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The genus Mycobacterium includes at least 54 species (Wayne, L. G., and
Kubica, G. P. 1986. Genus Mycobacterium. In "Bergey's Manual of Systematic
Bacteriology" (P. H. A. Sneath, N. Mair, and M. E. Sharp, eds.). Vol. 2,
pp. 1436-1457. Williams & Wilkins, Baltimore, Md.). Most of these species
are saprophytes and do not cause human or veterinary diseases. The
mycobacteria pathogenic to man which are the most important in terms of
morbidity and mortality are Mycobacterium tuberculosis and M. leprae,
which cause tuberculosis and leprosy. Tuberculosis remains one of the
major infectious diseases of the planet, with around 10 million new cases
and 3 million deaths per annum (Stylbo, K. 1983, Tuberculosis and its
control: Lessons to be learned from past experience and its implications
for leprosy control programmes. Ethiop. Med. J. 21:101-122 and World
Health Organisation. 1986. Results of a World Health
Organisation-sponsored workshop to characterize antigens recognized by
Mycobacterium-specific monoclonal antibodies. Infect. Immun. 51:718-720).
In Europe, Africa and North America, a recent trend towards an increase in
prevalence seems to be discernible, no doubt linked to the multiplication
of cases of AIDS (Stead, W. W., and Dutt, A. K. 1988. Changing faces of
clinical tuberculosis. In Mycobacterium tuberculosis. Interactions with
the Immune System" (M. Bendinelli and H. Friedman, eds.) pp. 371-388.
Plenum, N.Y.)
M. tuberculosis is taxonomically very close to M. bovis, M. africanum
(which also cause tuberculosis in man) and M. microti (tuberculosis of
certain rodents), so that these four species are collectively named the
"M. tuberculosis complex" (Wayne L. G. et al.). Among non-tuberculous
mycobacteria (sometimes called "atypical"), there should be mentioned the
increased incidence of mycobacteria belonging to the M.
avium-intracellulare complex in immunosuppressed patients (AIDS,
transplantations, cancer treatments, etc.) (Kielin, T. E., Edwards, F. F.,
Brannon, P., Tsang, A. Y., Maio, M., Gold, J. W. M., Whimby, E., Wong, B.,
McClatchy, J. K., and Amstrong, D. 1985. Infections caused by
Mycobacterium avium complex in immunocompromised patients: Diagnosis by
blood culture and fecal examination, antimicrobial susceptibility tests
and morphological and seroagglutination characteristics. J. Clin.
Microbiol. 21, 168-173. Macher, A. M., M. Kovacs, J. A. Gill, V., Roberts,
G. D., Ames, J., Parke, C. H., Strans, S., Lane, H. C., Parillo, J. E.,
Fanci, A. S., and Masur, H. 1983. Bacteremia due to Mycobacterium
avium-intracellulare in the acquired immunodeficiency syndrome. Ann.
Intern. Med. 99, 782-785).
This complex comprises the species M. avium, M. intracellulare (very
closely related to one another, whence the term M. avium-intracellulare),
M. paratuberculosis (the cause of Johne's disease in calves) and M.
lepraemurium (rat leprosy). Other species produce human infections of
lesser importance in terms of seriousness or morbidity, such as M.
kansasii (adentitis), M. ulcerans and M. marinum (skin ulcerations).
Virtually all the mycobacteria possess a characteristic and specific
antigen, termed 65-kD antigen, which has been completely sequenced and
identified.
The 65-kD antigen possesses numerous characteristics of interest which have
led to extensive studies. In the first place, this protein appears to be a
major mycobacterial antigen. The individuals or animals infected or
immunized with mycobacteria produce antibodies and T cells which recognize
this antigen in the large majority of cases, and this has made it
possible, moreover, to dissect the epitopes from it. Next, the 65-kD
antigen belongs to the family of "heat shock" proteins or thermal shock
proteins, which are also to be found with a high degree of conservation in
many prokaryotic and eukaryotic cells. These proteins function as a
"chaperon" in the post-translational assembling of certain proteins of
prokaryotes, chloroplasts and mitochondria (Ellis J. 1988. Nature 328:
378-9). Lastly, special interest also attaches to the 65-kD antigen
inasmuch as it has been associated with the pathogenesis of autoimmune
arthritis (Thole, J. E. R., and Van Der Zee, R. 1990. The 65-kD antigen:
molecular studies on a ubiquitous antigen. In: Molecular Biology of the
Mycobacteria, J. Mc Fadden Ed., Surrey University Press, London, pp.
37-67).
These numerous points of interest explain why this antigen has been one of
the very first to be cloned and sequenced in various mycobacteria
(Clark-Curtiss, J. E., Jacobs, W. R., Docherty, M. A., Richtie, L. R., and
Curtiss III, R. 1985. J. Bacteriol. 161, 1093-102. Young R. A., Blooms, B.
R., Grosskinsky, C. M., Ivangi, J., Thomas, D. and Davis R. W. 1985. Proc.
Natl. Acad. Sci. USA. 42:2583-7 Young, R. A., Mehra, V., Sweeetser, D.,
Buchanan, T., Clark-Curtiss, J., Dasvis, R. W., and Bloom, B. R. 1985.
Genes for the major protein antigens of the leprosy parasite Mycobacterium
leprae. Nature. 316:450-2. Lu, M. C., Lien, M. H., Becken, R. E., Heine,
H. C., Buggo, A. M., Lipovsek, D., et al. 1987. Infect. Imm. 55:23-82.
Husson R. N., and Young R. A., 1987. Proc. Natl. Acad. Sci. USA.
84:1679-83. Andersen, A. S., Worsaae, A. and Chaparas, S. D. 1988. Infect.
Imm. 56:1344-51. Shinnick, T. M. 1987. The 65-kilodalton antigen of
Mycobacterium tuberculosis. J. Bacteriol. 169:1080-88. Thols, J. E. R.,
Dauwesse, H. G., Das, P. K., Croothuis, D. G., Shouls, L. M. and Embden,
J. D. A. 1985. Cloning of Mycobacterium bovis BCG DNA and expression of
antigens in Escherichia coli. Inf. Imm. 50:800-6. Mehra, V., Sweetser D.,
and Young, R. A. 1986. Efficient mapping of protein antigenic
determinants. Proc. Natl. Acad. Sci. USA. 83:7013-17.).
The document WO-A-8800974, referred to in the reference of Young R. A. et
al., Nature, 1985, 376:450-2 describes a DNA sequence coding for the 65-kD
antigen of M. leprae, the document WO-A-8806591 describes a DNA sequence
coding for the 65-kD antigen of M. tuberculosis referred to in the
Shinnick et al. reference, and the publication of Thole et al, Infect.
Immunol., 1987, 55:1466-71 describes a DNA sequence coding for the 65-kD
antigen of M. bovis BCG.
However, all the techniques of qualitative and/or quantitative
characterization or identification of mycobacteria described in the prior
art, directly from their nucleic acids, still possess the following
drawbacks.
The genomic DNA sequence selected for the purpose of identifying virtually
constant regions (falling within the notion of homology) in the genus
Mycobacterium, and/or variable regions which are, respectively, specific
to the species belonging to said genus, is not conserved in most
mycobacteria known at present. It is, in general, conserved only for a few
species, thereby rendering the process of identification non-selective for
the whole of the genus Mycobacterium.
More recently, the document WO-A-9012875 described a 383-base pair
nucleotide sequence of the gene coding for the 65-kD antigen in M.
tuberculosis, M. bovis BCG, M. avium, M. paratuberculosis, M. fortuitum,
M. malmoense, M. leprae, M. kansaii and M. marinum, on the basis of which
sequence probes for the identification of some of the abovementioned
species or groups of species were determined; as well as primers for the
amplification of DNA fragments belonging to said gene.
Nevertheless, regions selected in the prior art are conserved only for a
few species, so that the primer or primers determined for amplifying these
regions do not hybridize with the genomic DNA of some species, as
demonstrated by the results obtained with the primers TB1 and TB2
according to WO-A-90 12875 and shown in Table 2.
The objective of the present invention is to remedy the above drawbacks.
More specifically, the subject of the invention is:
1) a sequence of the genomic DNA of mycobacteria, belonging to the gene
coding for the KD 65 mycobacterial antigen, comprising regions which are
virtually constant for the majority of species of mycobacteria;
2) any specific primer for amplification, by DNA polymerization, of the
sequence according to (1) which is virtually constant for the majority of
species belonging to the M tuberculosis and/or M avium-intracellulare
complex;
3) one or more detection probes, termed genus probes, which hybridize with
a portion of the sequence according to (1) which is virtually constant for
the majority of species belonging to the genus Mycobacterium;
4) one or more detection probes, termed species probes, which hybridize
with a portion of the sequence according to (1) which is virtually
constant for the majority of species belonging to the M tuberculosis
and/or M avium-intracellulare complex;
5) any reagent or reagents involving one or more isolated sequences
according to (1), and/or primers according to (2), and/or genus probes
according to (3), and/or species probes according to (4).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A-C shows a sequence comparison of a portion of the 64-kD antigen for
several Mycobacteria species.
The terms probes and/or primers as used in the present invention refer to a
natural DNA or RNA fragment, or a natural or synthetic oligonucleotide, or
a synthetic DNA or RNA fragment which is unmodified or comprises one or
more modified bases such as inosine, 5-methyldeoxycytidine, deoxyuridine,
5-dimethlaminodeoxyuridine, 2,6-diaminopurine, 5-bromodeoxyuridine or any
other modified base permitting hybridization, it being possible for the
DNA fragment to be single- or double-stranded.
According to the present invention, there is provided, in the first place,
a single-stranded DNA fragment, isolated or forming part of a
double-stranded DNA macromolecule, whose nucleotide sequence is included
in the gene of the species of the genus Mycobacterium, coding for the
so-called 65-kD mycobacterial antigen, containing regions which are
constant or homologous in practically all species of the genus
Mycobacterium, and one or more variable regions which are specific, that
is to say constant or homologous for a given species. According to the
invention, and on the basis of the sequencing of the abovementioned gene
established by Shinnick (Schinnick TM 1987, J. Bacteriol. 169: 1080-88),
said fragment is chosen from fragments whose nucleotide sequences possess
at least 70% homology, and preferably at least 85% homology, with a
predetermined or reference sequence, or homologous with the sequence
complementary to this chosen predetermined or reference sequence according
to the invention, said predetermined sequence beginning at nucleotide 438
and ending at nucleotide 751 of said gene coding for said 65-kD antigen of
all species of mycobacteria except for the species M. tuberculosis, M.
bovis BCG, M. avium, M. paratuberculosis, M. fortuitum, M. malmoense, M.
leprae, M. kansaii and M. marinum.
Before describing the invention, various terms used in the description and
claims are now defined:
according to the invention, a nucleotide fragment is a string of monomers
capable of hybridizing with a nucleotide fragment under predetermined
conditions, it being possible for the string to contain monomers of
different structures and to be obtained from a natural nucleic acid
molecule and/or by genetic recombination and/or by chemical synthesis,
thus, a monomer can be a natural nucleotide of nucleic acid whose
constituent elements are a sugar, a phosphate group and a nitrogenous
base; in DNA, the sugar is ribose, in RNA, the sugar is 2-deoxyribose;
depending on whether the nucleic acid is DNA or RNA, the nitrogenous base
is chosen from adenine, guanine, uracil, cytosine and thymine; or a
nucleotide which is modified in at least one of the three constituent
elements; as an example, the modification can take place in the bases,
generating modified bases such as incsine, 5-methyldeoxycytidine,
deoxyuridine, 5-dimethylaminodeoxyuridine, 2,6-diaminopurine,
5-bromodeoxyuridine and any other modified base promoting hybridization,
in the sugar, namely the replacement of at least one deoxyribose by a
polyamide (P. E. Nielsen et al., Science, 254, 1497-1500 (1991)), or in
the phosphate group, for example its replacement by esters chosen, in
particular, from diphosphate, alkyl- and arylphosphonate and
phosphorothioate esters,
complementary sequence is understood to mean any sequence which hybridizes
completely with the predetermined or reference sequence,
hybridization is understood to mean the process during which, under
suitable conditions, two nucleotide fragments having sufficiently
complementary sequences pair to form a double strand,
a probe is a nucleotide fragment comprising from 5 to 100 monomers, and
advantageously from 10 to 40 monomers, possessing a specificity of
hybridization under particular conditions to form a hybridization complex
with a nucleotide fragment having a nucleotide sequence included in the
genomic DNA of mycobacteria; a probe may be used for diagnostic purposes,
for instance capture and/or detection probes,
the capture probe may be immobilized on a solid support by any suitable
means, that is to say directly or indirectly, for example by covalent
bonding or passive adsorption,
the detection probe is labeled by means of a label chosen from radioactive
isotopes, enzymes chosen, in particular, from peroxidase and alkaline
phosphatase and those capable of hydrolyzing a chromogenic, fluorogenic or
luminescent substrate, chromophoric chemical compounds, chromogenic,
fluorogenic or luminescent compounds, nucleotide base analogs and biotin,
the probes used for diagnostic purposes of the invention may be employed in
all known hybridization techniques, and in particular the techniques
termed "DOT-BLOT" (MANIATIS et al, Molecular Cloning, Cold Spring Harbor,
1982), "SOUTHERN BLOT" (SOUTHERN. E. M., J. Mol. Biol., 98, 503 (1975),
"NORTHEN BLOT", a technique identical to the "SOUTHERN BLOT" technique but
which uses RNA as target, and the SANDWICH technique (DUNN A. R., HASSEL
J. A., Cell, 12, 23 (1977)); advantageously, the SANDWICH technique is
used in the present invention, comprising a specific capture probe and/or
a specific detection probe, it being understood that the capture probe and
the detection probe must possess an at least partially different
nucleotide sequence,
a primer is a probe comprising from 5 to 30 monomers, and preferably 15 to
25 monomers, possessing a specificity of hybridization under particular
conditions for the initiation of an enzymatic polymerization, for example
in an amplification technique such as PCR (Polymerase Chain Reaction), in
an elongation method such as sequencing, in a transcription method or the
like,
homology characterizes the degree of similarity of two nucleotide fragments
which are compared.
As an example of fragments according to the invention, the sequence of the
single-stranded fragment possesses at least 70% homology, and preferably
at least 85% homology, with any one of the sequences SEQ ID N01, SEQ ID
N02, SEQ ID N03, SEQ ID N04, SEQ ID N05 and SEQ ID N07 identified at the
end of the description.
By means of the single-stranded fragment according to the invention,
amplified with all suitable primers and detected with all genus and
species probes determined below, all the mycobacteria tested, namely 21
strains representing 19 species, responded positively to the test
performed, under circumstances where comparable tests, for example
according to the document WO-A-90 12875, enable only 15 strains out of 21
to be amplified, consequently leaving 6 strains of mycobacteria
undetected.
The invention also relates to any macromolecule of genomic or isolated DNA,
or of RNA, comprising or integrating a single-stranded fragment as defined
above. There corresponds to this definition, in particular, any
replication vector incorporating a said fragment, but also any
amplification product resulting from the labeling with suitable primers of
a nucleotide sequence corresponding to the predetermined sequence defined
above, to within at least 70% homology; in this case, a nucleotide
sequence the central portion of which corresponds, in single-stranded
form, to the predetermined sequence, flanked by the two amplification
primers, for example, is obtained.
On the basis of the predetermined sequence selected, the invention has
defined various specific primers for the amplification by polymerization
of the genomic DNA of a bacterium of the genus Mycobacterium. Generally
speaking, such a primer comprises a nucleotide sequence enabling it to
hybridize a so-called constant region of this predetermined sequence which
is homologous or identical for practically all species of the genus
Mycobacterium, in particular the region beginning at nucleotide 438 and
ending at nucleotide 457, and the region beginning at nucleotide 733 and
ending at nucleotide 751, again according to Shinnick's sequencing.
This primer advantageously comprises between 15 and 25 monomers.
As an example, this primer possesses a nucleotide sequence chosen from SEQ
ID N08 and SEQ ID N09, identified at the end of the description.
Any amplification technique may be used and, in particular, all pairs of
primers as defined above, for example all pairs of primers comprising at
least 10 bases of the sequences SEQ ID N08 and SEQ ID N09, will be
selected.
Still on the basis of the same single-stranded fragment according to the
invention, the latter also provides a so-called genus probe, capable of
hybridizing a constant region of said fragment which is homologous or
constant for practically all species of the genus Mycobacterium.
Preferably, this genus probe is capable of hybridizing an end region of the
single-stranded fragment according to the invention, corresponding to a
primer as defined above, integrated in or linked to said fragment.
However, this genus probe can hybridize any other constant region of the
same single-stranded fragment; advantageously, these probes possess a
nucleotide sequence chosen from SEQ ID N010 and SEQ ID N011, identified at
the end of the description.
Still on the basis of the same single-stranded fragment according to the
invention, other subjects of the latter are various species probes, each
comprising a nucleotide sequence enabling it to hybridize a so-called
variable region of said predetermined sequence, identified above, this
variable region being specific to at least one species of the genus
Mycobacterium.
According to an important feature, the present invention provides in
addition probes specific for groups of species corresponding to the M.
tuberculosis and M. avium-intracellulare complexes, respectively. These
probes comprise a nucleotide sequence enabling them to hybridize a
variable region of the fragment whose sequence is included in the gene
coding for 65-kD, said region being common to several species of the same
complex.
Preferably, these species or complex probes comprise from 10 to 40
monomers.
Advantageously, the nucleotide sequence of the probe is chosen from SEQ ID
N012 and SEQ ID N013 to SEQ ID N021, identified at the end of the
description.
According to the invention, a reagent or reagent kit is provided for
selectively detecting a bacterium of the genus Mycobacterium, belonging to
the M. tuberculosis and M. avium-intracellulare complexes, in a biological
sample. Such a kit comprises, where appropriate one or more primers as
described above, where appropriate one or more genus probes as described
above, and one or more species probes as described above.
Depending on the hybridization technique used, the probe or probes
according to the invention are in a liquid medium and/or bound directly or
indirectly to a solid support. As regards the solid support according to
the invention, in all suitable form such as tube, cone, well,
microtitration plate, sheet or soluble polymer, this is chosen from
polystyrenes, styrene/butadiene copolymers and styrene/butadiene
copolymers mixed with polystyrenes, polypropylenes, polycarbonates,
polystyrene/acrylonitrile copolymers, styrene/methyl methylmethacrylate
[sic] copolymers, from synthetic and natural fibers and from
polysaccharides and cellulose derivatives.
The invention also relates to a method for selectively detecting a
bacterium of the genus Mycobacterium in a biological sample. This method
entails the following steps:
preparation of samples in order to release the mycobacterial nucleic acid
hybridization of the genomic DNA and/or RNA of the bacterium, and/or of its
transcribed RNA, with at least one primer as defined above; multiplication
of the DNA or RNA fragment flanked by said primer or primers, to obtain a
multitude of single-stranded DNA and/or RNA fragments corresponding to the
definition according to the invention; these two steps are optional, their
object being to avoid culture of the bacteria
exposure of the fragment or fragments to at least one species probe and/or
at least one genus probe as defined above.
The present invention is now described according to Examples 1 to 5, and in
support of Tables 1 and 2 and of FIG. 1, which is divided into FIG. 1a, 1b
and 1c and which shows the alignment of the nucleotide sequences
(according to the numbering of Shinnick et al. 1987) over a 314-bp portion
of the gene coding for the 65-kD antigen of mycobacteria; the sequences of
the strains of the following species are taken from the literature:
M. tuberculosis (Document WO-A-8806591 and Shinnick, T. M. 1987. The
65-kilodalton antigen of Mycobacterium tuberculosis. J. Bacteriol.
169:1080-88)
M. bovis BCG (Thole et al. 1987. Characterization, sequence determination,
and immunogenicity of a 64-kilodalton protein of Mycobacterium bovis BCG
expressed in Escherichia coli K-12. Inf. Imm. 55:1466-1475).
M. avium, M. fortuitum and M. paratuberculosis (Document FR-A-2,645,878 and
Hance, A. J., Grandchamp, B., Levy-Frebault, V., Lecossier, D., Rauzier,
J., Bocart, D., and Gicquel, B. 1989. Detection and identification of
mycobacterium by amplification of mycobacterial DNA. Molecul. Microbiol.
3:843-9.)
Mycobacterium leprae (Young, R. A., Mehra, V., Sweeetser, D., Buchanan, T,
Clark-Curtiss, J., Davis, R. W., and Bloom, B. R. 1985. Genes for the
major protein antigens of the leprosy parasite Mycobacterium leprae.
Nature. 316:450-2)
The sequences of the following species were determined using strains
available to the Applicant: M. africanum, M. microti, M. chitae, M.
intracellulare 3324, M. intracellulare 83 2230, M. malmoense, M.
scrofulaceum.
According to FIGS. 1a to 1c, a dash means that the base is identical to
that identified on the first line.
Table 1 shows oligonucleotide sequences according to the invention,
exemplified below, and their specificity.
TABLE 1
__________________________________________________________________________
Synthetic oligonucleotides used as polymerization
primers or probe for detecting a portion of the gene for
the 65-kD antigen of mycobacteria, and whose specificity
is at the level of genus or of species of mycobacteria
(*) the figures in brackets indicate the position
of the oligonucleotide according to the numbering of
Shinnick et al., 1987; C. indicates a homology with the
complementary strand.
Oligonucleotide Sequence*
__________________________________________________________________________
Polymerization or amplification primers
SEQ ID NO8: GAT CCG TAC GAG AAG ATC GG
(438-457)
SEQ ID NO9: ACC TTG TCC ATC GCC TCG G
(C.733-751)
Genus hybridization probes:
SEQ ID NO11: CGC AAC GTC GCG GCC GGC GCC AAC CCG C
(561-588)
SEQ ID NO10: CCG AGG CGA TGG ACA AGG T
(733-751)
Hybridization probes for the avium-intracellulare complex:
SEQ ID NO21: TGC TCA AGT CGG CCA AGG
(640-657)
SEQ ID NO13: ACG GCA CGA CGA CGG (508-522)
SEQ ID NO14: CCA CGG TGC TSG CYC AGG
(523-540)
SEQ ID NO15: GAC CAG YSG ATC GGC GAC C
(708-726)
SEQ ID NO16: CCG CTG GGT CTS AA (585-598)
SEQ ID NO17: GCG TTG GTC CGC GAG GGC C
(540-558)
SEQ ID NO18: CGA CGA CGG CCA CGG TGC T
(514-532)
SEQ ID NO19: CCG CTG GGT CTS AAG CGC G
(585-603)
SEQ ID NO20: CCA AMC CGC TGG GTC TSA A
(580-598)
Hybridization probe for the tuberculosis complex:
SEQ ID NO12: GGT CAA AGA GGT AGC CAA G
(467-485)
__________________________________________________________________________
Table 2 shows the specificities of enzymatic amplification and of
hybridization of the bacterial strains tested. In this table:
as regards the source, the FIGS. 1 refer to strains available from the
Centre de Collection des Mycobacteries, [Collection Center for
Mycobacteria], CHUV at Lausanne, 2 to the Centre Medical Universitaire
[University Medical Center] in Geneva, 3 to the Institut d'Hygiene
[Hygiene Institute] in Geneva and Bio M to the laboratories of bioMerieux
TB1 and TB2 refere to primers according to the document WO-A-90 12875.
TABLE 2
__________________________________________________________________________
HYBRIDIZATION OF THE AMPLIFICATION
PRODUCT, and labeling of the
radioactive .sup.32 P probe
(.sup.32 P) or enzymatic detection
(cold)
AMPLIFICATION GENUS GENUS M. tuberculosis
SEQ ID NO8
SEQ ID NO
SEQ ID NO Complex SEQ ID
SPECIES SOURCE
TB1-TB2
SEQ ID NO9
11 (.sup.32 P)
10 (cold) N012
__________________________________________________________________________
(cold)
M. tuberculosis
2 + + + + +
M. microti 3 + + + + +
M. bovis 3 + + + + +
M. africanum La 1077
1 NP + + + +
M. kansasil 3 + + + + NP
M. marinum 2417
3 + + + + NP
M. simiae ATCC 25275
BloM NP + + + .
M. scrofulaceum
3 + + + + NP
M. gordonae BloM + + + + .
M. szulgai 3 + + + + NP
M. flavescens BloM NP + + + .
M. intracellulare 83-2230
2 . + + + NP
M intracellulare 83-3324
2 . + + + .
M. intracellulare ATCC 357 64
BolM NF + + + .
M. avium 2 + + + + NP
M. avium-intracellulare 4556
3 + + + + .
M. xenopi 4333 3 NF + + + NP
M. terrae ATCC 15755
BloM + + + + NP
M. malmoense 3 NP + + + NP
M. nonchlomogenicum
3 NP + + + .
M. triviale ATCC 23 292
BloM NP + + + NP
M. gastri 1 NP + NP + .
M. farcinogenos
3 NP + NP + NP
M. haomophilum 1 NP + NP + .
M. ulcerans 1 NP + NE + .
M. paratuberculosis
1 NP + + + .
M. chelonei 2 . + + + .
M. chelonei ATCC 14472
BloM NP + + + NP
M. fortuitum 83-3359
2 + + + + .
M. diernhoferi 3 + + + + NP
M. chitae 3 + + + + NP
M. chelonel-abscessus
3 NP + + + NP
M. senegalese 3 NP + + + NP
M. vaccae 3 . + + + NP
M. phlel 3 + + + + .
M. thermoresistibile
3 . + + + NP
M. smegmatis 3 . + + + NP
Nocardia asteroides 864
2 NP . . . NP
Nocardia asteroides 866
2 NP . . . NP
Nocardia asteroides 869
2 NP . . . NP
Nocardia asteroides 870
2 NP . . . NP
Nocardia caviae 861
2 NP . . . NP
__________________________________________________________________________
1 centre de collection des mycobacteries [Collection center for
mycobacteria], Lausanne, 2 Centre Medical Universitaire [University
Medical Center] in Geneva, 3 Institut d'hygiene [Hygiene Institute] In
Geneva
NP: not performed
EXAMPLE NO. 1
Definition of genus-specific amplification primers and their use
After various preliminary experiments, it was found that the sequences
provided in the literature as "universal" primers for the genus
Mycobacterium do not respond to some strains or species known at present;
thus, the primers termed TB1 and TB2 according to the document WO-A-90
12875 do not respond to some species, as indicated above.
A sequencing of the gene coding for the 65-kD antigen was hence carried out
in various species in which said gene had not yet been explored. By
alignment of the sequences thereby obtained with those already described
in the prior art, new primers which were discovered to hybridize with the
sequences conserved in practically all mycobacteria were then chosen; see
SEQ ID N08 and SEQ ID N09 in Table 1. This discovery was made by
amplification from a strain of mycobacteria. The technique used is
described below.
Extraction of genomic DNA was performed according to the protocol of
Sjobring (SjoSbring et al. 1990. Polymerase chain reaction for detection
of Mycobacterium tuberculosis. J.Clin. Microbiol. 28(10):2200-2204). 200
ml of liquid culture are centrifuged at 2600 rpm for 15 min. The
supernatant is discarded and the pellets are pooled. The pellet is washed
with pH 8.0 buffer (50 mM Tris base, 50 mM NaCl, 5 mMEDTA, pH 8.0). 0.1
volume of 10.times.digestion buffer (100 mM Tris pH 8.0, 200 mM EDTA, 10%
SDS) and proteinase K at a concentration of 5 mg/ml final are added. The
mixture is incubated at 60.degree. C. for 3 hours with stirring and heated
for 5 minutes to 100.degree. C. to inactivate the proteinase K. The DNA is
precipitated a first time by adding a solution of 0.4 volume of
cetyltrimethylammonium bromide solution (5% in 0.4M NaCl) and the mixture
is incubated for 15 minutes at temperature [sic] and then 15 min at
4.degree. C. It is transferred to Eppendorf tubes and centrifuged for 15
min at 12,000 rpm. The supernatant is discarded and the pellet is washed
in TE (Tris EDTA) buffer (10 mM Tris pH 7.5, 1 mM EDTA). The DNA is
precipitated a second time after extraction with phenol/chloroform
(1:1)/isoamyl alcohol (24:1). The DNA is precipitated with 2 volumes of
absolute ethanol and 0.1 volume of 3M sodium acetate.
The DNA is amplified according to the PCR technique of Saiki et al. (Saiki
et al. 1988. Primer-directed enzymatic amplification of DNA with a
thermostable DNA polymerase) using a Dri-Bock PCH-1 PCR apparatus (TECHNE,
Great Britain). The reaction medium consists of Tris.Cl 10 mmol/l;
MgCl.sub.2 1.5 mmol/l; KCl 50 mmol/l; gelatin 1 mg/ml; dATP, dCTP, dGTP,
dTTP 0.5 mmol/l each; pH 8.3; oligonucleotide TB1 (according to the
document WO-A-90 12875, 25 pmol; oligonucleotide TB2 (according to the
document WO-A-90 12875), 25 pmol; and 10 .mu.l of the bacterial DNA
preparation. After denaturation for 5 min followed by centrifugation, the
enzyme is added at a concentration of 1.5 U/reaction. PCR is performed
over 27 cycles with the parameters 96.degree. C./43.degree. C./74.degree.
C., for 1 min, 1 min and 0.7 min, respectively.
The amplified DNA is analyzed by electrophoresis on 0.8% agarose gel in TBE
(Tris Borate EDTA) buffer (89 mM Tris base, 89 mMboric acid, 2 mMEDTA).
The bands are visualized with ethidium bromide.
EXAMPLE NO. 2
Determination of nucleotide sequences of various species of mycobacteria
and sequence alignment.
Starting with the total DNA isolated as above, a portion of the gene coding
for the 65-kD antigen was amplified using the primers SEQ ID N08 and SEQ
ID N09 in Table 1. The amplification products obtained were sequenced
directly using the Gibco BRL kit (thermal cycling). The various sequences
obtained are presented in FIG. 1.
EXAMPLE NO. 3
Determination of genus- and species-specific oligonucleotide probes and
their use on DNA samples amplified according to Example No. 1.
The PCR amplification products obtained using the primers SEQ ID N08 and
SEQ ID N09 (Table 2) were tested with various species or genus probes
described in Table 1, according to the Dot-Blot technique.
The substrate DNA of the amplification reaction is extracted by a technique
different from that of Example 1: after centrifugation of an aliquot of
10.sup.7 mycobacteria in 0.5% Tween, the bacteria are suspended in a
Tris-HCl buffer (pH 8.0, 50 mM), and are then subjected to sonication for
10 min at 55.degree. C. in the presence of siliconed 10 .mu.m glass beads,
and then to boiling for 5 min. 10 .mu.l of the lysate are then amplified
according to the PCR protocol described in Example No. 1.
Hybridization of the species or genus oligonucleotides was performed
according to the protocol described by Ausubel FM, Brent R, Kingston RE,
Moore DD, Smith JA, Seidman JG and Struhl K (1987) Current protocols in
molecular biology, Green publishing Associated and Wiley intersciences,
New York. The oligonucleotides are labeled by kinasing with [gamma(or
[lacuna])-.sup.32 P]-ATP (5000 Ci/mol), and the temperatures and washing
conditions are as follows, for example for MYC3: 59.degree. C. 30 min in
1.times.SSC (Saline Sodium Citrate: 0.15M NaCl, 0.015M Na.sub.3 citrate (2
H.sub.2 O pH 7.0), 1% SDS.
EXAMPLE NO. 4
Species typing of mycobacteria by hybridization of a mycobacterial genus
amplification product using oligonucleotide probes, carried out using a
non-radioactive and semi-automated detection system described in the
document FR-A-2,663,040.
The amplification products obtained in Example No. 3 were tested again
according to the cold probe technology described below according to two
variants.
The first technique employs a microtitration plate format.
A solution of the capture oligonucleotide at a concentration of 1 ng/.mu.l
in 3.times.PBS (0.45M NaCl, 0.15M sodium phosphate, pH 7.0) is placed in a
microtitration plate (Nunc 439454). The capture probe or oligonucleotide
is chosen from the oligonucleotides described in Table 2 (SEQ ID N011, SEQ
ID N010, SEQ ID N012, SEQ ID N021 and SEQ ID N013 to SEQ ID N020) which
are specific for the genus or for various species of mycobacteria. Their
position in the sequence is indicated in FIG. 1. The plate is incubated
for 2 h at 37.degree. C. and then washed 3 times with 300 .mu.l of PBST
(1.times.PBS, 0.5% Tween 20 (Merck 822184)). The target consisting of 4
.mu.l of the amplified product is mixed with 76 .mu.l of salmon PBS buffer
(3.times.PBS +10 .mu.g/ml salmon sperm DNA (Sigma D9156) and 10 .mu.l of
2N sodium hydroxide. The mixture is neutralized 5 min later by adding 10
.mu.l of 2N acetic acid. The mixture is added into the well in addition to
50 .mu.l of a solution of the oligonucleotide-peroxidase conjugate at a
concentration of 0.5 ng/.mu.l of oligonucleotide in a horse PBS buffer
(3.times.PBS+10% horse serum (BioMerieux 55842). The
oligonucleotide-peroxidase conjugate constitutes the detection probe and
possesses the nucleotide sequence of SEQ ID N010. The plate is incubated
for 1 h at 37.degree. C. and washed with 3.times.300 .mu.l of PBST. Into
each well, 100 .mu.l of OPD substrate (ortho-phenylenediamine, Cambridge
Medical Biotechnology ref/456) are added in a suitable buffer (0.055M
citric acid, 0.1M Na.sub.2 HPO.sub.4, pH 4.93) at a concentration of 4
mg/ml, to which 30-volumes H.sub.2 O.sub.2 diluted to 1/1000 is added
immediately before use. After 20 min of reaction, the enzymatic activity
is blocked with 100 .mu.l of 1N H.sub.2 SO.sub.4 and reading is performed
on an Axia Microreader apparatus (Axia, BioMerieux registered trademark)
at 492 nm.
The specificity results are given in Table 2. They indicate that the probe
MYC 2-S [sic] is specific for the genus Mycobacterium, and that the probe
TUB 1-S [sic] is specific only for the species of the tuberculosis
complex. Moreover, the probe may be labeled using a radiactive isotope, a
suitable enzyme, a fluorochrome, a base analog or a compound involved in a
luminescence reaction. In addition, the probe can have a diphosphate,
alkyl- or acrylphosphorate or phosphorothioate ester skeleton or a
skeleton of the polyamide type.
The second technique employs the format of the VIDAS automated apparatus
(bioMerieux, France, registered trademark).
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 23
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single- stranded
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium bovis
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 461-728
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CGAGCTGGTCAAAGAGGTAGCCAAGAAGACCGATGACGTCGCCGGTGACGGCACCACGAC60
GGCCACCGTGCTGGCCCAGGCGTTGGTTCGCGAGGGCCTGCGCAACGTCGCGGCCGGCGC120
CAACCCGCTCGGTCTCAAACGCGGCATCGAAAAGGCCGTGGAGAAGGTCACCGAGACCCT180
GCTCAAGGGCGCCAAGGAGGTCGAGACCAAGGAGCAGATTGCGGCCACCGCAGCGATTTC240
GGCGGGTGACCAGTCCATCGGTGACCTG268
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single- stranded
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium microti
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 461-728
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CGAGCTGGTCAAAGAGGTAGCCAAGAAGACCGATGACGTCGCCGGTGACGGCACCACGAC60
GGCCACCGTGCTGGCCCAGGCGTTGGTTCGCGAGGGCCTGCGCAACGTCGCGGCCGGCGC120
CAACCCGCTCGGTCTCAAACGCGGCATCGAAAAGGCCGTGGAGAAGGTCACCGAGACCCT180
GCTCAAGGGCGCCAAGGAGGTCGAGACCAAGGAGCAGATTGCGGCCACCGCAGCGATTTC240
GGCGGGTGACCAGTCCATCGGTGACCTG268
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single- stranded
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium africanum
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 461-728
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CGAGCTGGTCAAAGAGGTAGCCAAGAAGACCAATGACGTCGCCGGTGACGGCACCACGAC60
GGCCACCGTGCTGGCCCAGGCGTTGGTTCGCGAGGGCCTGCGCAACGTCGCGGCCGGCGC120
CAACCCGCTCGGTCTCAAACGCGGCATCGAAAAGGCCGTGGAGAAGGTCACCGAGACCCT180
GCTCAAGGGCGCCAAGGAGGTCGAGACCAAGGAGCAGATTGCGGCCACCGCAGCGATTTC240
GGCGGGTGACCAGTCCATCGGTGACCTG268
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single- stranded
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium chitae
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 461-728
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CGAGCTGGTCAAGGAAGTAGCCAAGAAGACTGACGACGTCGCCGGCGACGGCACCACCAC60
CGCCACCGTTCTGGCCCASVCGCTGGTTCGCGAAGGTCTGCGCAACGTCGCGGCCGGCGC120
CAACCCGCTCGGCCTGAAGCGCGGCATCGAGAAGGCCGTCGAGACCGTCTCGGAGAACCT180
GCTCAAGTCGGCCAAGGAGGTCGAGACCAAGGAGCAGATCGCCGCCACCGCCGGGATCTC240
CGCGGGCGACAACACCATCGGTGACCTG268
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single- stranded
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium intracellulare
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 461-728
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CGAGCTGGTCAAGGAAGTCGCCAAGAAGACCGACGACGTTGCCGGTGACGGCACGACGAC60
GGCCACGGTGCTGGCCCAGGCGTTGGTTCGCGAGGGCCTGCGCAACGTCGCGGCCGGCGC120
CAACCCGCTGGGTCTGAAGCGCGGCATCGAGAAGGCCGTCGACAAGGTCACCGAGACCCT180
GCTCAAGTCGGCCAAAGAGGTCGAGACCAAGGACCAGATCGCTGCCACCGCGGCCATTTC240
GGCGGGCGACCAGTCGATCGGCGACCTG268
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 200 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single- stranded
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium intralcellulare
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 518-717
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GACGGCCACGGTGCTGGCTCAGGCGTTGGTCCGCGAGGGCCTGCGTAACGTCGCGGCCGG60
CGCCAAACCGCTGGGTCTCAAGCGCGGCATCGAGAAGGCCGTCGAGAAGGTCACCGAGAC120
CCTGCTCAAGTCGGCCAAGGAGGTCGAGACCAAGGACCAGATCGCTGCCACCGCGGCCAT180
TTCGGCGGGCGACCAGCGGA200
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single- stranded
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 461-728
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CGAGCTGGTCAAGGAAGTCGCCAAGAAGACCGACGACGTCGCCGGTGACGGCACGACGAC60
GGCCACGGTGCTGGCCCAGGCGCTGGTCAAGGAGGGCCTGCGCAACGTCGCGGCGGGCGC120
CAACCCGCTGAGCCTCAAGCGCGGCATCGAGAAGGCGGTCGAGAAGGTCACCGAGACCCT180
GCTCAAGTCGGCCAAGGAGGTCGAGACCAAGGACCAGATCGCCGCCACCGCGGCGATTTC240
GGCGGGCGACCAGTCGATCGGCGACCTG268
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single- stranded
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
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