The present invention relates to a method in which at least one target nucleic acid, present in a biological sample, is amplified by means of a method of transcriptional amplification which makes it possible to combine steps of different temperatures, namely the denaturation and the amplification per se.
The prior art is made up of a certain number of scientific papers which address the thermostabilizing effect of polyols on enzymes; this is in particular the case for:
It is therefore clear that, for about thirty years, a large number of researchers have been interested in the thermostabilizing effect of polyols on enzymes. Moreover a patent application was filed fifteen years ago, under number EP-A-0 821 058, which provides a method for improving an enzymatic activity at high temperature. The corresponding patent claims the use of polyols for thermostabilizing a polymerase and a restriction enzyme.
Despite the interest of scientists, it appears that no-one has attempted to adapt this approach to transcriptional amplification technologies of the NASBA, TMA, etc., type which, in order to operate, make use of several different enzymatic activities. The first step consists of the denaturation of the target, a nucleic acid, generally a ribonucleic acid (RNA), at 65° C. for 2 minutes, and the second step itself consists in adding enzymes required for isothermal amplification at 41° C. These two steps make the method technically restrictive for the user by virtue of the use of two successive temperatures.
Furthermore, the use of a single temperature (41° C.) currently poses various problems such as the amplification of targets rich in guanine and cytosine which have secondary structures that are difficult to amplify. The most well-known solution for making these secondary structures easy to amplify using transcriptional amplification techniques is the use of a fifth nucleotide in the amplification mixture, which is riboinosine triphosphate (K Nakahara et al. Nucleic Acids Res. 1998 Apr. 1; 26(7): 1854-1856).
Deoxyribonucleic acid (DNA) targets can be easily used with the NASBA technology. To do this, all that is needed is, for example, to apply beforehand to the treated sample a method according to EP-B-0 397 269 or according to patent application WO-A-02/070735, in order to enable the amplification of the DNA targets.
Although those skilled in the art have used this type of transcription of DNA targets with two steps of different temperatures for decades, they have not yet thought to try to improve the thermostability of the enzymes used in order to reduce the restrictions of this type of transcriptional amplification method.
The present invention therefore describes a simplification of the transcriptional amplification method which is then carried out in a single step by virtue of the simultaneous addition of the target nucleic acid and of the amplification reagents, such as buffers, enzymes or nucleotides, in the presence of thermostabilizing chemical additives, which enable the use of a higher amplification temperature with an alignment with the temperature of the step of denaturation of said targets. This is an effect which is particularly unexpected and therefore surprising.
The novelty lies in the simultaneous thermostabilization of all the enzymatic activities, in particular of the three enzymatic activities present in the NASBA amplification method, through the use of chemical additives such as polyols making it possible to preserve the activities of T7 RNA polymerase, RNAse H and AMV-RT at temperatures higher than 41° C., and thus making it possible to combine the experimental steps of denaturation and amplification.
The present invention provides a transcriptional amplification method in which:
According to one embodiment of the amplification method, the temperature at which the amplification is carried out is between 41 and 49° C.
According to another embodiment of the amplification method, the temperature at which the amplification is carried out is above or equal to 46° C.
Whatever the embodiment of the amplification method, the enzymatic activities provided by the enzymes are:
The RNAse H activity can be given by an independent enzyme (“individual” activity) or by an enzyme having another enzymatic activity (“combined” activity). This other activity combined with RNAse H may be given by a reverse transcriptase enzyme or an RNA polymerase or a different enzyme.
The enzymatic activities make it possible to carry out isothermal amplifications, such as:
Whatever the embodiment of the amplification method, the polyol(s) consist(s) of one of the compounds or a combination of the compounds which follow:
Whatever the embodiment of the amplification method, the concentration of polyol(s) is between 0.4 and 1.5 M.
The present invention also relates to a method for detecting amplicons obtained by means of the amplification method, as described above, which consists in adding, during step a), at least one type of detection probe per target nucleic acid which is sought and which may be present in the biological sample, and in carrying out the following additional step:
The present invention also relates to a method for pretreating the target nucleic acid(s) which is (are) sought and which may be present in the biological sample, and which must be amplified, as described above, consisting in carrying out the following additional step, before step a), in which said biological sample is subjected to a temperature below or equal to 65° C. for RNA and to a temperature below or equal to 95° C. for DNA.
The present invention also relates to a method for pretreating the target nucleic acid(s) which is (are) sought and which may be present in the biological sample, and which must be amplified, as described above, consisting in carrying out an additional step, before step a), in which said biological sample is subjected to a temperature below or equal to 49° C.
The present invention also relates to a method for the diagnosis, in vitro, of the presence of one type or of various types of target nucleic acids which are sought and which may be present in the biological sample, consisting:
According to one embodiment of the diagnosis method, all of the method is carried out in a single container.
According to a first embodiment variant of the above method, all of the method is carried out at a single temperature above 41° C.
According to a first embodiment variant of the above method, all of the method is carried out at a single temperature of between 46 and 49° C.
The figures appended hereto are given by way of explanatory example and have no limiting nature. They will make it possible to understand the invention more clearly.
The
Although the use of sugars and more generally of polyols for thermostabilizing the enzymes is a piece of information that could be found in the literature, their use at high concentrations in a transcriptional amplification, such as NASBA, for producing an isothermal amplification at more than 41° C., in particular at more than 44° C. and preferentially at more than 46° C., with a possible preincubation up to 49° C., is a technical advance which makes it possible to technically simplify this type of amplification for the end user.
Although it operates between 41 and 45° C., it is in particular necessary to increase the NASBA amplification temperature to 46° C. in order to facilitate the denaturation of structured targets, thus making it possible to improve detection performance levels.
The examples below use the NASBA (Nucleic Acid Sequence-Based Amplification) transcriptional and isothermal amplification method. However, the approach described in this document is also applicable to other isothermal amplification methods such as TMA (Transcription-Mediated Amplification) or 3SR (Self-Sustained Sequence Replication) for example (Gill and Ghaemi, 2008, Nucleosides, Nucleotides and Nucleic Acids, 27: 224-245; Leone et al. 1998, NAR, 26-9: 2150-2155).
NASBA technology is an alternative technology to PCR which allows, unlike the latter, the genetic detection of live microorganisms (bacteria, viruses, etc.) by RNA amplification. This amplification technology requires three enzymatic activities in order to operate, including T7 RNA polymerase, RNAse H and AMV-RT. Among these three enzymatic activities, T7 RNA polymerase is the most thermosensitive enzyme.
A set of compounds with thermostabilizing properties or assumed to have such properties was evaluated in a NASBA HIV-1 2.0 amplification test on a Nuclisens EasyQ™ amplification platform (bioMérieux, Marcy l'Etoile, France) according to the supplier's recommendations. 5 cps to 30 cps of an HIV-1 type B transcript were used as target in each reaction in the presence or absence of the compound to be evaluated.
The obtaining of an amplification in the presence of the compound at a temperature of 46° C. makes it possible to validate the compound as compatible and thermostabilizing for the NASBA reaction.
As shown by the examples of
It is demonstrated in this example that the T7 RNA polymerase denaturation temperature (T7 Tm) increases considerably in the presence of certain chemical additives, in comparison with that of the control without additive. T7 RNA polymerase was chosen as model enzyme since it is the most sensitive to thermal denaturation; the T7 Tm without additive is 48.5° C.
A UV spectrophotometry technique is used to measure the T7 Tm values. The change in the absorbance of the protein at λ=280 nm as a function of temperature is measured. When the enzyme is heated, the solution becomes cloudy, and aggregates form which correspond to the denatured form. The Tm corresponds to the temperature for which there is 50% of native form and 50% of denatured form (first derivative of the curve absorbance=f(temperature)).
In a polypropylene flask, 4 ml of 300 mM PBS phosphate buffer (Aldrich P-4417, St Quentin Fallavier, France) are mixed with 12μl of T7 RNA polymerase enzyme (bioMérieux, Marcy l'Etoile, France) at 17 mg/ml, i.e. a final protein concentration of 0.05 mg/ml. 500 μl of T7 RNA polymerase solution at 0.05 mg/ml and 500 μl of a concentrated solution of additives (Aldrich, St Quentin Fallavier, France) or of 300 mM PBS for the control are then placed in a quartz cuvette for UV spectrophotometry. After homogenization, the change in absorbance at λ=280 nm is measured as a function of temperature, between 30 and 65° C. at 1° C./min, in order to determine the T7 Tm as previously described (Cary UV spectrophotometer, Varian, Les Ulis, France).
Some representative results obtained according to the method previously described are reported in Table 1 below (the ΔTm values are reported as a function of the type of additive).
It is clearly observed that heating the T7 RNA polymerase enzyme in the presence of certain additives, such as sorbitol, sucrose or lactose, makes it possible to very significantly increase the denaturation temperature of the enzyme, by several degrees, thereby confirming its thermostabilization.
In this example, the values of T1/2 of T7 RNA polymerase are determined in the presence or absence of polyols. A description of the measurement method is given below, as are the various reagents used.
Solution W1:
Mix buffers B, C and D according to the following proportions with the following Nuclisens™ HIV-1 2.0 reagents (ref.: 285033, bioMérieux, Marcy l'Etoile, France) (for eight reactions):
Solution S (substrate mix):
Sequences used (5′-3′ orientation):
The results of measuring T1/2 of T7 RNA polymerase are described in Table 2:
According to Table 2, it is noted that all the chemical compounds evaluated have a thermostabilizing effect on the T7 RNA polymerase activity operating in an environment specific to NASBA. 1.48 M sorbitol generates the greatest thermostabilizing effect, while lactose has a weak thermostabilizing power on T7 RNA polymerase, even though it is visible and significant.
In this example, the maximum temperature that the T7 RNA polymerase could withstand for a preincubation of 3 minutes in the presence of thermostabilizing compounds is determined.
This step can be likened to a target predenaturation or preincubation phase. The protocol used in this example is similar to that of example 2, with the exception of the fact that the temperatures are variable and that the preincubation time was fixed at 3 minutes.
Firstly and as shown by
Description of the method for measuring the T7 RNA polymerase, RNAse H and AMV-RT activities for example 5:
Solution S for Measuring the T7 RNA Polymerase Activity:
Similar to example 3.
Solution S for Measuring the RNAse H Activity:
Solution S for measuring the DNA-dependent AMV-RT activity:
In this example, it is demonstrated that it is possible to carry out a NASBA amplification at 46° C. in the presence of thermostabilizing compounds and without a phase of denaturation of the HIV-1B transcripts used as targets at the concentration of 5 cps/reaction, which is the NASBA detection limit. The objective of this is to simplify the method by virtue of the simultaneous addition of the targets to the enzyme and amplification reagent mixes.
A Nuclisens™ HIV-1 2.0 amplification kit (ref.: 285033, bioMérieux, Marcy l'Etoile, France) was used to carry out the amplification experiments in the presence of trehalose, sucrose or sorbitol at the detection limit with an HIV-1 type B transcript at 5 cps/test. Each amplification is replicated 24 times in order to estimate the sensitivity of the test in the presence of thermostabilizing compounds at 46° C.
The sensitivity is expressed as percentage of positive signals determined by the EasyQ™ analysis system, relative to the total number of replicates.
Number | Date | Country | Kind |
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11 61758 | Dec 2011 | FR | national |
Filing Document | Filing Date | Country | Kind |
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PCT/FR2012/052934 | 12/14/2012 | WO | 00 |
Publishing Document | Publishing Date | Country | Kind |
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WO2013/088085 | 6/20/2013 | WO | A |
Number | Name | Date | Kind |
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20020132242 | Gerdes et al. | Sep 2002 | A1 |
Number | Date | Country |
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0 397 269 | Nov 1990 | EP |
0 821 059 | Jan 1998 | EP |
WO 0036112 | Jun 2000 | WO |
WO 02070735 | Sep 2002 | WO |
Entry |
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Number | Date | Country | |
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20140356870 A1 | Dec 2014 | US |