Difficile specific oligonucleotides

Details Difficile specific oligonucleotides Difficile specific oligonucleotides

1992-05-19

1995-10-17

Parr, Margaret

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

536 243, 536 2433, 435 912, C12Q 168, C07H 2100, C12P 1934

Patent

active

054590345

DESCRIPTION:

BRIEF SUMMARY
This invention relates to oligonucleotides specific to bacteria of the species Clostridium difficile and useful as C. difficile DNA probes or in the amplification of C. difficile DNA.
Clostridium difficile, a Gram positive anaerobic spore-forming organism, is recognized as the major cause of pseudomembranous colitis and is implicated in antibiotic-associated colitis and diarrhoea. The pathogenicity of the organism is related to the production of an enterotoxin, toxin A, and a potent cytotoxin, toxin B. The diagnosis of C. difficile-associated disease depends on the isolation and identification of the organism and/or the demonstration of toxins in faecal specimens of patients and mono-specific antibodies for that purpose are disclosed in EP-A-0153519 and EP-A-0154064. Such procedures are time consuming, and a more rapid diagnosis is essential to enable the initiation of prompt therapy.
The present invention aims to avoid these difficulties by providing oligonucleotides specific to C. difficile DNA and is a development of the work reported in J. Clin. Microbiol. 24, 3, pp 384-387 (Sep. 1986). In that paper, the results of immunoblot studies on various strains of C. difficile were reported to various immunogenic proteins were identified common to all nine C. difficile strains tested. In particular, three common antigens were potentially identified with molecular weights in the range 50 to 70 kDa.
Following on from those studies, further antigens have now been identified common to all C. difficile strains tested, and the genes encoding those antigens have been identified, cloned and, in one case, sequenced giving rise to the present invention, namely the construction, for the first time, of DNA probes and oligonucleotide primers specific to C. difficile DNA.


BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing the bands obtained by subjecting genetic DNA from C. difficile to PCR amplification using oligonucleotides according to the present invention as primers and subjecting the product of the CR reaction to separation on an agarose gel.
FIG. 2 is a similar photograph but showing the results obtained using a different pair of primers for the PCR reaction.
More specifically, in accordance with a first aspect of the present invention a 1947 bp DNA fragment encoding part of the A toxin gene of C. difficile has been identified, cloned and sequenced, the sequence being as set out in the Appendix I hereto as SEQ ID No.1 and which is to be taken as forming part of the present specification.
Analyzing that 1947 bp sequence in more detail, a continuous open reading frame is found containing four distinct groups of repeat nucleotide sequence with 88 to 100% identity within each group. These groups, identified herein as A, B, C, D (A=81 bp; B, C, D=63 bp) appear in the following arrangement:


__________________________________________________________________________ ##STR1## where A = 5' ATA GGG GTG TTT AAA GGA CCT AAA GGA TTT GAA TAT TTT GCA Ile Gly Val Phe Lys Gly Pro Lys Gly Phe Glu Tyr Phe Ala CCT GCT AAT ACT TAT AAT AAT AAC ATA GAA GGT CAG GCT 3' nucleotides Pro Ala Asn Thr Tyr Asn Asn Asn Ile Glu Gly Gln Ala 1-81 of Seq. I.D. No. 1 B = 5' ATA GTT TAT CAA AGT AAA TTC TTA ACT TTG AAT Ile Val Tyr Gln Ser Lys Phe Leu Thr Leu Asn GGT AAA AAA TAT TAC TTT GAT AAT AAC TCA 3' nucleotides Gly Lys Lys Tyr Tyr Phe Asp Asn Asn Ser 82-144 of Seq I.D. No. 1 C = 5' GAA GCA GCT ACT GGA TGG CAA ACT ATT GAT GGT Glu Ala Ala Tyr Gly Trp Gln Tyr Ile Asp Gly AAA AAA TAT TAC TTT AAT ACT AAC ACT GCT 3' nucleotides Lys Lys Tyr Tyr Phe Asn Tyr Asn Tyr Ala 208-270 of Seq I.D. No. 1 and D = 5' ATA GCT TCA ACT GGT TAT ACA ATT ATT AAT GGT Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly AAA CAT TTT TAT TTT AAT ACT GAT GGT ATT 3' nucleotides Lys His Phe Tyr Phe Asn Thr Asp Gly Ile 334-396 of Seq I.D. No. __________________________________________________________________________ 1
The repeating sequence of the A, B, C and D blocks given above account for 1935 bp of the 1947

REFERENCES:
patent: 4965188 (1990-10-01), Mullis et al.
Price et al., Curr. Microbiol. 16(1):55-60 (1987) "Cloning of the carbohydrate-binding portion . . . ".
Eichel-Streiber et al., J. Gen. Microbiol. 135(1):55-64 (1989) "Cloning and Characterization of Overlapping . . . ".
Kim et al., J. Clin. Microbiol. 27(6):1192-1196 (1989) "Etiology of Childhood Diarrhea in Korea".
Muldrow et al., FEB 213(2):249-253 (1987) "Molecular cloning of Clostridium Difficile . . . ".
Figueiredo et al., Biochem. Soc. Trans. 14(3):640 (1986) "Luminescent detection system for non-radiactively . . . ".
Wren et al., FEBS Letters, 225:82-86 (1987) "Molecular cloning and expression . . . ".
J. of Clinical Microbiology, Sep. 1986, pp. 384-387 vol. 24 No. 3 Heard et al. "Immunoblotting to Demonstrate Antigenic and . . . difficile ".
J. Clinical Microbiology Dec. 1988, pp. 2484-2488, vol. 26, No. 12 Wilson et al. "Species-Specific Oligonucleotide Probes for rNA of . . . Species".
J. Clinical Microbiology Aug. 1990 pp. 1808-1812 vol. 28, No. 8 Wren et al. "Identification of Toxigenic Clostridium difficile Strains . . . Probe".
J. Clinical Microbiology Jan. 1991 pp. 33-37, vol. 29, No. 1 Kato et al. "Identification of Toxigenic Clostridum difficule . . . Reaction".
FEBS Letters 04525 vol. 213, No. 2, pp. 249-253 Muldrow et al. "Molecular cloning of Clostridium difficile toxin A gene . . . ".
Dialog Information Service, File 5: Biosis Previews 69-91/ Mar. Biosis acc. no. 35037370 Diag Ass. No. 6171849 Fasching et al. "Oligonucleotide hybridization probe for detection of toxin A . . . ".

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