Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1994-10-14
1997-07-15
Scheiner, Toni R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 5, 435 31, 435 29, 435340, 5303884, G01N 33569, C07K 1612
Patent
active
056482272
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method of detecting the presence or absence of viable microorganisms in a sample.
More particularly, it relates to the use of suitable antibodies which specifically recognise and bind an epitope located on the surface of intact viable microorganisms.
There is a need to detect viable microorganisms in food samples, water, air, industrial products, human clinical and animal samples as rapidly as possible. It is also desirable to identify genus and species at the same time.
A number of ways to improve upon the traditional culture methods have more recently been developed. Thus, to accelerate the bacterial identification process, highly specific polyclonal and preferably monoclonal antibodies have been developed that can be used in immunodiagnostic tests, such as an ELISA test. In the ELISA test a sample of culture medium is added directly to an antibody coated tube and the specific microorganisms in the culture medium are thus immobilised onto the surface of the coated tube, where they can be detected by means of a second, labelled antibody.
The ELISA test suffers a disadvantage compared to the extended culture procedure discussed below. The sensitivity of the best ELISA tests currently available is about 10.sup.4 to 10.sup.5 organisms per milliliter. At microorganism concentrations below this range the test will be negative and the microorganisms will not be detected. In contrast, culture procedures should be able to detect a single bacterium. Since the ELISA tests currently available are a relatively less sensitive technique, they still require one or more culture steps in order to enrich the number of microorganisms present in the sample before the ELISA test is carried out. Often these additional culture steps can take 16 hours or more.
The traditional culture techniques are slow and are now being superseded by rapid detection methods based particularly on ultra-sensitive DNA probe and DNA amplification techniques. DNA amplifiers, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR) and the DNA/RNA systems, such as NASBA or 3SR, can theoretically detect a single microorganism, and can provide a species identification at the same time.
In a production process, such as the preparation of a pharmaceutical or a foodstuff, there is often incorporated a sterilisation step to kill the microorganisms present in the product. Sterilisation steps commonly employ heat, extremes of pH, salt, bactericidal chemicals, irradiation or exposure to ethylene oxide. Less aggressive conditions are also commonly used, such as pasteurisation. Any microorganisms present in, or on, the product will not be viable after sterilisation and a subsequent culture procedure will report a negative result. However, the culture procedure is slow, and alternatives, such as ultrasensitive DNA probes and DNA amplifiers, cannot distinguish between live and dead cells and also cannot distinguish between live cells and extraneous nucleic acid from disrupted (dead) cells. Hence, these detection techniques are undesired because they are tedious and slow or because they can report false positives after sterilisation procedures, which has serious commercial implications in the food and manufacturing industries and serious medical implications in healthcare.
One way of capturing microorganisms in a dispersed food sample, in a clinical sample, e.g. blood, urine, or in a wash eluent from a surface is to pass a volume of the fluid through a controlled pore or depth filter membrane, to wash the membrane and then to detect the entrapped microorganisms. The disadvantages of this approach are that until now intact viable and intact dead cells could only be distinguished by culture techniques and, as a secondary issue, dispersed particulate samples such as foodstuffs, red cells in whole blood, or tissue material in urine can block the filter rapidly.
It is well known that microorganisms are disrupted on heating to high temperatures e.g. boiling, as this is often used to release the nucleic acid from the
REFERENCES:
patent: 5077192 (1991-12-01), Liang et al.
Nair, et al., International Journal of Leprosy and Other Mycobacterial Diseases, vol. 58, No. 3, pp. 540-547, Sep. 1990.
Amdex A/S
Scheiner Toni R.
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