Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1996-02-07
1999-06-22
Housel, James C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 5, 435 34, 435 36, 435291, 435292, 435293, 426 34, 426 41, 426 43, G01N 33554, A23C 912, C12M 132
Patent
active
059142405
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method of detection, identification and/or quantification of bacteriophage (phage or phages), and to test kits for use in carrying out that method. Particularly the method enables detection of bacteriophages specific to particular bacterial genus, species or serotype, whether in isolated form or as contaminants in environmental or forensic samples, or in foodstuffs.
Although often undesirable, bacteria also have many industrial applications. Increasingly important is the role of bacteria in the area of genetic engineering and particularly in the large scale production of protein products as genetically engineered bacteria. More traditionally they have been used in production of natural products in fermentation, notably in production of cheese and milk fermentation products. The rapid fermentation of lactose to lactic acid is the principal reaction in the manufacture of such milk derived products and is initiated by the addition of starter cultures of species of lactic acid bacteria to the milk substrate. On occasion, for various reasons, these starter cultures fail and normal acid development fails to begin or is not maintained.
One of the most important reasons for starter culture failure is the presence in the milk of bacteriophage, often originating from the dairy environment. These are specific viral agents that attack and kill bacteria, in this case those of the starter culture. The bacteriophage reproduce parasitically in their bacterial hosts, resulting in a progeny of new phage particles which are liberated into the environment upon lysis of the bacterial cells. A bacteriophage infection in a dairy plant results in a serious decrease and, in some cases, a total failure in the production of lactic acid by the starter cultures. For many years the problem of phage has been the most serious one confronting the cheesemaker because of the economic losses it entails. These include the time lost in manufacture, loss of the raw material and of substandard product (see Klaenhammer (1984) Adv. Appl. Microbiol. 46, 313-25 and Heap & Lawrence (1988) Developments in Food Microbiology, Elsevier). Conventional techniques used in the dairy industry for bacteriophage detection are based upon traditional microbiological technology, and are both labour intensive and time consuming. These include observation of bacterial plaques, ie. clear areas in a background of bacteria, produced on lawns of starter culture bacteria in petri dishes and measuring the rate of lactic acid production in cultures, both inoculated with environmental samples. It is clear that the development of a rapid yet simple bacteriophage detection technique would be of immense benefit to the industry.
The present inventor has now provided such a technique, which in its preferred form uses bioluminescent techniques to provide a light signal indicative of the presence and amount of a specific bacteriophage or bacteriophage type in a sample under investigation, giving a positive result within about 4 to 5 hours, as opposed to 24 hours or more for existing methods.
The method of the invention is based upon the release of cellular components from bacteria infected with bacteriophage, particularly when the bacteriophage undergoes lytic cycle replication. In this cycle the bacteriophage take over the metabolism of the cell and replicate themselves, whereby at the end of the cycle the bacterial cell walls rupture to release progeny and substantially the entire bacterial cell contents. By measuring one or more particular components found associated with the bacterial cell that are made accessible to reagents in an incubation medium by the phage it is possible to measure infection, eg. via lysis, and using calibration curves or other statistical techniques, it is thus possible to estimate the amount of phage in the original sample. By exposing the sample to a bacteria that is a specific target of the phage which is the subject of a test, and providing incubation conditions that would allow infection of that bacteria by the sp
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Sigma Biochemicals, Organic Compounds Catalogue, "Adenosine 5'-Triphosphate (ATP) Bioluminescent Assay Kit", p. 51, 1992.
Housel James C.
Swartz Rodney P
The Minister of Agriculture Fisheries & Food in Her Brittanic Ma
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