Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Patent
1989-05-30
1992-02-25
Wax, Robert A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
435 29, 435 12, 435 10, 435 24, 435870, 435 14, 435 30, C12Q 120, C12Q 102
Patent
active
050913077
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a procedure for the counting, detection and identification of mycoplasms in general and urinogenital mycoplasms in particular. An object of the invention is also a biological medium specially adapted to this effect.
BACKGROUND OF THE INVENTION
It is known that mycoplasms are bacteria without walls endowed with enzymatic properties (urease for Ureaplasma urealyticum and arginine decarboxylase for Mycoplasma hominis and fermantans).
These enzymatic properties are used for the identification and counting of the strains in the urinogenital samples.
In effect, these micro-organisms are commensal bacteria (present on perfectly healthy individuals' mucous membrane at a rate slightly lower or equal to 10.sup.3 CCU/ml--unit of colour change/ml--). When an infection breaks out or when a mucous membrane is rendered fragile (viral infection, bacteria, hormonal imbalance, etc.), they can proliferate and lead to states of chronic superinfection which can result: etc.).
In all the cases in question, they are present at a supra-pathological rate higher or equal to 10.sup.4 CCU/ml.
Until now two techniques were used for the counting of urinogenital mycoplasms:
1. The number of colonies per microscopic field were counted on an isolating gelose.
This technique can be compared to that used for the counting of bacteria in urinary infections (Kass).
However, this type of counting has the inconvenience of systematically using a solid gelose, which is expensive, and is not well adapted for the systematic research of urinogenital mycoplasms. Furthermore, the technique necessitates an incubation in an anaerobic jar.
2. A counting based on the dilution in a series of the sample to be analysed in a liquid medium (U.sub.9 for U. urealyticum, M.sub.4 2 for M. hominis) and on the end-point of the appropriate colour indicator of the phenol red type contained in the dilution medium.
However, this technique using the enzymatic properties of urinogenital mycoplasms has several inconveniences:
the urea, in complex liquid medium, is unstable. Because of this its concentration (representing the enzymatic substrate) can vary from one series of tests to another, inducing the risk of a lack of reproductibility;
a reading is not possible until after the colour changes of the indicator have stabilised for at least 24 hours, which involves a minimum response waiting period of 72 hours;
the technique of dilutions in a series is cumbersome and furthermore is marred by a margin of error due to the manipulation;
no commercial kit exists containing all of the necessary reagents as well as other materials.
SUMMARY OF THE INVENTION
The present invention proposes to avoid these inconveniences and provides a process for the counting and detection of mycoplasms using enzymatic kinetics.
Although this last technique, in effect, is known to be used in biochemistry to assay enzyme activity, its use has never been suggested or envisaged to be used for the assay of bacteria, as is the case in the process of the present invention.
The Applicant has found, in effect, that the speed of the enzymatic response was proportional to the quantity of mycoplasms present in the sample to be analysed.
This process is essentially characterized by the fact that the enzymatic reactions are carried out under anaerobic conditions between a liquid growth medium for mycoplasms serving as a dilution medium of the sample of fluid to be analysed, a first substrate comprising dehydrated urea in the presence of a pH colour indicator also in dehydrated form and a second substrate comprising arginine in dehydrated form in the presence of a pH colour indicator. The speed of the enzymatic response is followed by noting the corresponding time for the colour-change of the indicators, the respective quantities of urea and arginine, on the one hand, and the concentration and the nutritious components of the growth and dilution media, on the other hand, being first selected and standardised in such a way that for the Ureaplasma urealyt
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Escarguel Claude
Grosso Marie-Helene
Laconi Patrick
Diffusion Bacteriologie du Var-D.B.V.
Preston David R.
Wax Robert A.
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