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  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6851—Quantitative amplification

Definitions

• the present invention relates to a method of detection and quantification in various biological media of adenovirus nucleic acids by the real-time measurement of polymerase chain reaction (PCR). It further relates to oligonucleotides for the implementation of this process.
• PCR polymerase chain reaction
• Adenoviruses which are naturally responsible for generally mild infections, are among the vectors used because of their many advantages.
• This method is therefore intended to identify subgroups and serotypes and not to detect and quantify all of the adenoviruses present in a sample.
• the sensitivity of this method is low and therefore unsuitable for precise dosages, such as those required when monitoring patients treated by gene therapy.
• Crawford-Miksza et al. Journal of Clinical Microbiology 1999, Vol. 37, pp. 1107-1 1 12
• nine separate primers are described.
• the present invention therefore aims to solve these problems by providing a sensitive technique and making it possible to detect the majority of adenovirus serotypes.
• the applicants have surprisingly shown that such advantages are obtained by amplifying in real time a determined sequence of DNA coding for hexon, using primers having a limited degeneration, and by highlighting the product. amplification using a non-degenerate probe.
• the present invention relates first of all to a method for detecting and / or quantifying adenovirus nucleic acids in a biological sample in which:
• an adenovirus nucleotide sequence is amplified by real-time PCR using a pair of degenerate sense and antisense primers, consisting of mixtures of oligonucleotides having at least 80% homology with a sequence between nucleotides 21000 and 22000 of the sequence of the adenovirus type 5, corresponding to the sequence SEQ ID No. 4, or with its complementary sequence, and
• the product of the amplification reaction is detected using a non-degenerate probe, consisting of one or more oligonucleotides having a homology of at least 80% with a sequence between nucleotides 21000 and 22000 of the adenovirus type 5 sequence, corresponding to the sequence SEQ ID No. 4, or with its complementary sequence, during a sufficient number of cycles to allow obtaining a measurable amount of amplification product.
• a non-degenerate probe consisting of one or more oligonucleotides having a homology of at least 80% with a sequence between nucleotides 21000 and 22000 of the adenovirus type 5 sequence, corresponding to the sequence SEQ ID No. 4, or with its complementary sequence, during a sufficient number of cycles to allow obtaining a measurable amount of amplification product.
• Real-time PCR is understood to mean any amplification technique making it possible to follow the progress of the amplification reaction in progress.
• the sequences of the primers and / or of the probe have homology of at least 80% with a sequence between nucleotides 540 and 780 of the sequence SEQ ID No. 4, or with its complementary sequence.
• the two primers are respectively chosen on the sense and antisense strands, and so as to allow the amplification of a DNA fragment.
• the probe is chosen so as to hybridize with the DNA fragment resulting from the amplification.
• the PCR primers in accordance with the present invention used to amplify the target adenovirus nucleic acid of a sample are located in a constant zone for the hexon gene of human adenoviruses and the site of hybridization of the probe in accordance with the present invention lies between the two primers.
• the adenoviruses are human tropism.
• said oligonucleotides comprise at least 15 nucleotides and the probe has a theoretical melting temperature Tm approximately 10 ° C. ⁇ 0.5 higher than the theoretical Tm of the primers.
• Tm melting temperature
• the amplification product is preferably hybrid to the HEX probe described below.
• the subject of the present invention is in particular an oligonucleotide characterized in that it comprises at least 10 consecutive nucleotides of the sequence SEQ ID No. 1 below or of a sequence having a sequence homology of at least 80%, and preferably of 90% and even more preferably 95%, with said sequence:
• said oligonucleotide comprises between 15 and 30 nucleotides.
• the present invention further relates to a sense primer HEX1 consisting of a mixture of oligonucleotides meeting this definition.
• the primer HEX1 exhibits three degenerations in position 1, 10 and 19 making it possible to cover the majority of the serotypes.
• the present invention also relates to a second oligonucleotide characterized in that it comprises at least 10 consecutive nucleotides of the following sequence SEQ ID No. 2 or of a sequence having a sequence homology of at least 80%, and preferably 90% and even more preferably 95%, with said sequence:
• said oligonucleotide comprises between 15 and 30 nucleotides.
• the present invention further relates to a HEX2 antisense primer consisting of a mixture of oligonucleotides meeting this definition.
• the primer HEX2 has three degenerations in position 6, 9 and 21 making it possible to cover the majority of the serotypes sequenced.
• said primers comprise at least two oligonucleotides, and even more preferably three or four oligonucleotides.
• the positions of the sequences SEQ ID N ° 1 and 2, located in the 3 ′ part of the ORF coding for the protein Hexon, are, taking as reference the complete sequence of the adenovirus type 5 (deposit number M73260) , respectively 21565 (HEX1) and 21656 (HEX2). They correspond to positions 21048 and 21139 of the sequence Ad5CMVp53 (length 35308 bp).
• the concentration of the primers is a critical parameter of real-time PCR. As a result, the degeneration of the primers HEX1 and HEX2 was limited.
• the present invention further relates to a third oligonucleotide characterized in that it comprises at least 10 consecutive nucleotides of the following sequence SEQ ID No. 3 or of a sequence having a sequence homology of at least 80%, and preferably 90% and even more preferably 95%, with said sequence, or with a sequence complementary to said sequence:
• said oligonucleotide comprises between 20 and 35 nucleotides.
• the present invention further relates to a HEX probe comprising at least one oligonucleotide having this sequence.
• the probe comprises a molecule or a revealing system of molecules.
• Said revealing system preferably consists of a reporter dye and a fluorescence quenching dye, respectively attached to the 5 ′ and 3 ′ ends of the probe.
• the revealing system is constituted by the pair "reporter / quencher ”represented by 6-carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) fixed respectively 5 'and 3' of the probe.
• FAM 6-carboxyfluorescein
• TAMRA 6-carboxytetramethylrhodamine
• a particularly preferred probe according to the invention consists of: 5 'FAM- CAC CAG CCA CAC CGC GGC GTC ATC GA -TAMRA 3'.
• Such a revealing system can also be a so-called “tailing” revealing system.
• "Tailing” consists of including at one end of the probe a tail (tail) which can self-pair. This auto-pairing is highlighted using a marker which is specifically fixed on the sequence in this configuration. If there is a target, there is a mismatch and a signal is emitted. In the absence of target the signal is not emitted.
• Other revealing systems can also be used.
• the sense and antisense primers may include a molecule or a developer molecule system.
• the PCR product obtained by the method which is the subject of the present invention using the primers of sequences SEQ ID No. 1 and SEQ ID No. 2 preferably has a size of 1 14 bp and has a% GC of approximately 58.8%.
• the polymerase preferably used in the present invention is Taq polymerase, but it can be any other enzyme exhibiting polymerase activity and usable under the operating conditions of PCR.
• the present invention also relates to a reagent kit for amplification reaction of the real-time PCR type for detection of adenovirus, characterized in that it comprises a pair of sense and antisense primers and a probe as described. above.
• Said kit advantageously further comprises two negative controls and two positive controls.
• the present invention further relates to a PCR method for the detection and / or quantification of adenovirus in a test sample capable of containing it, characterized in that it consists of a real-time PCR using a kit according to the invention.
• said two positive controls are extracted in parallel with a series of samples to be assayed and consist of a standard consisting of a purified and titrated adenovirus solution and a calibrator adapted to the type of sample to be assayed.
• the method according to the invention finds an advantageous application in the detection and / or in the quantification of adenoviruses present in samples of suspension or supernatant type from culture, plasma, urine, oropharyngeal washings, lymphocytes, seminal fluids, tumor biopsies or no, rectal swabs, faeces, ascites.
• the present invention also relates to a method of selecting adenoviruses useful as vector candidates by evaluation of the replicative performance of adenoviruses by application of a detection and / or quantification method according to the invention to the monitoring of kinetics of adenoviral multiplication.
• the present invention also relates to a method for diagnosing the adenovirus serotype present in a test sample consisting in implementing a method of the invention and then in sequencing the PCR product obtained.
• the nucleotide sequences of the various serotypes encoding the Hexon protein were obtained from databases or were sequenced.
• the serotypes Ad3, Ad7, Ad 12, Ad 14, Ad 16 and Ad21 have an A residue at position 9 of the HEX probe.
• the serotypes Ad5, Adl, Ad2, Ad6, Adl3, Ad40 and Ad48 have a residue at this same position G while the serotypes Ad4 and Ad41 have a residue C.
• the type A sequence is more common than the type C sequence.
• a / C mismatches (Ad5CMNp5i and serotypes 5, 1, 2, 6, 13, 40 and 48) or A / G (serotypes 4 and 41) result in a decrease in the Rn delta without significant modification of the Ct (Rn delta is the difference in fluorescence detected between the measured fluorescence of the background noise and the detected fluorescence of the test sample; Ct is defined as the number of fractional cycles in which the fluorescence generated by cleavage of the probe significantly exceeds - generally 10 times - background noise).
• Serotypes Ad5, Ad4, Ad 12 and Ad 14 and Ad5CMV / 53 show additional differences in position o 5 for serotype Ad5 and Ad5CMV 75J o 24 for serotypes Ad4 and Adl2 o 3, 12 and 19 for Adl4 serotype
• One of the advantages of the present method of detecting and / or quantifying human adenoviruses compared to the methods described in the prior art is a remarkable detection sensitivity with a detection threshold of 10 particles or 1 pfu ( range unit) by PCR reaction.
• Another advantage lies in the universality of the method since 17 different serotypes of human adenoviruses, representative of all of the subgroups A to F, could be detected and quantified.
• the analysis of the PCR products is carried out directly at the end of the PCR cycles by reading the fluorescence obtained during the cycles. It is therefore not necessary to work with PCR products which are at risk of contamination for subsequent analyzes.
• the quantification of the number of targets initially used in the reaction is very reliable and reproducible.
• the detection of the PCR product is done during PCR cycles using a fluorescent probe. This is necessary to detect the PCR product and takes place in the full exponential phase of PCR and not at the end point; this detection principle is therefore more sensitive and more specific.
• Another advantage lies in the fact that non-specific amplifications are avoided, thanks to the “hot start” principle, real-time PCR being carried out in the presence of a thermostable DNA polymerase which activates at the first denaturation.
• the present invention is illustrated by nonlimiting examples and with reference to the appended figure which represents a standard curve of a hexon PCR method according to the invention.
• Ad2 and Ad5 Some titrated wild adenoviruses (Ad2 and Ad5) come from UMR No. 1592 (Emmanuelle Vigne, Vectorology and Gene Transfer Unit, Gustave-Roussy Institute).
• the Ad5CMV /? 55 titrated comes from the Production Unit of RPR Gencell, Vitry (Didier Faucher). The titles are provided in number of pfu (“plaque forming unit”). This unit will remain the reference unit for quantification.
• the cell line 293 comes from ATCC (CRL 1573).
• the cell line A549 (ATCC: CCL 185) was kindly provided by E. Vigne and A. Fallourd, RPR Gencell.
• the MRC5 cell line comes from bioMérieux (ref. 84002, France).
• Clinical samples • The clinical samples (plasma, urine, oropharyngeal lavage, faeces) come either from healthy volunteers or from patients hospitalized at the Gustave-Roussy Institute.
• Some plasmas come from the Transfusion Center, either by purchasing a bag from around 200 donors, or bags that can no longer be used for medical use.
• Adenovirus collections and clinical samples are transported in dry ice and, upon receipt, stored at -80 ° C.
• Cookies and switchboard There are two types of negative cookies:
• a negative control PCR or NTC (non template control) which consists of the PCR buffer with distilled water (without target). The value of Ct must be 50 since 50 PCR cycles are performed.
• the second type of negative control is the negative extraction control which is put in place from the extraction stage. In general, it consists of 200 ⁇ l of physiological water and it is extracted in parallel with a series of extracts (generally 1 in 10). The value of Ct must also be 50. There are two types of positive "controls":
• the standard which consists of a purified and titrated adenovirus solution ( ⁇ 5.10 3 pfu), is extracted in parallel with a series of samples to be assayed.
• the nucleic acid extract is diluted (generally 10 to 10) to constitute the different standard points for the construction of the standard curve (Figure).
• the second type of positive control is the calibrator.
• calibrators There are different types of calibrators depending on the type of samples to be diagnosed. It consists of 200 ⁇ l medium closest to the sample and a known amount of adenovirus lying in the middle of the standard range (in general 5.10 3 pfu).
• the media used for the manufacture of the calibrators are physiological water for oropharyngeal washes, a "pool" of plasma from 200 healthy donors for plasmas, urine from healthy subjects for urine and fresh culture medium. for culture supernatants. These calibrators verify that the extraction technique is correct. The quantization value compared to the expected theoretical value will allow, by simple rule of three, to readjust the final quantification obtained with the samples to be tested.
• a CDD camera collects fluorescence emissions approximately every 6 seconds for each well.
• S.D.S. Sequence Detector System TM
• the quantification is based on the principle of real-time PCR. Indeed, the PCR product is characterized by the moment of the PCR cycle when the amplification is detectable by the degradation of the probe linked to the accumulation of PCR products. The higher the number of target starting copies, the fewer PCR cycles it will take to detect a significant increase in fluorescence.
• the number of target copies of a sample is quantified by measuring the Ct value and using a standard curve ( Figure).
• Figure if the PCR works at 100%, it takes 3.22 PCR cycles (Ct) to multiply by 10 the number of targets. In general, a factor of 10 of the number of targets gives a difference in Ct values between 3.4 to 3.6.
• the second parameter (Delta Rn) is important to check to confirm the positivity of the PCR signal.
• the Rn delta is the difference in fluorescence detected between the measured fluorescence of the background noise (generally measured between cycle 3 and cycle 15 of the PCR on the entire plate) and the detected fluorescence of the sample to be analyzed.
• RNA Isolation Kit All the reagents used for this preparation come from the kit called High Pure RNA Isolation Kit, and marketed by Boehringer / Roche. To prepare this sample proceed as follows:
• washing buffer I Add 500 ⁇ l of washing buffer I and centrifuge for 1 min. at 8,000 rpm.
• the nucleic acid extracts are stored at 4 ° C until the PCR step.
• n ° 1 illustrating the extraction techniques optimized for the detection of adenovirus coming from different types of samples including that of the present example.
• concentrations of all the reagents used in the amplification reactions in particular the concentrations of primers sense “HEX1”, antisense “HEX2”, probe “HEX”, and MgCl ; and other reagents of the TaqMan TM PCR Core Reagents Kit (Perkin-Elmer Applied Biosystems) are given in table 2.
• the standard curve of a PCR detection method according to the invention is shown in the figure.
• One of the applications of the hexon PCR method according to the invention is the detection of adenovirus in culture supernatants.
• the presence of an adenovirus in cell culture is conventionally visualized by a cytopathic effect in the cell layer.
• a cytopathic effect is due to an adenovirus an antigen specific to the adenovirus is detected (by ELISA, eg Ref. K6021, Dako Diagnostic LTD, Denmark House, England).
• Extracts of nucleic acids from the main cell lines (Hep2, MRC5, 293 and A549, described above in the “materials and methods” section) used for the cellular diagnosis of human adenoviruses were tested to verify the negativity in PCR hexon according to the present invention. All DNA extracts evaluated were negative demonstrating the specificity of detection.
• Ad40 has a mismatch for the HEX2 antisense primer of the invention and a mismatch for the HEX probe of the invention.
• the hexon gene sequence for serotype 18 is not available. It is likely that these results obtained with Adl ⁇ are also due to mismatches.
• Example 3 Sensitivity of the Method According to the Invention 3.1. Estimation of the detection threshold in number of particles.
• Table 3 presents the results of the 17 adenovirus serotypes with Ct values obtained as a function of the number of particles per ml measured by the HPLC technique.
• the Ct values are in the range of 8.1 to 9.1 for a number of particles from 1.1 to 8.5.10'7ml .
• the detection threshold can be estimated from 10 to 80 particles per PCR reaction.
• 25 cm 2 culture flasks of cells were infected with adenovirus dilutions of between 10 3 and 1 pfu.
• the culture medium (5 ml) was removed, the cells were infected with different concentrations of virus in a volume of 2 ml for 2 hours (at 37 ° C). After two hours, the medium was changed. After infection, every day (D0 to Dl 1), the culture flasks were removed: 250 ⁇ l for the ELISA test (detection of Hexon antigen) and 200 ⁇ l for the detection of PCR according to the invention. 450 ⁇ l of fresh medium were reintroduced into the culture flask.
• the appearance of the CPE (cytopathic effect) (table 4) is between 4 days for the 10 'point and 8 days for the 10 pfu point. There is a relationship between the amount of virus inoculated in the culture flask and the time to positivity.
• the positivity threshold is 10 pfu / culture flask.
• the Ct values for the negative controls (T) are slightly positive (37.6-37.8). This result can be explained either by slight aerosol contamination at the time of inoculation of the culture flasks, or by slight contamination at the time of extraction. The first hypothesis seems to be more likely since on D10, the values of Ct increased to 31.2 (difference of 6.5 Ct) which represents an increase by a factor of 100 in the number of copies of adenovirus.
• the hexon PCR method of the invention shows the existence of a viral multiplication (significant decrease in Ct) in the absence of PCE and detection of the Hexon antigen.
• the hexon PCR method of the invention reveals, in a completely surprising manner, from 2 to 4 "waves" of viral multiplication.
• the hexon PCR method of the invention is positive sooner than the ECP or the ELISA hexon. Also, the hexon PCR method conforms to the present invention is at least 10 times more sensitive than conventional cell culture tests (10-day culture + ELISA test).
• the hexon PCR method according to the invention can advantageously contribute to the selection of vector candidates for biotechnological applications, in particular in gene expression and / or therapy.
• Example 5 Repeatability and reproducibility of the method according to the invention.
• the variation in Ct is also greater, rarely exceeding a value of 2 Ct between two different extracts except for urine No. 2403 which has a difference of 3 Ct (factor 8 in quantification).
• some urines nos. 2029, 2626, 3736, 3763, 3765 and 5399
• the standard curve is usually generated using 12 points of 1 to 10 5 pfu per PCR reaction tested in duplicate. For each repetition, the amplification curves and the PCR cycle values are very reproducible (see figure). On the other hand, as a final point, the fluorescence signals are very variable (up to a factor of 10), hence the advantage of analyzing the PCR products at the time of the appearance of the signal (PCR cycle: Ct) and not at the end point as in "classic" PCR.
• the standard curve obtained with these standard points is shown in the figure.
• the correlation coefficient of 0.999 indicates excellent linearity. In the vast majority of cases, this coefficient is between 0.98 and 1.
• Manipulations using a wider standard range 0.5 to 10 6 pfu have shown similar correlation coefficients (not shown) thus confirming the supplier information describing a linearity of quantification on more than 6 logarithms.
• Y-intercept represents the number of PCR cycles deduced by the standard curve to detect an Ad5CMVp5J pfu. According to standard curves, this Ct value is between 35 and 39.
• the value of the slope is related to the yield of the PCR. It is recalled that for a PCR at 100% yield, this value must be - 3.22. For the hexon PCR method according to the invention, the value of the slope is between - 3.4 to - 3.9 representing an average yield of 90%.
• Example 8 Quantification on clinical samples: The examples of quantification obtained with the hexon PCR on clinical samples (Table 7) were selected on two main criteria. The first criterion is that the sample was analyzed by two completely independent serial PCR extractions (see Table 6). The second criterion is that the PCR results have Ct values less than or equal to 36 corresponding well to a quantifiable status for the sample. Six clinical samples meet these two criteria (Table 7). The quantification results are very reproducible and the level of variation rarely exceeds a factor of 2 for the same sample (Table 7). It is particularly interesting to note for urine No.
• Table n ° 1 Optimized extraction techniques for the detection of adenovirus from different types of samples.
• Table 3 Time to onset of the PCE (day urs) - * OD value in Elisa, Ct value (PCR hexon) and fineness in HPLC after inoculation of A549 cells a supernatant of MRC5 cells harvested after infection with wild adenoviruses from the collection.
• Table 5 Ct values obtained with 1 pfu of Ad5CMV 7J3 in the PCR reaction (101 tests).
• a The average was calculated from the values of Ct indicated in table 6.
• b The standard used is known in number of pfu of Ad5CMVp.53. This quantification is reported per milliliter of initial clinical samples.

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