Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-05-14
2002-10-01
Leary, Louise N. (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S004000, C435S286100, C435S286500, C435S283100
Reexamination Certificate
active
06458553
ABSTRACT:
The invention relates to an analysis card making it possible to count and characterize microorganisms or any other biological material, such as a nucleic acid or antibody, in a biological sample. The invention also relates to a method of filling such a card as well as to the use of this card.
The terms [sic] “biological sample” should be understood to mean a liquid sample resulting from the physical treatment, for example homogenization, or dissolution in a solvent, or from the biological treatment, such as enzymatic digestion, of solid or liquid products, possibly consisting of food products, of water, of blood.
Apart from the conventional counting method in Petri dishes, nonautomated methods are found in the prior art for quantifying microorganisms; these methods are mainly based on determining the Most probable number (MPN).
U.S. Pat. No. 5,700,655 describes an apparatus for quantification of biological material in a liquid sample. This apparatus consists of a plastic sheet obtained by thermoforming, the sheet itself consisting of analysis cavities of the same size and of a cover making it possible to maintain sterility. The method consists in firstly depositing the sample to be analyzed in the center of the sheet and then the reaction mixture supplied with the apparatus. The apparatus thus prepared is agitated so as to fill all the cavities in the identical manner and then the excess reaction mixture is eliminated. Finally, the sheet is incubated for a time long enough to reveal the presence of microbes in one or more wells.
However, this apparatus, like the conventional Petri dishes, only allows the sample to be diluted once. Knowing that the sample has to be diluted several times in order to be sure of being able to determine the concentration of germs contained in said sample, it will be necessary to repeat this operation at least once.
In addition, this apparatus has another major drawback. This drawback consists of the need to eliminate the surplus reaction mixture. Since this operation is carried out by tilting the sheet, certain cavities may lose a part of their content. The amount of liquid in the cavities will therefore no longer be identical, and consequently the determination of the amount of germs will be false.
Furthermore, eliminating the surplus reaction mixture results in the production of potentially contaminated waste, which has to be reprocessed. Likewise, this elimination of the surplus contaminates the outside of the apparatus, which then becomes a source of biological contamination for the operator and all other surfaces which come into contact with him.
Finally [sic], since the cavities are arranged concentrically, the read-out also takes place concentrically, which may be a source of error, especially as the positive cavities are often dispersed over the sheet.
Finally, the abovementioned apparatus still has the basic drawback of Petri dishes, namely the fact that it must absolutely be handled with care and horizontally while the medium is gelling, if this gelling function is provided, so as to prevent a sample from leaking or passing from one cavity to another, which results in handling artifacts.
U.S. Pat. No. 5,518,892 discloses another apparatus for quantification of microorganisms in a liquid medium. It comprises a bag, made in a material and obtained by thermoforming, which has an opening on one of its sides. The method consists in forming, by heating, impermeable compartments or cavities on the lower face of the bag, allowing various amounts of the sample to be separated. Once the wells have been formed, the reaction mixture is prepared by adding the sample to be analyzed to the reaction mixture supplied with the apparatus. The reaction mixture is then poured into the bag via the opening provided for this purpose. A check is made that all the cavities are properly filled and then, after heat sealing, the bag is incubated for a time long enough to reveal the presence of microorganisms in one or more cavities. The cavities may be of the same size or three series of different sizes. It is thus possible to use a single bag in order to simulate seeding according to several dilutions.
However, this system has certain drawbacks. Firstly, and unlike an automated and very precise industrial injection molding system, this apparatus cannot guarantee optimum molding quality making it possible to obtain cavities of perfectly identical capacity, since basically simple plastic sheets are used which are formed by thermoforming. This is particularly important when a molding is produced with series of cavities of various volumes, especially when these are small volumes. Secondly, the reaction mixture is introduced by hand and the distribution in the cavities therefore takes place randomly. Thus, there may be no certainty that the volume of reaction mixture delivered into each cavity is the same, even if the size of the cavities is perfectly identical. The results may therefore again be false. Thirdly, the cavities are isolated by heat sealing at temperatures above 180° C., which carries the risk of irreversibly impairing (destroying the biological material to be analyzed and/or evaporating all or some of the liquid containing said material) the sample fraction contained in the cavities. This is all the more sensitive the smaller the size of the cavities and the smaller the amount of liquid they contain. This handling may therefore falsify the results of the counting and of the characterization.
According to the present invention, the analysis card proposed helps to overcome all of the abovementioned drawbacks.
This card makes it possible to fill all the cavities, whatever their size, which is proportional from one cavity to another, and which does not require positioning precautions to be taken when filling it. By “proportional from one cavity to another” is meant that when two cavities have the same dimensions, the proportion will then be substantially 1 for 1, or when two cavities have different dimensions, the proportion is different from 1 for 1. Moreover, there is no risk of contamination, on the one hand, of the specimen with the outside, since the liquid remains confined, and, on the other hand, between the various cavities of the card, since an isolating separation is also provided between each cavity of said card.
For this purpose, the present invention relates, according to a first preferred embodiment, to an analysis card which comprises a body consisting of:
a region for injecting a biological liquid to be analyzed into the card,
a main feed channel for the biological liquid coming from the injection region,
at least two secondary channels lying in the extension of the main channel, and
at least one well corresponding to each secondary channel,
characterized in that each well constitutes a means of delivering a predetermined volume of said biological liquid into at least two terminal analysis cavities, via a terminal channel for each analysis cavity, and in that the total volume to be analyzed is less than the total volume of all of the analysis cavities.
According to a second preferred embodiment of the invention, the analysis card which comprises a body consisting of:
a region for injecting a biological liquid to be analyzed into the card,
at least two feed channels for the biological liquid coming from the injection region, and
at least one well corresponding to each feed channel,
[lacuna] characterized in that each well constitutes a means of delivering a predetermined volume of said biological liquid into at least two terminal analysis cavities, via a terminal channel for each analysis cavity, and in that the volume to be analyzed is less than the total volume of all of the analysis cavities.
According to the above two embodiments, the delivery of the biological liquid into the terminal analysis cavities takes place in parallel.
According to a third preferred embodiment of the invention, the analysis card which comprises a body consisting of:
a region for injecting a biological liquid to be analyzed into the card
Colin Bruno
Paris Cécile
Sofia Thierry
Terrot Claude
Bio Merieux
Leary Louise N.
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