Measurement of a population of nucleic acids, in particular by real time PCR
The invention relates to a method for measuring the amount of nucleic acids of a target sequence in a sample of interest. The method comprises several steps, and in particular subjecting the sample of interest to an amplification treatment, in the presence of at least one nucleic acids label specific for said target sequence; measuring a physical quantity representative of the evolution of the label; calculating a parameter F 0 representative of the physical quantity of the nucleic acids label before any amplification cycle; estimating the initial population size of nucleic acids of the target sequence using a conversion law, comprising contextual parameters, and applied to said parameter F 0 . The contextual parameters are pre-recorded reference parameters which are substantially independent of at least a part of the experimental conditions. The invention is also directed towards an apparatus comprising units organized for implementing a method for measuring the amount of nucleic acids of a target sequence in a sample of interest.
1 . Method for measuring the amount of nucleic acids of a target sequence in a sample of interest, comprising the steps of:
a. subjecting the sample of interest to an amplification treatment, with a batch of reagents comprising at least one nucleic acid(s) label specific for said target sequence, said amplification treatment comprising successive amplification cycles (i; from 1 to n),
b. measuring a physical quantity (F i ) representative of the evolution of the label for at least a part of the amplification cycles,
c. expressing a parameter (F 0 ) representative of the physical quantity of the nucleic acid(s) label before any amplification cycle, using the measurements carried out in step b.,
d. estimating the initial population size (N 0 sam ) of nucleic acid(s) of the target sequence using a conversion law, comprising contextual parameters, and applied to said parameter (F 0 ) expressed in step c.,
characterized in that:
in step d., said contextual parameters are pre-recorded reference parameters, these parameters being substantially independent of at least a part of the experimental conditions.
2 . Method according to claim 1 , characterized in that the reference parameters are determined in advance based on the behaviour of at least one reference sample of the same biological format as the sample of interest and of known initial population size N 0 ref of nucleic acid(s).
3 . Method according to claim 1 , characterized in that the conversion law in step d. is applied directly, without the need for correction by the execution in parallel of steps a., b. and c. for at least one calibrator.
4 . Method according to claim 1 , characterized in that step d. also comprises a correction of the estimated initial population size (N 0 sam ) this correction being based on steps a., b. and c. being carried out in advance for at least one calibrator (EQC) of known initial population size of nucleic acid(s) and of known parameter (F 0 EQC ) representative of the physical quantity of the nucleic acid(s) label before any amplification cycle.
5 . Method according to claim 4 , characterized in that the correction of the estimated initial population size (N 0 sam ) comprises the steps of:
establishing a corrective law between:
said known parameter (F 0 EQC ) representative of the physical quantity of the nucleic acid(s) label before any amplification cycle, firstly, and
the effectively expressed parameter (F 0 EQCmeasured ) representative of the physical quantity of the nucleic acid(s) label before any amplification cycle, of the calibrator,
applying said corrective law to said conversion law comprising the contextual parameters of step d., in order to estimate said initial population size (N 0 sam ) of nucleic acid(s) of the target sequence, present in the sample of interest.
6 . Method according to claim 4 , characterized in that said calibrator (EQC) of known initial population size is based on a biological substance able to form a positive control for the sample of interest.
7 . Method according to claim 1 , characterized in that the expression of the parameter (F 0 ) involves at least:
one parameter relating to the switch between a first phase of constant amplification yield and a second phase of non-constant amplification yield,
one parameter relating to the constant amplification yield during said first phase, and
one parameter relating to the non-constant amplification yield during said second phase.
8 . Method according to claim 1 , characterized in that the nucleic acids label is a fluorescent label.
9 . Method according to claim 1 , characterized in that the sample of interest comprises a biological specimen that may comprise a pathogenic agent.
10 . Method according to claim 1 , characterized in that the amplification reaction is a real time polymerase chain reaction (PCR).
11 . Method according to claim 1 , characterized in that said reference parameters are independent of at least one of the elements of the group of following experimental conditions: apparatus used, type of apparatus used, operator, period of validity of the batch of reagents, method of extraction of the nucleic acids of the target sequence.
12 . Method according to claim 1 , characterized in that the sample of interest comprises several distinct target sequences.
13 . Apparatus (AP) for measuring the amount of nucleic acids of at least one target sequence in a sample of interest, comprising:
a. a carrier element (U.SUP) for carrying at least one sample (PROBE) comprising a batch of reagents and the target sequence with at least one nucleic acid(s) label specific for said target sequence,
b. an amplification unit (U.AMP) for subjecting the sample of interest to an amplification treatment comprising successive amplification cycles (i; from 1 to n),
c. a measuring unit (U.MES) for measuring a physical quantity (F i ) representative of the evolution of said label for at least a part of the amplification cycles,
d. a processing unit (U.TRT) comprising a memory and organized so as:
i. to express a parameter (F 0 ) representative of the physical quantity of the nucleic acid(s) label prior to any of the amplification cycles, on the basis of measurements of physical quantity,
ii. to estimate the initial population size (N 0 sam ) of nucleic acid(s) of a target sequence present in the sample of interest using a conversion law, on the basis of contextual parameters,
e. a controller (CNTR) organized so that, when a sample of interest (PROBE) is received on the carrier element (U.SUP), it applies the amplification unit (U.AMP) and the measuring unit (U.MES) to said sample (PROBE) received, and calls up the processing unit (U.TRT) with the measurements obtained by said measuring unit (U.MES),
characterized in that said contextual parameters are reference parameters which are substantially independent of at least a part of the experimental conditions and are pre-recorded in the processing unit (U.TRT).
14 . Computer program product comprising instructions for implementing the method according to claim 1 , and intended to be stored in the memory of the processing unit (U.TRT) in the apparatus (AP) according to claim 13 .
15 . Data storage support, characterized in that it comprises instructions for implementing the method according to claim 1 .