Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-02-29
1999-12-14
Marschel, Ardin H.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 911, 435 912, 536 221, 536 2433, C12Q 168, C12P 1934, C07H 1900, C07H 2104
Patent
active
060015659
DESCRIPTION:
BRIEF SUMMARY
CROSS REFERENCE TO RELATED APPLICATION
This application claims priority to International Application PCT/GB94/01967, filed Sep. 9, 1994, which designates the United States of America.
FIELD OF THE INVENTION
This invention relates to the detection and speciation of Campylobacter bacteria, for example in clinical, environmental and food samples. In particular, this invention relates to a method of detecting whether a sample contains Campylobacter and to a method of differentiating between the main Campylobacter species jejuni, coli, upsaliensis and lari.
BACKGROUND OF THE INVENTION
Campylobacter species are recognised as the most frequent cause of bacterial gastroenteritis in the United Kingdom and many other countries throughout the world. In the U.K. approximately 90% and 10% of case isolates are identified as Campylobacter jejuni and Campylobacter coli respectively, plus a small number of other species such as Campylobacter upsaliensis and lari. The majority of the infections are sporadic the source of which remains largely unknown although the importance of several vehicles is now recognised.
There is a known desire to be able to detect and differentiate species of Campylobacter. However, it is also known that present Campylobacter enrichment culture techniques lack sensitivity, making detection difficult. Campylobacter jejuni does not multiply in foodstuffs and low numbers may be present together with a high background of indigenous microflora. Also, surface viable counts of Campylobacter can decrease rapidly and cells that are potentially culturable are often lost before samples reach a laboratory for analysis. Another factor making detection problematic is that antibiotics used in culture enrichment media may damage already weakened Campylobacter.
There are currently available assays for detection of a variety of food and water-borne pathogens; L. pneumophila, V. vulnificus, enteroinvasive E. coli, Shigella; but no satisfactory method of detecting Campylobacter or distinguishing between the four main Campylobacter species is known.
A method of detecting Campylobacter has been published by Giesendorf, B A J, et al in Applied and Environmental Microbiology, December 1992, pages 3804-3808. The method detects the species jejuni, coli and lari, and produces similar results to conventional methods but in a reduced time. The method suffers from a number of drawbacks. It does not enable detection of the species upsaliensis. Further, the method employs polymerase chain reaction (PCR) techniques but nevertheless requires a short enrichment culture before the PCR can be employed. Further still, the primer used for the PCR does not have the precise homology with DNA sequences in the three Campylobacter species that can be detected using the method.
Another method for detecting Campylobacter jejuni and Campylobacter coli is known from Wegmuller, B E et al, Applied Environmental Microbiology, vol. 59, part 7, 1993 pages 2161-2165. The described method detects only the species jejuni and coli.
In addition to the above-identified problems with detection and speciation of Campylobacter, recent work on Campylobacter jejuni suggests that in certain circumstances it enters a "non-culturable, viable form" when subjected to environmental stresses, such as pH or temperature extremes, increased oxygen tension or nutrient depletion. In this form, Campylobacter infectivity is maintained but-the organisms cannot be cultured. Thus there exists a need for the improvement of methods of detection of non-culturable forms of Campylobacter.
SUMMARY OF THE INVENTION
It is an object of this invention to provide a method of testing for the presence of Campylobacter that enables more efficient detection and eliminates or mitigates the problems with existing techniques. It is a further object to provide a method of distinguishing the Campylobacter species jejuni, coli, upsaliensis and lari.
DETAILED DESCRIPTION OF THE INVENTION
Accordingly, in a first aspect the present invention provides a method of testing for the presence o
REFERENCES:
New England Biolabs Inc., Beverly, MA pp. 7-31, 1986.
Alm, R.A., et al., "Distribution and Polymorphism of the Flagellin Genes from Isolates of Campylobacter coli and Campylobacter jejuni," J. Bacteriol. 175(10):3051-3057 (May 1993).
Birkenhead, D., et al., "PCR for the Detection and Typing of Campylobacters," Lett. Appl. Microbiol. 17(5):235-237 (Nov. 1993).
Bolton, F.J., et al., "Development of a Blood-free Campylobacter Medium: Screening Tests on Basal Media and Supplements, and the Ability of Selected Supplements to Facilitate Aerotolerance," J. Appl. Bacteriol. 54(1):115-125(1983).
Giesendorf, B.A.J., et al., "Rapid and Sensitive Detection of Campylobacter spp. in Chicken Products by Using the Polymerase Chain Reaction," Appl. Env. Microbiol. 58(12):3804-3808 (Dec. 1992).
Nachamkin, I., et al., "Flagellin Gene Typing of Campylobacter jejuni by Restriction Fragment Length Polymorphism Analysis," J. Clin. Microbiol. 31(6):1531-1536 (Jun. 1993).
Wegmuller, B., et al., "Direct Polymerase Chain Reaction Detection of Campylobacter jejuni and Campylobacter coli in Raw Milk and Dairy Products," Appl. Env. Microbiol. 59(7):2161-2165 (Jul. 1993).
English-language abstract for European Patent Office Publication No. EP 0 350 392 (Ref. AL1), Derwent WPI Accession No. 90-010125/02.
Fox Andrew John
Jones Dennis Mackay
Marschel Ardin H.
Public Health Laboratory Service Board
Riley Jezia
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