Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-05-30
1998-07-28
Scheiner, Toni R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 71, 435 72, 435 732, 435 772, 435 8, 436501, 436518, 436829, 536 221, 5303913, C12Q 168, G01N 33569, G01N 3358, G01N 33532
Patent
active
057861510
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a methods and apparatus for the detection, identification and/or quantification of materials that are capable of being bound by specific binding agent, particularly biological materials such as antigens, microorganisms and nucleic acids. Particularly the methods enable detection of low numbers of microorganisms of specific genus, species or serotype, in isolated form or as contaminants in foodstuff, environmental or forensic samples.
There are many requirements for methods of screening for specific substances in low quantity in specific environments, such as for microorganisms such as bacteria, for example, for the detection of human bacterial pathogens in contaminated foods. Public health and quality control bodies demand rapid bacterial detection methods which have suitable levels of specificity and sensitivity, but few satisfactory methods exist.
In recent years, food poisoning has become a major topic of both public and scientific debate. This has been of great concern to the food producing industries and has led to increased demands for rapid bacterial food screening procedures to ensure product quality and allow early release for sale. If pathogenic bacteria are present in commercially prepared products they are likely to occur in very low numbers and it is this fact which makes bacterial detection a slow process, often taking days to complete.
Current bacterial detection techniques need the presence of high numbers of bacterial cells (10.sup.5 -10.sup.8) at the final stage before detection is possible. The increase in cell numbers is achieved by laborious and time consuming procedures involving selective enrichment and isolation steps.
U.S Pat. No. 4,704,355 (Bernstein) discloses the use of sensitised liposomes containing ATP which may be used in assays for antigens and DNA probes. Unfortunately, by their very nature, liposomes can only be used effectively to contain fluids and can not be isolated or stored in dry powder form. This can lead to problems with the stability and retention of the fluid and solute contained therein. Such problems are particularly common during even quite short periods of storage during which fluid may be rapidly lost to the storage medium.
The development of commercially viable, rapid and specific bacterial detection techniques has been worked on world-wide by many companies, often with the ultimate aim of producing an instant `dip stick` type of test which would specifically detect very low numbers of bacteria, e.g. Salmonella spp and which would be stable enough to be stored without significant loss of effectivess. The present invention is a test of this type, eliminating the need for lengthy enrichment stages and is able to rapidly detect very low amounts of antigenic material, such as specific microorganisms. The method can also be developed to any antibody/antigen assay systems, particularly where binding components are able to be fixed to a surface, wherein it can provide high sensitivity detection.
A sensitive and rapid bioluminescent ATP based detection system for bacterial pathogens is the subject of the applicant's copending application No PCT/GB93/01989. The existing patented method relies on the specific release of bacterial ATP by a bacteriophage and its subsequent detection by measuring the light produced after the addition of luciferin and luciferase. However this technique is limited by the amount of ATP present in viable bacterial cells, the sensitivity of the luciferin/luciferase detection system being about 1 pg, equivalent to about 1000 `average` bacterial cells.
Thus in a first aspect, the present invention provides a method of labelling a biological material comprising microencapsulating a nucleotide in microcapsules substantially impermeable to the nucleotide and linking the resulting microcapsules to the bioloigcal material. Preferably the nucleotide is an adenine nucleotide eg. ATP and is in crystalline form. By encapsulating the nucleotide in this form, very high quantities of ATP can be stored, thereby allowing h
REFERENCES:
patent: 4604364 (1986-08-01), Kosak
patent: 4704355 (1987-11-01), Bernstein
Scheiner Toni R.
The Minister of Agriculture Fisheries & Food in her Britannic Ma
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