Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-11-08
1999-02-23
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 4353201, 536 237, C12Q 168, C12P 1934, C07H 2104
Patent
active
058742204
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to primers for the amplification of genes coding for the enterotoxin and the lecithinase, also called phospholipase C, of Clostridium perfringens.
Another object of the invention is the application of these primers for the detection and the numeration of C. perfringens.
DESCRIPTION OF THE PRIOR ART
Clostridium perfringens type A is widely distributed (soil, sewage, intestinal tracts of humans and animals), and is a common causative agent of bacterial food poisoning outbreaks worldwide. The symptoms, predominantly diarrhea and abdominal pain, appear 6 to 24 hours after ingestion of contaminated food. Vomiting and fever are unusual. Death occurs occasionally among debilitated persons, particularly the elderly.
C. perfringens enterotoxin CPE which is produced during the sporulation phase has been shown to produce the symptoms associated with C. perfringens food poisoning. The illness is caused by the ingestion of food that contains larger number of vegetative enterotoxigenic C. perfringens (more than 10.sup.5 organisms per g). These bacteriae multiply and sporulate, releasing CPE into the intestine.
A C. perfringens count of more than 10.sup.6 /g in fecal samples of ill persons is indicative of C. perfringens food poisoning. In addition, CPE detection directly in fecal samples is a valuable method confirming the diagnosis.
The epidemiological investigations involve C. perfringens numeration in suspected foods. The characterization of the enterotoxigenic C. perfringens strains is not used routinely, since C. perfringens sporulates poorly in usual culture media.
Recently, CPE and phospholipase C gene sequences have been determined (VAN DAMME et al. 1989, Ant. Van Leeuwen 56, 181-190; TSO J. and SIEBEL, 1989. Infect. Immun. 57: 468-476, TITBALL et al. 1989 Infect. Immun. 57:367-376). The phospholipase C gene is located on a variable region of the chromosomal DNA in all C. perfringens toxinotypes whereas the distribution of CPE gene is restricted. Only 6% of the C. perfringens isolates from various origins showed the presence of CPE gene by DNA--DNA hybridization. This ratio is higher (59%) among C. perfringens strains isolated from confirmed outbreaks of food poisoning. In the standard methods the enterotoxin detection by biological or immunological tests requires previously the sporulation of C. perfringens. Several specific medium and protocols for C. perfringens sporulation have been described, but they are time consuming and many C. perfringens strains do not sporulate or very poorly (DUCAN and STRONG, 1968. Appl. Microbiol. 16: 82; PHILIPS, 1986, Lett. Appl. Microbiol 3: 77-79), which impairs the CPE detection.
A method for the detection of C. perfringens by polymerase chain reaction (PCR) has yet been disclosed during the third Congress of the French Society of Microbiology (Apr. 21-24, 1992) through a poster of FACH et al.
The method consisting of an amplification of the parts of the genes encoding the .alpha.-toxin, also called lecithinase, and the enterotoxin of C. perfringens by using oligonucleotidic primers which were choosen in these genes. However, the sequence of the primers used for carrying out this method was not disclosed and the sensitivity mentioned by the authors was low, i.e. from 500 to 5000 bacteriae per gram of feces.
Moreover, the results were obtained on feces artificially contaminated and not on feces from contaminated patients or on contaminated food.
The inventors have thus sought to elaborate a sensitive and reliable method allowing the detection of low quantities of bacteriae in samples of different origins, such as in feces or food, in a raw form.
They have surprisingly shown that it is necessary to choose the primers in some well determined regions of the genes, as well as in the gene of the enterotoxin that is one of the lecithinase.
Besides, they have carried out a process for the treatment of foods samples, allowing a specific, sensitive and reliable determination of the presence of the C. perfringens contained in these foods.
SUMMARY OF TH
REFERENCES:
Saito et al., Int. J. Food Microbiol. 17(1), 47-55 (1992).
Havard et al., FEMS Microbiol. Lett. 97, 77-82 (1992).
Saint-Joanis et al., Mol. Gen. Genet. 219, 453-460 (1989).
Czeczulin et al., Infec. Immun. 61(8), 3429-3439(1993).
Fach et al., J. Appl. Bacteriol. 74,61-68 (01 Jan. 1993).
Fach et al, J. Appl. Bacteriol, (1993 Jan.) 74 pp.61-66.
Daude, et al, J. Appl. Bacteriol, (1994 Dec) 77 pp.650-655.
Titball et al, Infection and Immunity, vol. 57, No. 2, Feb. 1989 pp. 367-376.
Okabe et al, Biochem. Biophys. Res. Commun. vol. 160 (1989) pp. 33-39.
Leslie et al, Mol. Microbiol. 3:383-392 (1989).
Fach et al, Med. Mal. Infect., (1993) 23/2 (70-77).
Fach Patrick
Guillou, deceased Jean-Pierre
Popoff Michel
Centre National D'Etudes Verterinaires et Alimentairescneva
Horlick Kenneth R.
Institut Pasteur
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