Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1998-07-28
2001-05-01
Bui, Phuong T. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S004000, C435S007100, C435S007200, C435S239000, C436S063000, C436S518000, C436S546000
Reexamination Certificate
active
06225046
ABSTRACT:
TECHNICAL FIELD
The present invention relates to methods for detecting the presence of microorganisms in a sample rising particles bearing a ligand reactive to the microorganisms and fluorescent labelled ligands. The methods are suitable for flow cytometric detection of microorganisms.
BACKGROUND ART
Testing samples for the presence of microorganisms. in particular human pathogens. is an important part of monitoring samples including biological samples, foods, drinks, the environment and water supplies. In order to obtain immediate results testing often involves the direct analysis of samples for specific microorganisms. This can be labour intensive and routine for the technician involved. In particular. there is an increasing need to monitor water supplies to ensure they meet strict standards for human consumption. This often involves the testing of large volumes of water in order to detect relevant numbers of microbial contaminants which is time consuming and expensive. Automated methods and apparatus are being developed to assist in the large scale testing of samples for microbial contamination. The methods presently in use are often insensitive and do not allow the identification of specific microorganisms present in the samples being tested.
Flow cytometric detection of specific microorganisms relies on labelling the target organism with highly specific probes attached to fluorochrome molecules. To enable accurate detection. two or more different fluorescent labels need to be attached to the target organism (Vesey et al. 1994A). The types of probes available for these techniques are monoclonal and polyclonal antibodies, lectins and oligonucleotides. The range of fltiorochromes that can be coupled to these probes is limited. For example, many flow cytometers utilise a 488 nm laser to illuminate the sample, and accordingly, the choice of fluorochromes is limited to those which can be excited at 488 nm for those machines.
For a flow cytometer to distinguish one fluorochrome from another, the fluorochromes must emit at different wavelengths. There are only three types of fluorochromes presently available that excite at 488 nm and emit at wavelengths different enough to be distinguished by flow cytometry: green fluorochromes such as fluorescein isothiocyanate (FITC); red fluorochromes such as phycoerythrin (PE) and tandem fluorochromes. Unfortunately, the tandem fluorochromes are often not bright enough to be used in many applications. Therefore, flow cytometry is often limited to the detection of two fluorochromes. In applications such as the detection of specific microorganisms in a range of sample types, this poses a problem if there are only a small number of sites available for recognition on the microorganism. The level of sensitivity that can be achieved with two fluorochromes is often not good enough for these applications.
The present inventors have developed methods of detecting microorganisms in a fluid sample utilising particles and fluorescent labelled ligands reactive to microorganisms.
DISCLOSURE OF THE INVENTION
Accordingly. the present invention consists in a method of detecting the presence of microorganisms of a predetermined type in a sample containing the microorganisms. the method comprising the steps of:
(a) treating the sample with at least one detectable particle, each particle bearing a ligand reactive to the microorganisms of the predetermined type, the sample being treated for a period of time sufficient to allow the microorganisnms of the predetermined type in the sample to bind to the particle via the ligand;
(b) further treating the sample with at least one ligand labelled with a fluorescent marker, the ligand being reactive to the microorganisms of the predetermined type, the sample being treated for a period of time sufficient to allow the at least one ligand to bind to the microorganisms of the predetermined type; and
(c) analysing the sample so as to detect the presence of a particle associated with one or more of the fluorescent markers, the ligands being so selected that such an association is indicative of the presence of microorganisms of the predetermined type in the sample.
In a preferred embodiment of the present invention the particle is a fluorescent particle and more preferably a fluorescent latex bead. The beads preferably have a nominal diameter from 10 nanometres to 0.1 millimetres. The beads are preferably detectable by virtue of being fluorescently labelled. More than one type of particle can be used with each type bearing a ligand reactive to the same or different type of microorganism to be detected. It would, however, be within the scope of the invention to detect the bead by magnetism, by charge, by density difference or in any other suitable manner.
In a further preferred embodiment of the present invention the ligand is selected from the group consisting of antibody, lectin and oligonucleotide. Preferably, at least one of the ligands is a monoclonal antibody. When the particle is a fluorescent particle, the fluorescent markers attached to the at least one ligand have different fluorescent spectra to that of the fluorescent particle.
In a still further preferred embodiment. the analysing of the treated sample is by flow cytometry. the microorganisms being detected by the presence of fluorescence of the labelled ligand or in combination with the size of the particle, or more preferably, fluorescence of both the marker and the particle. With regard to the detection of the size of the particle, this includes either detecting the known size of the particle or detecting or measuring for an increased size caused by the binding of microorganisms to the particle.
In a still further preferred embodiment of the present invention, the particle is labelled with several ligands reactive to the same or different microorganisms. Furthermore, several different particles can also be used having the same or different ligands bound thereto. For example, in step (b) several different ligands reactive to the same or different microorganisms but provided with different fluorescent markers are utilised to allow the possible detection of more than one type of microorganism bound to the particle.
The method according to the present invention preferably uses one or more fluorescent markers that are excited at 488 nm and emit at wavelengths ranging from green to infra-red. It will be appreciated by one skilled in the art that fluorescent markers that are excited at other wavelengths are also suitable for the present invention.
The present invention is suitable for detecting multiple forms of the same species of microorganism or detecting several different microorganisms from the same sample. The microorganisms bound to the particle may be further treated or analysed after being detected by the method of the present invention.
The number of particles used in the present methods will depend on the type of particle, the type of sample being tested, and the number and type of microorganisms in the sample. It will be appreciated that the microorganism must come in contact with a particle to allow binding. Therefore, the number of particles should be in excess to the number microorganisms in a given sample to ensure detection of the microorganisms of interest. In order to assist in this regard usually at least 10
3
particles per ml, preferably between 10
4
to 10
7
particles per ml are used. When a sample has a lot of particulate material present then usually a higher number of detectable particles is used in order to increase the possibility that the microorganisms present in the sample will come into contact with the particles and bind. The present invention has the advantage that the number of microorganisms in a sample can also be estimated by adding a known number of detectable particles to the sample and counting all of those particles to determine the number that have bound microorganisms. Furthermore, by adding a known number of particles to the sample it is also possible to confirm that the sample was correctly analysed by enumerating the numb
Veal Duncan
Vesey Graham
Williams Keith
Bui Phuong T.
Macquarie Research Ltd.
Morgan & Lewis & Bockius, LLP
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