Chemistry: molecular biology and microbiology – Apparatus – Involving lysis of a microorganism by means other than...
Reexamination Certificate
2001-03-02
2003-10-14
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Apparatus
Involving lysis of a microorganism by means other than...
C435S259000, C435S283100
Reexamination Certificate
active
06632662
ABSTRACT:
The present invention relates to a novel device for the lysis of micro-organisms making it possible, by completely or partially destroying the membrane, to release at least one intracellular biological component. It also relates to a lysis method employing a device as defined hereinabove.
The state of the art describes a method which is generally used for cell lysis, and which essentially consists in placing, in a container, a biological sample in liquid medium, comprising the micro-organism to be lysed. Next, at least one material in the form of particles, relatively hard and substantially inert with respect to the biological components is added to the container and finally the mixture consisting of the biological sample and of the material in the form of particles is subjected to a vortex-type motion.
This method used exhibits certain disadvantages. Thus, it has been noticed that the lysis method commonly used is not sufficiently efficient, and that, in particular, the cell lysis proves insufficient in terms of quantity and in terms of quality.
In addition, the method employed does not always allow the lysis of cells which are deemed to be lysis-resistant, like Gram-positive cells such as mycobacteria, for example.
Likewise, implementation of the method often entails adding additional reagents, particularly enzymes and/or detergents.
The applicant company has filed a patent application FR97/12164 dated Sep. 23, 1997, regarding a method for the lysis of micro-organisms.
This lysis method is intended to release at least one biological compound contained in the micro-organism, comprising a lysis step wherein:
a biological sample in a liquid medium, comprising the micro-organism that is to be lysed, is placed in a container,
at least one material in the form of particles, relatively hard and substantially inert with respect to the biological compounds, is placed in said container, and
the mixture of the biological sample and of the material in the form of particles is subjected to a vortex-type motion.
The method uses a material in the form of particles which comprises beads with a diameter of between 90 and 150 &mgr;m, and which satisfies the following equation:
Ve=&bgr;Vb,
where Vb is the apparent volume of the beads, Ve is the volume of the liquid sample and a is a number between 1.4 and 10 when the container is of tubular shape, and less than or equal to 2.1 when said container is of diskoid shape.
These characteristics are still particularly advantageous and the content of that patent application is to be considered as being contained in the description of the present invention.
Document U.S. Pat. No. 5,643,767 proposes a container to allow cellular components such as ribonucleic acids (RNAs) to be isolated from cells present in a liquid solution. To do that, this container contains two types of bead, which differ in terms of their diameter. The first type, of small size, is made of glass or metal with a diameter of between 0.1 and 1 millimeter. The second type, of large size, is made of the same material, glass or metal, with a diameter of between 3 and 5 millimeters.
The essential disadvantage with this device lies in the fact that whether the vortex is mechanical or magnetic, the motion of the beads is homogeneous.
However, the applicant company, through sustained effort and additional work, kept up these studies into the field of vortex-type-motion magnetic lysis and succeeded in determining characteristics allowing vortex-type agitation in a container without the container needing to be mechanically agitated, as is the case with a mechanical vortex. Thus, by adding beads of essentially two different diameters, it is possible to considerably increase the efficiency of said lysis, improving the release of the intracellular components. It is also possible, for the same efficiency of lysis, to reduce the time taken to achieve this lysis, which is something which may prove particularly beneficial in the case of diagnostic tests which have to be performed quickly.
To this end, the present invention relates to a device for the lysis of at least one species of micro-organism, so as to release at least one intracellular biological component, which comprises a container in which there are, on the one hand, a biological sample in liquid medium, which contains the micro-organism to be lysed and, on the other hand, at least one material in the form of particles, which is relatively hard and substantially inert with respect to the biological compounds of the sample, characterized in that the material in the form of particles comprises at least two types of grinding means of different sizes:
at least one large-sized magnetic means which is slaved to the movement of a magnetic field, and
at least one small-sized means which is set in motion by the large-sized grinding means.
The material in the form of particles comprises, by way of large-sized grinding means, at least one large-diameter bead and, by way of small-sized grinding means, at least one small-diameter bead.
The ratio between the sizes of the small-sized grinding means and the size of the large-sized grinding means is between 1/10 and 1/100, preferably between 1/30 and 1/60 and, more specifically, this ratio is 1/40.
In addition, the ratio between the size of the micro-organism to be lysed and the size of the small-sized grinding means is between 1/10 and 1/100, preferably between 1/30 and 1/60, and more specifically is 1/50.
Another characteristic: the ratio between the total volume of the small-sized grinding means and the volume of the biological sample containing the micro-organism to be lysed is between 1/2 and 1/5 and is preferably 1/3.
The number of large-sized grinding means is between 1 and 10 and preferably between 1 and 4.
In a first preferred embodiment, the container is of tubular shape.
In a second preferred embodiment, the container is of diskoid shape.
Whatever the embodiment used, each large-sized magnetic grinding means is moved by the rotary motion about the tube of at least one permanent magnet.
In addition, each large-sized magnetic grinding means is moved by the rotary motion under the tube of at least one permanent magnet.
When the container is of diskoid shape, each large-sized magnetic grinding means collaborates with a roughly circular runway located at the periphery of the container.
The present invention also relates to a method for the lysis of at least one species of micro-organism in a biological sample so as to release at least one intracellular biological component, which uses, in a container, at least one material in the form of particles, forming a grinding means which is relatively hard and substantially inert with respect to the biological compounds in the sample, characterized in that it consists in:
placing at least one large-sized magnetic grinding means, at least one small-sized non-magnetic grinding means and a biological sample containing the micro-organism that is to be lysed in the container,
placing the magnetic grinding means in a moving magnetic field so as to generate a vortex motion in the mixture, and
stopping the effect of the magnetic field on said magnetic grinding means.
In a first mode of operation, the magnetic field can rotate about the container at the height of the biological sample.
In a second mode of operation, the magnetic field can rotate under the container at the height of the biological sample.
Whatever the mode of operation, the rotary magnetic field applies to the magnetic grinding means located in the biological sample a field which is alternately strong and weak.
REFERENCES:
patent: 5643767 (1997-07-01), Fischetti et al.
patent: 2 768 743 (1999-03-01), None
Melendres et al., A kinetic analysis of cell disruption by bead mill, 1991, Bioseparation, vol. 2, pp. 231-236.
Broyer Patrick
Cleuziat Philippe
Colin Bruno
Paris Cécile
Santoro Lyse
Bio Merieux
Ketter James
Sullivan Daniel M.
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