. tuberculosis as well as distinguishing between M. tuberculosis, M. bovis and BCG vaccine organism. The DNA probes can be used for the detection of M. tuberculosis in clinical samples such as sputum, urine or cerebrospinal fluid by the techniques of dot blot analysis, solution hybridization or polymerase chain reaction.

Description

DEOXYRIBONUCLEIC ACID PROBES FOR DIAGNOSIS OF TUBERCULOSIS This invention relates to deoxyribonucleic acid probes ("DNA probes") for diagnosis of tuberculosis.

Laboratory diagnosis of tuberculosis is unsatisfactory because several weeks may be required to obtain a definite result and the detection of small numbers of Mycobacterium tuberculosss bacteria in heavily contaminated samples is often difficult. The specific identification of mycobacteria is also difficult, and especially the differentiation between the members of the M. t ubercul osis complex: M. tuberculosis itself, the bovine strain M. bovis and the vaccine strain BCG (which may cause disease in immunologically suppressed individuals). The use of DNA probes can provide a sensitive method for the rapid detection of small numbers of specific bacteria in the presence of other organisms.

DNA probes for dentifying strains of the M. tuberculosis complex are commercially available but depend on detecting ribosomal R.NA and require the bacteria to be cultivated first.

Although they are capable of identifying the M. tuberculosis complex they are not suitable for detecting bacteria in a specimen of sputum.

DNA probes derived from chromosomal DNA of M tuberculosis have been described (K.D. Eisenach, J. T. Crawford a J.H. Bates.

1988. Repetitive DNA sequences as probes for Mycobecterf um tuberculosis. Journal of Clinical Microbiology 26 : 2240-2245).

The existing probes are not capable of distinguishing between closely related members of the M. tuberculosis complex.

It has now been found that DNA probes having potential applications to the diagnosis of tuberculosis can be derived from deoxyribonucleotide sequences capable of hybridizing with those present in a naturally occurring plasmid.

As part of an investigation into antibiotic resistance, the presence of plasmids in M. tuberculosis was sought by hybridizing the total DNA from three clinical isolates with DNA of a naturally occurring plasmid known to exist in M. fortuftum (A. Labidi, C. Dauguet, K. S. Goh & H. L. David. 1984. Flasmid profiles of Mycobacterium fortultum complex isolates. Current Microbiology 11 : 235-240). Plasmids have not hitherto been found in M tuberculosis, and it was hoped that they would be revealed by the use of the plasmid DNA as a probe.

Surprisingly, while this did not reveal the presence of any plasmids in M. tuberculosis, it did show that there were chromosomal DNA fragments capable of hybridizing with the plasmid DNA. Moreover, in total DNA from the three clinical isolates digested with restriction endonuclease BamHI or PvuII, the size of the hybridizing fragments was not the same for each strain.

According to the invention there is provided a DNA probe for diagnosis of tuberculosis which comprises a deoxyribonucleotide sequence capable of hybridizing with DNA of a plasmid occurring in M. fortuitum and with chromosomal DNA of M. tuberculosis.

The hybridizing sequence is preferably one occurring in a DNA fragment of approximately 5000 base pairs which is present in DNA of M. tuberculosis cut with restriction endonuclease EcoRI and which detects banding patterns on Southern blotting distinguishing different strains of M. tuberculosis as well as distinguishing between M. tuberculosis, M. bovis and BCG vaccine organism.

DNA probes according to the invention can be used for the detection of M. tuberculosis in clinical samples such as sputum, urine or cerebrospinal fluid by the techniques of dot blot analysis, solution hybridization or polymerase chain reaction.

A probe having a hybridizing sequence occurring in the DNA fragment of approximately 5000 base pairs, when used in combination with Southern blotting of restriction-endonucleasedigested DNA from such clinical material or from bacteria isolated from it, can distinguish M. tuberculosis from M. bovis and BCG, and is of value for epidemiological purposes because it is also able to differentiate between clinical isolates of H. .t ubercul osf s.

Although the fragment of approximately 5000 base pairs can itself be used as a probe, the effective nucleotide sequence is believed to comprise only part of this fragment. This effective sequence may be identified and isolated, and if not too large may be synthesized, by methods known per se.

The specific descr ption which follows is given by way of illustration.

METHOD OF ISOLATION OF THE PROBES As part of an investigation into the possible presence of plasmids in clinical isolates of M. tuberculosis, total DNA from three such isolates was subjected to Southern blotting and probed with a naturally occurring plasmid from M. fortuf tum This plasmid, referred to as pSU300, was obtained from M. fortuitum strain CIPT 14-041-0003 in the Collection de 1' Institut Pasteur, Tuberculose, Paris, France. The results showed that there were chromosomal DNA fragments in the strains of H. tuberculosis capable of hybridizing to this plasmid, and also that in material digested with BamHI or PvuII the size of the hybridizing fragments was not the same for each strain.

In order to isolate these hybridizing fragments, a total DNA library from a clinical isolate of M. tuberculosis (strain 50401) was constructed in the lambda phage vector EMBL4 by ligation of a partial Sau3AI digest of the H. tuberculosis DNA with BdHI-digested EMBL4. The library was screened with a DNA probe derived by labelling a recombinant plasmid pSU3Oi. This plasmid was constructed by ligating an EcoRI digest of plasmid pSU300 with an EcoRI digest of the Escherichia coli plasmid vector pUCi9. Positive plaques were purified through further rounds of plaque screening. The probes described below were obtained from the recombinant phage, referred to as EMBL4/A-3, of one of the positive plaques obtained by this procedure.

The DNA from this recombinant phage was extracted and digested with EcoRI. Agarose gel electrophoresis and Southern blotting demonstrated that EcoRI-digested EMBL4/A-3 contained two fragments of approximate sizes 9000 base pairs (9 kb) and 5000 base pairs (5 kb) which hybridized with the plasmid pSU300.

These fragments were each excised from the gel and are referred to as probe A3/1 (the 9 kb fragment) and probe A3/2 (the 5 kb fragment) respectively.

PROPERTIES OF PROBE A3/1 Probe A3/1 was radioactively labelled with 32P using the Multiprime random primer extension method (Amersham) and hybridized to Southern blots of PvuII-digested total DNA from eight clinical isolates of M. uberculosis (isolate numbers 50410, 60925, 61066, 61104, 61125, 61267, 61377, 61513) as well as M. bovis (field strain, Central Veterinary Laboratory) and BCG (NCTC 5692). After agarose gel electrophoresis, the DNA fragments were transferred to a Hybond-N filter and fixed by baking at 80-C for 1 hour. The filter was prehybridized at 68 C in hybridization buffer. Hybridization with the probe was carried out in the same buffer at 68-C overnight.

The hybridization buffer consisted of 5x Denhardt1s solution, 5x SSPE buffer, 0.2% sodium dodecyl sulphate (SDS) and 100 Fg./ml. sonicated salmon sperm DNA. The Denhardt's solution and the SSPE buffer were made up as stock solutions as follows.

50x Denhardt's solution: 0.5 g. Ficoll (mw 400,000); 0.5 g. polyvinylpyrrolidone (mw 40,000); 0.5 g. bovine serum albumin. The ingredients were dissolved in sterile deionized distilled water to a final volume of one litre which was dispensed into aliquots and stored at -20iC.

20x SSPE buffer: 3. 6M Nazi; 20mM ethylenediaminetetraacetic acid (EDTA); O.2M NaH2P0/Na2HP04, pH 7.7. The ingredients were dissolved in one litre of deionized distilled water and autoclaved.

The filter was then washed twice with 2x SSC, once with 2x SSC containing 0. 1% SDS and once with 0. 1X SSC containing O. 1X SDS. All washes were done at 68-C. The SSC was made up as a stock solution as follows.

20x SSC: 3M Nail; O.3M sodium citrate. The ingredients were dissolved in distilled water to a final volume of one litre and autoclaved after the pH had been adjusted to 7.0.

The filter was covered with Saran wrap and exposed tb X-ray film (RX, FuJi) for 16 hours at room temperature.

Each strain of M. tuberculosis hybridized to probe A3/1 exhibited several hybridizing bands; some elements of this pattern varied from strain to strain while others remained constant. M. bovis and BCG also hybridized to probe A3/1 with a pattern which retained the conserved features of the M. tuberculosis pattern.

The following species of mycobacteria (one strain each except where indicated) did not hybridize with probe A3/1 to any significant extent: M. paratuberculosis, M. intracellulare, M. scrofulaceum, M. phlei, M. fortuitum (three strains) and M. chelonei (two strains).

Probe A3/1 was therefore specific for the M. tubercu2oss complex (which includes M. bovis and BCG > , with some ability to differentiate between strains.

PROPERTIES OF PROBE A3/2 Probe A3/2 was tested in a similar way, with nine strains of M. tuberculosis as well as M. bovis and BCG. The conditions were the same as described above for probe A3/1, except that autoradiography was for 6. 5 hours at room temperature.

Each M. tuberculosis strain showed between five and fifteen strongly hybridizing fragments, as well as a number of weaker bands. The pattern of bands was markedly different for each strain. The number of bands and the strength of the signal, as well as the variation between strains, indicated the presence of a randomly inserted repetitive DNA element in the chromosome of these strains.

M, bowls and BCG showed a simpler pattern of two and three major bands respectively. These organisms could therefore be easily distinguished from M. tuberculosis and from each other.

The following species of mycobacteria (one strain each except where indicated) did not hybridize with probe A3/2: H. paratuberculosis, M. intracellulare, M. scrofulaceum, M. phlei, M fortuitum (three strains) and M. chelonei (two strains > .

Probe A3/2 was therefore specific for the M. tuberculosis complex and was in addition able to distinguish between M. tuberculosis, H. bovis and BCG, and to distinguish between strains of M. t tuberculosis.

Claims (3)

1. DNA probe for diagnosis of tuberculosis which comprises a deoxyribonucleotide sequence capable of hybridizing with DNA of a plasmid of Mycobacterium fortuitum and with chromosomal DNA of Mycobacterium tuberculosis.

2. DNA probe as claimed in claim 1 in which the sequence is one occurring in a DNA fragment of approximately 5000 base pairs which is present in DNA of Mycobacterium tuberculosis cut with restriction endonuclease EcoRI and which detects banding patterns on Southern blotting distinguishing different strains of Mycobacterium tuberculosis as well as distinguishing between Mycobacterium tuberculosis, Mycobacterium bovis and BCG vaccine organism.

3. Process for the detection of Mycobacterium tuberculosis in clinical samples such as sputum, urine or cerebrospinal fluid comprising the use of a DNA probe as claimed in claim 1 or claim 2 by the techniques of dot blot analysis, solution hybridization or polymerase chain reaction.