Patent EA-000613-B1 (EA)
Title: Microorganism quantitation and detection method and kit
Inventor: ANDRIEU JEAN-MARIE
IPC: C12Q1/68
Filed: 1996-11-05
Abstract: 1. A method using an external standard for quantitation and detection of any DNA or ARN containing microorganisms, characterized in that comprises (1) use or determination of standard concentration of a microorganism or DNA or ARN concentration containing in this microorganism defined as a standard microorganism in the form of interval of concentrations of the standard microorganism obtained by a successive dilution of a microorganism preliminary standartized by the DNA or ARN concentration in it, forming an external standard, (2) comparison of quantity of a product of a reverse transcription and/or DNA or ARN amplification obtained from an unknown concentration of a microorganism with quantities of DNA or ARN amplificate of the known concentration of the standard microorganism, wherein a target microorganism or its genome, which is to be determined, is identical to the standard microorganism or its genome, treated parallel, and a method comprises also an internal control of efficiency of amplification or reverse transcription. 2. A method of Claim 1, characterized in that a step (2) consists essentially of comparative detection based on comparison of concentration of standard microorganism with concentration of the target microorganism preferably determined by a method of branched ADN or by any other method, which do not amplify ADN or ARN of the standard or target microorganisms. 3. A method of Claim 1, characterized in that comprises: 1) sorting out and purification of a predetermined amount of the target microorganism from samples, obtained from a human or animal organisms or from vegetables or cellular culture wherein a quantity of a microorganism must be sufficient for such concentrations of target ADN or ARN, which can be measured by direct methods; 2) an extraction of ADN or ARN and measuring the obtained extracted quantity; 3) determination of a number of nucleotidic bases of the target microorganism; 4) calculation by an estimated method a summed molecular mass of ADN or ARN of said microorganism based of said nucleotidic bases and mean known molecular mass of one nucleotide; 5) calculation by an estimated method ADN or ARN quantity of each microorganism using Avogadro number; 6) preparation and/or utilisation of concentration interval of the standard microorganism obtained by successive dilution of the microorganism, preliminary standartized by the ADN or ARN concentration in it, forming an external standard for which (a) ADN or ARN concentration and (b) microorganisms concentration is known for each dilution; 7) an extraction of ADN or ARN of the standard and target microorganisms with subsequent simultaneous their detection in the extractsby a method of the branched ADN or by any other method, which do not amplify ADN or ARN of the standard or target microorganisms and/or subjected to the reverse transcription and/or, at least one amplification and registration on the obtained results; 8) comparison of ADN or ARN concentrations of the standard and target microorganism or the products of amplification of the target microorganism with the products of amplification of the external standard to determine ADN and ARN concentration or a summed concentration of the microorganismin each sample of the target microorganism. 4. A method of Claim 3, characterized in that steps (2)-(5) are performed by a simple direct measurement of a number of microorganisms. 5. A method of any one of the preceeding Claims, characterized in that comprises an internal control in the form of a heterologic plasmide ADN or the ARN transcriptions, the sequence and primers of which are not associated with the ADN and ARN of the target microorganism for control efficiency of amplification or reverse transcription-amplification. 6. A method of Claim 1 or 2, characterized in that preparation of the known concentrations of the microorganism are performed by preparing the known concentrations -the same inactive microorganism, ADN and ARN of which can be revealed or quantitatively detected using the method of the branched ADN and/or the method of reverse transcription and/or amplification, identical in case of ADN or ARN natif virus -complete nucleotidic sequence extracted from the same microorganism, or -complete nucleotidic seqience obtained through synthesis. 7. A method of any one of the preceeding Claims, characterized in that the known standard concentrations of the microorganism of the type which needs to be quantitatively detected or reveal and which is completely ADN or ARN, or prepare concentrations of ADN or ARN genome of the said microorganism corresponding to the known concentrations of this microorganism for their storing till requested by a user. 8. A method of any one of the preceeding Claims, characterized in that a number of microorganisms in any media or samples are determined or revealed a minimum measured number of microorganisms or a greater number, than the minimum measured number. 9. A kit assembly of means for quantitation and detection of any DNA or ARN containing microorganisms, characterized in that contains the following components appropriately placed in containers and/or in other appropriate means: -a series of known standard concentrations of a microorganism of a type which is to be quantitatively detected or revealed and which is completely ADN or ARN, or ADN or ARN concentrations, corresponding to the known concentrations of the above-said microorganism; -a means for extraction, detection and/or reverse transcription and/or amplification using mainly polymerase chain reaction, reverse transcription-polymerase chain reaction or NASBA, ADN or ARN concentrations of the standard and target microorganisms; -a means for analysis of ADN or ARN concentrations of the standard and target microorganisms or detection of -the amplification products; and -a means for internal control. 10. A kit assembly of Claim 9, characterized in that comprises means for a simple direct measurement of a number of microorganisms. 11. A kit assembly of Claim 9 or 10, characterized in that the means for the internal control is in the form of a heterologic plasmide ADN or the ARN transcriptions, the sequence and primers of which are not associated with the ADN and ARN of the target microorganism for control efficiency of amplification or reverse transcription-amplification.