Primers and probes for detecting mycobacteria

Abstract

The present invention relates to a set of primers for amplifying by polymerase chain reaction (PCR) the sequence of any mycobacterial rrs gene present in a sample, the set of primers comprising at least three primer pairs and allowing the amplification of a sequence from the rrs gene of any mycobacterial species. The present invention also relates to probes related to the primer pairs, allowing the specific detection of the mycobacterial rrs sequence amplified by the primer pairs. The present invention also relates to different methods and uses of these primers and probes for comprehensive and specific detection of the presence of mycobacteria in any product, in particular pharmaceutical products such as vaccines.

Description

[Technical field]

[0001] The present invention is particularly in the area of ​​detection of mycobacteria in biological samples and pharmaceutical products.The present invention also relates to safety release testing for mycobacteria detection, which is required for all products expressed in or based on eukaryotic cells, particularly vaccines.The present invention also relates to oligonucleotides, particularly primers and probes, useful for the specific and comprehensive detection of mycobacteria in a sample. [Background technology]

[0002] Mycobacteria are part of the family Mycobacteriaceae. The phylogenetics and nomenclature of mycobacteria is rapidly evolving. Prior to February 2018, all species of mycobacteria were classified into a single genus, namely Mycobacterium. Since then and in the publication by Gupta R.et al, 2018, over 188 species of mycobacteria have been referenced and grouped into five different genera, namely Mycobacterium, Mycobacteroides, Mycolicibacterium, Mycolicibacter and Mycolicibacillus.

[0003] The nomenclature has recently been further modified slightly (see Riojas MA et al, 2018). It is now recognized that there are 200 identified species of mycobacteria, 6 of which include subspecies or variants, thus including 217 identified species, subspecies or variants of mycobacteria. Subsequent changes have altered the classification of mycobacteria reconstituting a single genus Mycobacterium, but have not changed the strains or the number considered to be mycobacteria (Meehan CJ et al., 2021).

[0004] Of these different species, some are pathogenic to humans, such as M. tuberculosis, the causative agent of tuberculosis, and M. leprae, the causative agent of leprosy.

[0005] Other mycobacteria, termed nontuberculous mycobacteria (NTM), can be opportunistic pathogens in humans associated with both pulmonary and extrapulmonary infections (as M. avium and M. marinum, respectively). However, other species are commensal bacteria.

[0006] The growth rate of mycobacteria varies greatly and is much slower than that of most other bacteria; in fact, fast growing mycobacteria have a reproductive cycle of about 48-72 hours, whereas in slow growing species it may be 10 days to 2 months. By comparison, the growth rate of E. coli is 20 minutes.

[0007] Mycobacteria are widely distributed in various environments such as water and soil, can have several hosts (human, bovine, protozoan), and their detection is very difficult (Haig et al., 2018). During the manufacturing process, mycobacteria can potentially contaminate biopharmaceuticals such as vaccines through raw materials of animal or human origin used in the process, such as cells and serum. Contamination can also be introduced by the operator or the environment, such as water.

[0008] Mycobacteria are therefore screened in vaccine products, particularly non-inactivated viral vaccine products, as well as in cell and viral seed banks used in vaccine production.

[0009] Mycobacteria detection is a safety release test that may be required for any biological or pharmaceutical product expressed in or based on eukaryotic cells. It is required for viral vaccines in viral seed lots and viral harvests by the European Pharmacopoeias, equivalent requirements can be found in the Japanese and Chinese Pharmacopoeias, and mycobacteria testing is also performed in the United States to ensure drug safety.

[0010] The current method described in the European Pharmacopoeia for safety release testing is based on microbial culture (see European Pharmacopoeia. Chapter 2.6.2 and Technical Report Series-TRS 978 of WHO). Two suitable solid media (Loewenstein-Jensen medium and Middlebrook 7H10 medium are considered suitable solid media) and one suitable liquid medium should be inoculated with samples in triplicate. All media are incubated at 36.5°C ± 1.5°C for 56 days.

[0011] The main problems and limitations of the above mentioned official culture-based assays are, among others, the availability and potential shortage of specific media, the very long time to results (56 days) and the lack of detection of some mycobacteria such as intracellular mycobacteria such as M. intracellulare. An important human origin species, namely M. leprae, an obligate intracellular parasite, is not detected by the above mentioned tests. Thus, there is also an additional safety risk associated with the lack of detection of this species by the official methods. Culture-based assays are also unable to detect fastidious species with specific growth requirements such as M. genavense, which requires longer incubation times (i.e. 8-12 weeks) or M. haemophilum, which requires iron medium supplementation.

[0012] Several alternative methods based on PCR (polymerase chain reaction) or qPCR (quantitative PCR) targeting different regions of the mycobacterial genome have been disclosed in the art. These methods may overcome at least some of the limitations of culture-based techniques, since they target bacterial DNA and therefore should not be constrained by the need to culture the microorganism.

[0013] However, some of these alternative techniques developed to detect mycobacteria based on a qPCR step still required at least one month for completion because they were combined with days of culture. Thus, these methods do not solve either the time limitations of the official culture-based assays or the problem of detecting intracellular mycobacteria.

[0014] Other methods have been developed using qPCR targeting ITS and 16S sequences but to the previous taxonomy, Mycobacteria (2018). These methods generally show a lack of sensitivity and / or susceptibility, especially when applied to the new taxonomy (2018).

[0015] Another technique has been developed based on bulk filtration (water samples), immunocapture and qPCR targeting the hsp65 gene. However, this technique does not guarantee sufficient sensitivity, since not all mycobacteria are detected according to this method.

[0016] Indeed, given the lack of data on corresponding sequences across mycobacterial species, it is strongly suggested that the target sequences in the methods disclosed in the prior art do not allow for comprehensive detection of mycobacteria, which poses a significant safety risk.

[0017] Finally, most of the sequences targeted by these alternative methods are not specific for mycobacteria, and therefore non-mycobacterial sequences are also amplified, resulting in false positives and unreliable results.

[0018] Moreover, it is emphasized that most of the PCR-based detection methods disclosed in the art are aimed at diagnostic purposes, at detecting only mycobacteria pathogenic to humans, and coverage of the genus Mycobacterium was not even an objective, nor was it specific. Similarly, most of the methods are intended for detection in samples obtained from patients, which may present significant concentrations of mycobacteria, and therefore the sensitivity is not tested and / or is too low to be compatible with the detection of contamination in biopharmaceuticals, which generally requires detection at concentrations lower than 100 viable mycobacteria per mL.

[0019] Thus, no prior art technology has provided the necessary safety, efficiency, and robustness to meet the requirements of safety release testing. Thus, there remains a significant need for alternatives to compendial methods that provide improved comprehensiveness and faster. Thus, there remains a significant need for alternatives to compendial methods that provide the same specificity, but with improved comprehensiveness, efficiency, robustness, and faster. Thus, there remains a significant need for alternatives to compendial methods that provide the same specificity, but with improved comprehensiveness, efficiency, robustness, and faster, and can be as sensitive as compendial methods.

[0020] Using genomic analysis and bioinformatics applied to the new taxonomy, the inventors unexpectedly found a primer combination that is compatible with PCR and detects all known strains and species of mycobacteria. Thus, a method based on this combination allows the detection of mycobacterial species in a sample in a comprehensive manner, i.e., regardless of the classification of mycobacteria, regardless of whether they are human pathogens or not, regardless of whether they are fast-growing or slow-growing mycobacteria, regardless of whether they are in high concentrations as in human body fluid samples or in very low concentrations as contaminants, all species of mycobacteria potentially present are detected.

[0021] Furthermore, the inventors have also found a primer-probe combination that is capable of specifically detecting all known strains and species of mycobacteria, and thus a method based on this combination allows the specific detection of mycobacterial species in a sample in a comprehensive and specific manner, i.e. any species, subspecies or variants of mycobacteria are detected, but none of the other bacteria, especially the phylogenetically close species, are detected. Summary of the Invention [Means for solving the problem]

[0022] The present invention relates to a set of primers for amplifying by polymerase chain reaction (PCR) the sequence of any mycobacterial rrs gene present in a sample, said set comprising at least the three following primer pairs: First pair: Forward primer F1 having the sequence: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) or a modification thereof; Reverse primer R1 having the sequence: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO:2) or a modification thereof; Second pair: Forward primer F3 having the sequence: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) or a modification thereof; Reverse primer R3 having the sequence: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO:5) or a modification thereof; Third pair: Forward primer F4 having the sequence: 5'-CCCTTATGTCCAGGGCTTCA-3' (SEQ ID NO: 7) or a modification thereof; Reverse primer R4 having the sequence: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8) or a modification thereof Including, The modification is an addition and / or deletion of 1 to 4 nucleotides at the 3' and / or 5' end and / or a substitution of 1 or 2 nucleotides, The three primer pairs allow the amplification of sequences from the rrs gene of any mycobacterial species.

[0023] According to a particular embodiment, the first primer pair comprises: 1) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-1: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO:2); 2) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13); 3) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-5: 5'-ACGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO: 12); 4) Forward primer F1-2: 5'-CCTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-1: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO:2); 5) Forward primer F1-2: 5'-CCTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-3: 5'-CACGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO:27); 6) Forward primer F1-2: 5'-CCTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-5: 5'-ACGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO: 12); 7) Forward primer F1-2: 5'-CCTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13); and 8) Forward primer F1-3: 5'-CTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 11) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13) is selected from.

[0024] According to certain embodiments, the second primer pair comprises: 1) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-1: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO:5); 2) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-1: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO:5); 3) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-2: 5'-CATGCACCACCTGCACACAG-3' (SEQ ID NO: 15); 4) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-3: 5'-ATGCACCACCTGCACACAG-3' (SEQ ID NO: 16); 5) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-2: 5'-CATGCACCACCTGCACACAG-3' (SEQ ID NO: 15); 6) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-3: 5'-ATGCACCACCTGCACACAG-3' (SEQ ID NO: 16); 7) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-4: 5'-TGCACCACCTGCACACAG-3' (SEQ ID NO: 17), and 8) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-4: 5'-TGCACCACCTGCACACAG-3' (SEQ ID NO: 17) is selected from.

[0025] According to a particular embodiment, the third primer pair comprises: 1) Forward primer F4-1: 5'-CCCTTATGTCCAGGGCTTCA-3' (SEQ ID NO: 7); Reverse primer R4-1: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); 2) forward primer F4-3: 5'-CCCCTTATGTCCAGGGCTTC-3' (SEQ ID NO: 18); Reverse primer R4-1: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); 3) Forward primer F4-5: 5'-GCCCCTTATGTCCAGGGC-3' (SEQ ID NO: 19); Reverse primer R4-1: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); 4) Forward primer F4-1: 5'-CCCTTATGTCCAGGGCTTCA-3' (SEQ ID NO: 7); Reverse primer R4-3: 5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); 5) Forward primer F4-1: 5'-CCCTTATGTCCAGGGCTTCA-3' (SEQ ID NO: 7); Reverse primer R4-5: 5'-GCATCGCAGCCCTTTGTA-3' (SEQ ID NO:28); 6) Forward primer F4-3: 5'-CCCCTTATGTCCAGGGCTTC-3' (SEQ ID NO: 18); Reverse primer R4-3: 5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); 7) Forward primer F4-5: 5'-GCCCCTTATGTCCAGGGC-3' (SEQ ID NO: 19); Reverse primer R4-3: 5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); and 8) Forward primer F4-5: 5'-GCCCCTTATGTCCAGGGC-3' (SEQ ID NO: 19); Reverse primer R4-6: 5'-GGCATCGCAGCCCTTTGTA-3' (SEQ ID NO: 21) is selected from.

[0026] The present invention also relates to a kit for amplifying by PCR a mycobacterial rrs gene sequence in a sample, said kit comprising a set of primers as defined above.

[0027] According to one embodiment, the kit is for detecting mycobacterial rrs gene sequences amplified by PCR in a sample and includes the following relevant probes for detecting the amplification products: - a probe S1 having the sequence 5'-CGGTGGGTACTAGGTGTG-3' (SEQ ID NO: 3) or a modification thereof, for detecting the sequence amplified by said primers F1 and R1; - a probe S3 having the sequence 5'-TCGGTTCCCTTGTGGC-3' (SEQ ID NO: 6) or a modification thereof, for detecting the sequence amplified by said primers F3 and R3; a probe S4 having the sequence 5'-ACATGCTACAATGGCCGG-3' (SEQ ID NO: 9) or a modification thereof, for detecting the sequence amplified by said primers F4 and R4; wherein the modification is an addition and / or deletion of 1 to 2 nucleotides at the 3' and / or 5' end.

[0028] According to one embodiment, the probe comprises: a) For probe S1, Probe S1-0: 5'-CGGTGGGTACTAGGTGTG-3' (SEQ ID NO: 3), Probe S1-1: 5'-CGGTGGGTACTAGGTGT-3' (SEQ ID NO: 22), and Probe S1-2: 5'-CGGTGGGTACTAGGTG-3' (SEQ ID NO: 23) The group consisting of When probe S1 is S1-1, the first primer pair is 1) forward primer F1-1 (SEQ ID NO: 1) and reverse primer R1-1 (SEQ ID NO: 2); 2) forward primer F1-1 (SEQ ID NO: 1) and reverse primer R1-6 (SEQ ID NO: 13); 3) forward primer F1-1 (SEQ ID NO: 1) and reverse primer R1-5 (SEQ ID NO: 12); 4) forward primer F1-2 (SEQ ID NO: 10) and reverse primer R1-1 (SEQ ID NO: 2); and 5) Forward primer F1-3 (SEQ ID NO: 11) and reverse primer R1-6 (SEQ ID NO: 13) is selected from When probe S1 is S1-2, the first primer pair is 1) forward primer F1-1 (SEQ ID NO: 1) and reverse primer R1-1 (SEQ ID NO: 2); 2) forward primer F1-1 (SEQ ID NO: 1) and reverse primer R1-6 (SEQ ID NO: 13); 3) forward primer F1-1 (SEQ ID NO: 1) and reverse primer R1-5 (SEQ ID NO: 12); 4) forward primer F1-2 (SEQ ID NO: 10) and reverse primer R1-1 (SEQ ID NO: 2); 5) forward primer F1-2 (SEQ ID NO: 10) and reverse primer R1-5 (SEQ ID NO: 12); and 6) Forward primer F1-3 (SEQ ID NO: 11) and reverse primer R1-6 (SEQ ID NO: 13) selected from the group; b) For probe S3, Probe S3-0: 5'-TCGGTTCCCTTGTGGC-3' (SEQ ID NO: 6), Probe S3-1: 5'-ATCGGTTCCCTTGTGGC-3' (SEQ ID NO: 24), and Probe S3-2: 5'-CGGTTCCCTTGTGGC-3' (SEQ ID NO: 25) The group consisting of When probe S3 is S3-1, the second primer pair is 1) forward primer F3-1 (SEQ ID NO: 4) and reverse primer R3-1 (SEQ ID NO: 5); 2) forward primer F3-3 (SEQ ID NO: 14) and reverse primer R3-1 (SEQ ID NO: 5); 3) forward primer F3-1 (SEQ ID NO: 4) and reverse primer R3-2 (SEQ ID NO: 15); 4) forward primer F3-3 (SEQ ID NO: 14) and reverse primer R3-2 (SEQ ID NO: 15); and 5) Forward primer F3-3 (SEQ ID NO: 14) and reverse primer R3-3 (SEQ ID NO: 16) is selected from When probe S3 is S3-2, the second primer pair is 1) forward primer F3-1 (SEQ ID NO: 4) and reverse primer R3-1 (SEQ ID NO: 5); 2) forward primer F3-3 (SEQ ID NO: 14) and reverse primer R3-1 (SEQ ID NO: 5); 3) forward primer F3-1 (SEQ ID NO: 4) and reverse primer R3-2 (SEQ ID NO: 15); 4) forward primer F3-1 (SEQ ID NO: 4) and reverse primer R3-3 (SEQ ID NO: 16); 5) forward primer F3-3 (SEQ ID NO: 14) and reverse primer R3-4 (SEQ ID NO: 17); and 6) Forward primer F3-3 (SEQ ID NO: 14) and reverse primer R3-3 (SEQ ID NO: 16) selected from the group; c) For probe S4, Probe S4-0: 5'-ACATGCTACAATGGCCGG-3' (SEQ ID NO: 9), and Probe S4-1: 5'-ACATGCTACAATGGCCGGT-3' (SEQ ID NO: 26) The group consisting of When probe S4 is S4-1, the third primer pair is 1) forward primer F4-1 (SEQ ID NO: 7) and reverse primer R4-1 (SEQ ID NO: 8); 2) forward primer F4-3 (SEQ ID NO: 18) and reverse primer R4-1 (SEQ ID NO: 8); 3) forward primer F4-5 (SEQ ID NO: 19) and reverse primer R4-1 (SEQ ID NO: 8); 4) forward primer F4-1 (SEQ ID NO: 7) and reverse primer R4-3 (SEQ ID NO: 20); 5) forward primer F4-3 (SEQ ID NO: 18) and reverse primer R4-3 (SEQ ID NO: 20); 6) forward primer F4-5 (SEQ ID NO: 19) and reverse primer R4-3 (SEQ ID NO: 20); and 7) Forward primer F4-5 (SEQ ID NO: 19) and reverse primer R4-6 (SEQ ID NO: 21) Selected from the group:

[0029] In one embodiment, the kit is for amplification by quantitative polymerase chain reaction (qPCR) or digital polymerase chain reaction (dPCR).

[0030] The present invention also relates to a method for amplifying and detecting sequences of the rrs genes of any mycobacteria present in a sample, which comprises the following steps, performed simultaneously or sequentially: a) performing in vitro PCR on nucleic acid extracted from the sample using at least the above three sets of primer pairs; b) detecting the presence or absence of an amplification product Includes.

[0031] The amplification step a) can for example be carried out by quantitative PCR or digital PCR.

[0032] According to one embodiment, the detection step b) comprises the use of the following related probes: i. A probe S1, Probe S1-0: 5'-CGGTGGGTACTAGGTGTG-3' (SEQ ID NO: 3), Probe S1-1: 5'-CGGTGGGTACTAGGTGT-3' (SEQ ID NO: 22), and Probe S1-2: 5'-CGGTGGGTACTAGGTG-3' (SEQ ID NO: 23) is selected from the group consisting of When probe S1 is S1-1, the first primer pair is 1) forward primer F1-1 (SEQ ID NO: 1) and reverse primer R1-1 (SEQ ID NO: 2); 2) forward primer F1-1 (SEQ ID NO: 1) and reverse primer R1-6 (SEQ ID NO: 13); 3) forward primer F1-1 (SEQ ID NO: 1) and reverse primer R1-5 (SEQ ID NO: 12); 4) forward primer F1-2 (SEQ ID NO: 10) and reverse primer R1-1 (SEQ ID NO: 2); and 5) Forward primer F1-3 (SEQ ID NO: 11) and reverse primer R1-6 (SEQ ID NO: 13) is selected from When probe S1 is S1-2, the first primer pair is 1) forward primer F1-1 (SEQ ID NO: 1) and reverse primer R1-1 (SEQ ID NO: 2); 2) forward primer F1-1 (SEQ ID NO: 1) and reverse primer R1-6 (SEQ ID NO: 13); 3) forward primer F1-1 (SEQ ID NO: 1) and reverse primer R1-5 (SEQ ID NO: 12); 4) forward primer F1-2 (SEQ ID NO: 10) and reverse primer R1-1 (SEQ ID NO: 2); 5) forward primer F1-2 (SEQ ID NO: 10) and reverse primer R1-5 (SEQ ID NO: 12); and 6) Forward primer F1-3 (SEQ ID NO: 11) and reverse primer R1-6 (SEQ ID NO: 13) Selected from: probe S1; ii. A probe S3, Probe S3-0: 5'-TCGGTTCCCTTGTGGC-3' (SEQ ID NO: 6), Probe S3-1: 5'-ATCGGTTCCCTTGTGGC-3' (SEQ ID NO: 24), and Probe S3-2: 5'-CGGTTCCCTTGTGGC-3' (SEQ ID NO: 25) is selected from the group consisting of When probe S3 is S3-1, the second primer pair is 1) forward primer F3-1 (SEQ ID NO: 4) and reverse primer R3-1 (SEQ ID NO: 5); 2) forward primer F3-3 (SEQ ID NO: 14) and reverse primer R3-1 (SEQ ID NO: 5); 3) forward primer F3-1 (SEQ ID NO: 4) and reverse primer R3-2 (SEQ ID NO: 15); 4) forward primer F3-3 (SEQ ID NO: 14) and reverse primer R3-2 (SEQ ID NO: 15); and 5) Forward primer F3-3 (SEQ ID NO: 14) and reverse primer R3-3 (SEQ ID NO: 16) is selected from When probe S3 is S3-2, the second primer pair is 1) forward primer F3-1 (SEQ ID NO: 4) and reverse primer R3-1 (SEQ ID NO: 5); 2) forward primer F3-3 (SEQ ID NO: 14) and reverse primer R3-1 (SEQ ID NO: 5); 3) forward primer F3-1 (SEQ ID NO: 4) and reverse primer R3-2 (SEQ ID NO: 15); 4) forward primer F3-1 (SEQ ID NO: 4) and reverse primer R3-3 (SEQ ID NO: 16); 5) forward primer F3-3 (SEQ ID NO: 14) and reverse primer R3-4 (SEQ ID NO: 17); and 6) Forward primer F3-3 (SEQ ID NO: 14) and reverse primer R3-3 (SEQ ID NO: 16) Selected from: probe S3; iii. A probe S4, Probe S4-0: 5'-ACATGCTACAATGGCCGG-3' (SEQ ID NO: 9), and Probe S4-1: 5'-ACATGCTACAATGGCCGGT-3' (SEQ ID NO: 26) is selected from the group consisting of When probe S4 is S4-1, the third primer pair is 1) forward primer F4-1 (SEQ ID NO: 7) and reverse primer R4-1 (SEQ ID NO: 8); 2) forward primer F4-3 (SEQ ID NO: 18) and reverse primer R4-1 (SEQ ID NO: 8); 3) forward primer F4-5 (SEQ ID NO: 19) and reverse primer R4-1 (SEQ ID NO: 8); 4) forward primer F4-1 (SEQ ID NO: 7) and reverse primer R4-3 (SEQ ID NO: 20); 5) forward primer F4-3 (SEQ ID NO: 18) and reverse primer R4-3 (SEQ ID NO: 20); 6) forward primer F4-5 (SEQ ID NO: 19) and reverse primer R4-3 (SEQ ID NO: 20); and 7) Forward primer F4-5 (SEQ ID NO: 19) and reverse primer R4-6 (SEQ ID NO: 21) Selected from the probe S4 This is done using

[0033] The method may include the initial step of extracting nucleic acid from the sample before performing step a).

[0034] The present invention also relates to a method for detecting the presence of mycobacterial contamination in a pharmaceutical product without any culture step, which comprises the following simultaneous steps: a) a step of in vitro amplification by quantitative PCR (qPCR) or dPCR carried out on nucleic acids extracted from the product sample using at least the three primer pairs described above, said primers hybridizing to the rrs genes; b) The following probes: i. A probe S1, Probe S1-0: 5'-CGGTGGGTACTAGGTGTG-3' (SEQ ID NO: 3), Probe S1-1: 5'-CGGTGGGTACTAGGTGT-3' (SEQ ID NO: 22), and Probe S1-2: 5'-CGGTGGGTACTAGGTG-3' (SEQ ID NO: 23) is selected from the group consisting of When probe S1 is S1-1, the first primer pair is 1) forward primer F1-1 (SEQ ID NO: 1) and reverse primer R1-1 (SEQ ID NO: 2); 2) forward primer F1-1 (SEQ ID NO: 1) and reverse primer R1-6 (SEQ ID NO: 13); 3) forward primer F1-1 (SEQ ID NO: 1) and reverse primer R1-5 (SEQ ID NO: 12); 4) forward primer F1-2 (SEQ ID NO: 10) and reverse primer R1-1 (SEQ ID NO: 2); and 5) Forward primer F1-3 (SEQ ID NO: 11) and reverse primer R1-6 (SEQ ID NO: 13) is selected from When probe S1 is S1-2, the first primer pair is 1) forward primer F1-1 (SEQ ID NO: 1) and reverse primer R1-1 (SEQ ID NO: 2); 2) forward primer F1-1 (SEQ ID NO: 1) and reverse primer R1-6 (SEQ ID NO: 13); 3) forward primer F1-1 (SEQ ID NO: 1) and reverse primer R1-5 (SEQ ID NO: 12); 4) forward primer F1-2 (SEQ ID NO: 10) and reverse primer R1-1 (SEQ ID NO: 2); 5) forward primer F1-2 (SEQ ID NO: 10) and reverse primer R1-5 (SEQ ID NO: 12); and 6) Forward primer F1-3 (SEQ ID NO: 11) and reverse primer R1-6 (SEQ ID NO: 13) Selected from: probe S1; ii. A probe S3, Probe S3-0: 5'-TCGGTTCCCTTGTGGC-3' (SEQ ID NO: 6), Probe S3-1: 5'-ATCGGTTCCCTTGTGGC-3' (SEQ ID NO: 24), and Probe S3-2: 5'-CGGTTCCCTTGTGGC-3' (SEQ ID NO: 25) is selected from the group consisting of When probe S3 is S3-1, the second primer pair is 1) forward primer F3-1 (SEQ ID NO: 4) and reverse primer R3-1 (SEQ ID NO: 5); 2) forward primer F3-3 (SEQ ID NO: 14) and reverse primer R3-1 (SEQ ID NO: 5); 3) forward primer F3-1 (SEQ ID NO: 4) and reverse primer R3-2 (SEQ ID NO: 15); 4) forward primer F3-3 (SEQ ID NO: 14) and reverse primer R3-2 (SEQ ID NO: 15); and 5) Forward primer F3-3 (SEQ ID NO: 14) and reverse primer R3-3 (SEQ ID NO: 16) is selected from When probe S3 is S3-2, the second primer pair is 1) forward primer F3-1 (SEQ ID NO: 4) and reverse primer R3-1 (SEQ ID NO: 5); 2) forward primer F3-3 (SEQ ID NO: 14) and reverse primer R3-1 (SEQ ID NO: 5); 3) forward primer F3-1 (SEQ ID NO: 4) and reverse primer R3-2 (SEQ ID NO: 15); 4) forward primer F3-1 (SEQ ID NO: 4) and reverse primer R3-3 (SEQ ID NO: 16); 5) forward primer F3-3 (SEQ ID NO: 14) and reverse primer R3-4 (SEQ ID NO: 17); and 6) Forward primer F3-3 (SEQ ID NO: 14) and reverse primer R3-3 (SEQ ID NO: 16) A probe S3 selected from the group consisting of iii. A probe S4, Probe S4-0: 5'-ACATGCTACAATGGCCGG-3' (SEQ ID NO: 9), and Probe S4-1: 5'-ACATGCTACAATGGCCGGT-3' (SEQ ID NO: 26) is selected from the group consisting of When probe S4 is S4-1, the third primer pair is 1) forward primer F4-1 (SEQ ID NO: 7) and reverse primer R4-1 (SEQ ID NO: 8); 2) forward primer F4-3 (SEQ ID NO: 18) and reverse primer R4-1 (SEQ ID NO: 8); 3) forward primer F4-5 (SEQ ID NO: 19) and reverse primer R4-1 (SEQ ID NO: 8); 4) forward primer F4-1 (SEQ ID NO: 7) and reverse primer R4-3 (SEQ ID NO: 20); 5) forward primer F4-3 (SEQ ID NO: 18) and reverse primer R4-3 (SEQ ID NO: 20); 6) forward primer F4-5 (SEQ ID NO: 19) and reverse primer R4-3 (SEQ ID NO: 20); and 7) Forward primer F4-5 (SEQ ID NO: 19) and reverse primer R4-6 (SEQ ID NO: 21) Selected from the probe S4 Detection of the presence or absence of an amplification product by Includes.

[0035] The amplification step a) may, for example, be carried out simultaneously using said three primer pairs and said associated probe in a single reaction mixture.

[0036] According to yet another embodiment, the present application also relates to a process for producing a biopharmaceutical, comprising a step of testing for mycobacterial contamination by carrying out the method of the present invention disclosed above for detecting the sequence of any mycobacterial rrs gene. The step of testing for mycobacterial contamination can be carried out at any stage during the production of a biopharmaceutical, for example on the raw material, such as on the cell substrate, and / or on the virus seed, and / or on the culture medium, and / or on the cell culture, and / or on the harvest of the biopharmaceutical and / or on the formulated biopharmaceutical, and / or on the filled biopharmaceutical. If the raw material or biopharmaceutical (i.e. a sample from the raw material or biopharmaceutical) is detected as contaminated by mycobacteria according to the method of the present invention disclosed above, the raw material or biopharmaceutical is discarded from the manufacturing process. The product, e.g. raw material or biopharmaceutical, determined to be devoid of mycobacterial contamination (i.e. the sample is determined to be devoid of mycobacterial contamination) by testing according to the method of the present invention disclosed above is maintained in the manufacturing process. Such a process for manufacturing a biopharmaceutical may further include the step of releasing a biopharmaceutical that passes testing based on the absence of detection of an amplification product using the three primer pairs and associated probes of the present invention.

[0037] The process for producing a biopharmaceutical may be a process for producing a biopharmaceutical free of mycobacterial contamination and may include the steps of providing a biopharmaceutical and performing the method of the present invention disclosed above to detect any mycobacterial rrs gene sequence for the biopharmaceutical.

[0038] A biopharmaceutical product may be a biological or pharmaceutical product, the preferred product being a vaccine.

[0039] Additional primer and probe based uses and methods are also part of the present invention.

[0040] Definition: Mycobacteria: Mycobacteria according to the present invention are bacteria of the family Mycobacteriaceae according to the February 2018 classification (Gupta et al, 2018) completed by newly identified species. Currently, as of the date of the present invention, this family includes 217 species, subspecies or variants from the previously defined genera Mycobacteroides, Mycolicibacterium, Mycolicibacter, Mycolicibacillus and Mycobacterium, representing 217 different sequences. These different species and subspecies or variants are detailed in Table 4, which correspond to mycobacteria whose sequences have been verified by publication, and these bacteria are considered mycobacteria for the purposes of the present invention, regardless of their classification within the mycobacteria family.

[0041] Mycobacteria-specific amplification of a target sequence refers to the amplification of the target sequence in all related species of mycobacteria (i.e., 217 species, subspecies or variants).

[0042] The mycobacterial species below refer to any of these 217 species, subspecies or variants. [Brief description of the drawings]

[0043] [Figure 1A-1B]Comparison of amplification curves in simplex and duplex amplification. These figures represent the level of fluorescence at the wavelength corresponding to the fluorophore FAM (FIG. 1A), i.e. the fluorophore of the probe S1-0, and at the wavelength corresponding to the fluorophore of the probe for the internal control (IC), as a function of the number of PCR cycles for amplification / detection carried out with a fixed concentration of the internal control (IC) in simplex (using only F1-1 / R1-1 / S1-0 or only primers / probes of the internal control IC) or duplex (using both primers / probes of F1-1 / R1-1 / S1-0 and the internal control IC), for samples containing DNA extracted from M. tuberculosis var BCG at different concentrations. The X-axis represents the number of PCR cycles and the Y-axis represents the fluorescence level in a logarithmic scale. Figure 1A: Top panel: Amplification in simplex (diamonds ◆) with primers / probe of internal control (simplex IC) and in duplex (circles ●) with primers / probe of internal control and F1-1 / R1-1 / S1-0. Samples are spiked with DNA of internal control and M. tuberculosis var BCG at different concentrations. Bottom panel: Amplification in simplex (diamonds ◆) with primers / probe of F1-1 / R1-1 / S1-0 (simplex 16S) and in duplex (circles ●) with primers / probe of internal control and F1-1 / R1-1 / S1-0. Samples are spiked with DNA of internal control and M. tuberculosis var BCG at different concentrations. Figure 1B: Amplification in simplex (internal control primer / probe) and duplex (internal control primer / probe and F1-1 / R1-1 / S1-0) of samples spiked with (star ★) and unspiked (square ■) internal control and M. tuberculosis var BCG DNA. [Diagram 2]Comparison of amplification curves in duplex and quadrupleplex amplification. The figure represents the fluorescence level at the wavelength corresponding to the fluorophore FAM, i.e. the fluorophores of probes S1-0, S3-0 and S4-0, as a function of PCR cycle number for amplification / detection performed in duplex or quadruple on samples containing different concentrations of DNA of M. tuberculosis var BCG with an internal control. The X-axis represents the PCR cycle number and the Y-axis represents the fluorescence level in logarithmic scale. Figure 2A: Amplification curves of quadrupleplex, duplex compared with the first system (F1-R1 / S1 corresponding to F1-1 / R1-1 / S1-0) only and with the internal control and associated primers and probes (IC). Figure 2B: Amplification curves of quadruple and duplex were compared to the third system (F3-R3 / S3 corresponding to F3-1 / R3-1 / S3-0) alone and with the internal control and associated primers and probe (IC). Figure 2C: Amplification curves of quadruple and duplex were compared to the fourth system (F4-R4 / S4 corresponding to F4-1 / R4-1 / S4-0) alone and with the internal control and associated primers and probe (IC). [Figure 3A-3F] Amplification curves of the rrs genes of different mycobacteria (detected by FAM). Shown is the curve obtained with the extracted DNA "pure" as well as with the different dilutions 10-1, 10-2 and 10-3. Figures 3A-3C: Six slow-growing mycobacteria: M. tuberculosis var BCG and M. hiberniae (Figure 3A), M. avium and M. terrae (Figure 3B), M. xenopi and M. kansaii (Figure 3C). Figures 3D-3F: Six fast-growing mycobacteria: M. flavescens and M. smegmatis (Figure 3D), M. abscessus and M. fortuitum (Figure 3E), M. phlei and M. chelonae (Figure 3F). [Figure 4] Amplification curves of the rrs gene in different samples containing M. tuberculosis var BCG at 100 viable mycobacteria per mL (positive control) and different actinomyces bacteria at 100 colony forming units (CFU) per mL (detected by FAM). The X-axis represents the cycle number of the PCR and the Y-axis represents the fluorescence level in logarithmic scale. Figure 4A: Comparison of amplification curves obtained with M. tuberculosis var BCG at 100 CFU / mL and N. asteroides. No amplification of N. asteroides by mycobacteria qPCR (cycle threshold >40). Figure 4B: Comparison of amplification curves obtained with M. tuberculosis var BCG at 100 CFU / mL and D. papillomatosis. No amplification of D. papillomatosis by mycobacterial qPCR (cycle threshold >40). [Diagram 5]Amplification curves (detected by FAM) of the rrs gene in different matrices spiked or not with M. tuberculosis var BCG at different concentrations (105-10 viable mycobacteria / mL). The X-axis represents the PCR cycle number and the Y-axis represents the fluorescence level in logarithmic scale. Figure 5A: Comparison of amplification curves obtained with matrices corresponding to the supernatant of CHO cells to be used for CMV production, spiked (stars ★) or not (circles ●) with M. tuberculosis var BCG at different concentrations (10, 100, 103, 104 and 105 viable mycobacteria per mL, 2 replicates per concentration). Figure 5B: Comparison of amplification curves obtained with matrices corresponding to crude harvests of drug substance from HAV produced on MRC5 cells spiked (stars ★) or not (diamonds ◆) with M. tuberculosis var BCG at two different concentrations (10 and 105 viable mycobacteria / mL, 6 replicates / concentration). Figure 5C: Comparison of amplification curves obtained with matrices corresponding to crude harvests of drug substance from Yellow Fever Virus vaccine produced on Vero cells spiked (stars ★) or not (squares ■) with M. tuberculosis var BCG at two different concentrations (10 and 105 viable mycobacteria / mL, 6 replicates / concentration). [Figure 6]Amplification curves (detected by FAM) of the rrs genes of different mycobacteria with 1 or 0.1 viable mycobacteria per mL of matrix (Vero cells). The X-axis represents the number of cycles of PCR and the Y-axis represents the fluorescence level in logarithmic scale. Figure 6A: Comparison of amplification curves obtained with a matrix corresponding to Vero cells spiked (star ★) or not (circle ●) with 1 viable mycobacteria / mL M. avium. Figure 6B: Comparison of amplification curves obtained with a matrix corresponding to Vero cells spiked (star ★) or not (circle ●) with 1 viable mycobacteria / mL M. hiberniae. Figure 6C: Comparison of amplification curves obtained with a matrix corresponding to Vero cells spiked (star ★) or not (circle ●) with 0.1 viable mycobacteria / mL M. phlei. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The inventors have unexpectedly found a primer combination that is compatible with PCR and results in mycobacteria-specific amplification of the target gene, i.e. the rrs gene. Thus, a method based on this primer combination allows the detection of mycobacteria species, i.e. all known strains and species of mycobacteria, in a comprehensive manner in a sample, i.e. any species, subspecies or variants of mycobacteria potentially present are detected. The minimal primer combination further comprises only six primers to allow simultaneous amplification with all primers.

[0045] According to a first aspect, the present invention relates to a set of primers for amplifying the sequence of the rrs gene of mycobacteria by polymerase chain reaction (PCR), more specifically a set of primers for amplifying the rrs gene of any potential bacterium classified as mycobacteria. The set of primers therefore covers the entire mycobacteria family according to the February 2018 classification (Gupta et al., 2018) and also novel bacteria classified as mycobacteria, as well as all mycobacteria of the genus Mycobacterium according to the latest classification (Meehan CJ et al., 2021).

[0046] The set of primers of the present invention covers the entire mycobacteria family, ie 217 species, subspecies or variants of Table 4 whose sequences have been verified by publication.

[0047] The set of primers therefore allows the amplification by PCR of the rrs genes of any mycobacteria present in the sample. A set of primers according to the invention comprises at least the three following primer pairs: First pair: Forward primer F1 having the sequence: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) or a modification thereof; Reverse primer R1 having the sequence: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO:2) or a modification thereof; Second pair: Forward primer F3 having the sequence: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) or a modification thereof; Reverse primer R3 having the sequence: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO:5) or a modification thereof; Third pair: Forward primer F4 having the sequence: 5'-CCCTTATGTCCAGGGCTTCA-3' (SEQ ID NO: 7) or a modification thereof; Reverse primer R4 having the sequence: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8) or a modification thereof It consists of or comprises:

[0048] The set of primers of the present invention may only include these three primer pairs, i.e., six primers in total, or may include additional primer pairs or alternative forward or reverse primers. Thus, the minimum number of primers is six in an exhaustive manner for amplifying rrs genes of any mycobacterial origin, i.e., whether the mycobacterial strain is pathogenic or not, whether it infects humans or not, whether it originates from the tuberculosis complex or not. This very small number of primers is particularly advantageous, since it requires fewer reagents and allows simultaneous amplification with all six primers. Thus, the inventors have defined a minimal combination of primers that allows mycobacteria-specific amplification of rrs genes.

[0049] The inventors have in fact designed three primer pairs, corresponding to the first primer pair SEQ ID NO:1 and SEQ ID NO:2, the second primer pair SEQ ID NO:4 and SEQ ID NO:5 and the third primer pair SEQ ID NO:7 and SEQ ID NO:8, making it possible to amplify the rrs gene of any mycobacteria species. According to a particular embodiment, the set of primers thus comprises the following three primer pairs: First pair: Forward primer F1-1 having the sequence: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO:1); reverse primer R1-1 having the sequence: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO:2); Second pair: Forward primer F3-1 having the sequence: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4); reverse primer R3-1 having the sequence: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO:5); Third pair: Forward primer F4-1, having the sequence: 5'-CCCTTATGTCCAGGGCTTCA-3' (SEQ ID NO: 7); Reverse primer R4-1 having the sequence: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8) It consists of or comprises:

[0050] The inventors further demonstrated that modifications of some of the sequences of these primers do not change the properties of the primer pairs, i.e., they still amplify the same mycobacterial rrs genes, such that the three primer pairs collectively allow amplifying the rrs genes of any mycobacterial species.

[0051] Thus, the present invention also relates to modifications of SEQ ID NO:1 and SEQ ID NO:2 for the first primer pair, SEQ ID NO:4 and SEQ ID NO:5 for the second primer pair and SEQ ID NO:7 and SEQ ID NO:8 for the third primer pair, to the extent that the resulting three primer pairs still allow the amplification of the rrs gene of any mycobacterial species. Example 7 in the experimental section below details how to design suitable modifications of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:8 while maintaining comprehensiveness.

[0052] The modification is especially an addition and / or deletion of 1 to 4 nucleotides at the 3' and / or 5' end and / or a substitution of 1, 2 or 3 nucleotides. According to a particular embodiment, the deletion is of 1 to 4 nucleotides at the 3' end, the 5' end or the 3' and 5' ends. The substitution is especially a substitution of 1 or 2 nucleotides, especially not at the 3' and 5' ends, especially not at the 3' end, more usually not at the last 5 nucleotides at the 3' end.

[0053] According to one embodiment, the modification is an addition and / or deletion of 1 to 4 nucleotides at the 3' and / or 5' end of the primer sequence.

[0054] Considering the modifications detailed above, according to a different embodiment of the invention, the first primer pair has the following sequence: a) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1), corresponding to the unmodified first primer pair, i.e. the original first primer pair Reverse primer R1-1: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO:2); b) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13); c) forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1); Reverse primer R1-5: 5'-ACGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO: 12); d) Forward primer F1-2: 5'-CCTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-1: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO:2); e) Forward primer F1-2: 5'-CCTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-3: 5'-CACGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO:27); f) Forward primer F1-2: 5'-CCTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-5: 5'-ACGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO: 12); g) Forward primer F1-2: 5'-CCTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13); or h) Forward primer F1-3: 5'-CTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 11) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13) The present invention may have any one of the following features.

[0055] As demonstrated in the experimental section of this application, all of these different primer pairs allow the amplification of the same mycobacterial species as the original first primer pair. Furthermore, the combination of any one of these primer pairs with the primer pairs corresponding to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8 or variants thereof allows the amplification of the sequence of the rrs gene of any mycobacterial species.

[0056] Notwithstanding the above, the selection of the sequences of the first primer pair, particularly the forward and reverse primers of this set, is made independently of the selection of the sequences of the second and third primer pairs.

[0057] Taking into account the modifications detailed above, according to another independent embodiment of the invention, the second primer pair has the following sequence: a) The unmodified second primer pair, i.e. corresponding to the original second primer pair, forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-1: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO:5); b) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-1: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO:5); c) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-2: 5'-CATGCACCACCTGCACACAG-3' (SEQ ID NO: 15); d) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-3: 5'-ATGCACCACCTGCACACAG-3' (SEQ ID NO: 16); e) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-2: 5'-CATGCACCACCTGCACACAG-3' (SEQ ID NO: 15); f) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-3: 5'-ATGCACCACCTGCACACAG-3' (SEQ ID NO: 16); g) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-4: 5'-TGCACCACCTGCACACAG-3' (SEQ ID NO: 17); or h) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-4: 5'-TGCACCACCTGCACACAG-3' (SEQ ID NO: 17) The present invention may have any one of the following features.

[0058] As demonstrated in the experimental section of this application, all of these different primer pairs allow amplification of the same mycobacterial species as the original second primer pair. Furthermore, the combination of any one of these primer pairs with the primer pairs corresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7 and SEQ ID NO:8 or variants thereof allows amplification of the sequence of the rrs gene of any mycobacterial species.

[0059] Notwithstanding the above, the selection of the sequences of the second primer pair, particularly the forward and reverse primers of this set, is made independently of the selection of the sequences of the first and third primer pairs.

[0060] According to yet another independent embodiment of the invention, the third primer pair has the following sequence: a) the unmodified third primer pair, i.e. corresponding to the original third primer pair, forward primer F4-1: 5'-CCCTTATGTCCAGGGCTTCA-3' (SEQ ID NO: 7); Reverse primer R4-1: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); b) forward primer F4-3: 5'-CCCCTTATGTCCAGGGCTTC-3' (SEQ ID NO: 18); Reverse primer R4-1: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); c) forward primer F4-5: 5'-GCCCCTTATGTCCAGGGC-3' (SEQ ID NO: 19); Reverse primer R4-1: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); d) forward primer F4-1: 5'-CCCTTATGTCCAGGGCTTCA-3' (SEQ ID NO: 7); Reverse primer R4-3: 5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); e) forward primer F4-1: 5'-CCCTTATGTCCAGGGCTTCA-3' (SEQ ID NO: 7); Reverse primer R4-5: 5'-GCATCGCAGCCCTTTGTA-3' (SEQ ID NO:28); f) forward primer F4-3: 5'-CCCCTTATGTCCAGGGCTTC-3' (SEQ ID NO: 18); Reverse primer R4-3: 5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); g) forward primer F4-5: 5'-GCCCCTTATGTCCAGGGC-3' (SEQ ID NO: 19); Reverse primer R4-3: 5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); or h) forward primer F4-5: 5'-GCCCCTTATGTCCAGGGC-3' (SEQ ID NO: 19); Reverse primer R4-6: 5'-GGCATCGCAGCCCTTTGTA-3' (SEQ ID NO: 21) The present invention may have any one of the following features.

[0061] As demonstrated in the examples, all of these different primer pairs allow amplification of the same mycobacterial species as the original third primer pair. Furthermore, the combination of any one of these primer pairs with the primer pairs corresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:5 or variants thereof allows amplification of the sequence of the rrs gene of any mycobacterial species.

[0062] Notwithstanding the above, the selection of the sequences of the third primer pair, and particularly the forward and reverse primers of this set, is made independently of the selection of the sequences of the first and second primer pairs.

[0063] Regardless of the above-mentioned modifications of the primer sequence, which are modifications in the sequence that hybridizes with the target sequence to be amplified, the primer of the present invention may also contain additional sequences or moieties that are not directly involved in the interaction with the target sequence. The disclosed primers may, inter alia, contain loops that are required in the design of primers compatible with the LAMP (loop-mediated isothermal amplification) technology. Thus, the sequences of the primers disclosed herein relate to the sequence that hybridizes with the target mycobacteria sequence.

[0064] The three primer pairs disclosed above allow for amplifying sequences from the rrs gene of any mycobacteria species, i.e. regardless of mycobacteria species, subspecies or variants. The three primer pairs do not target the same part of the rrs gene, but rather target sequences of the rrs gene that are conserved between different species of mycobacteria or subgroups of mycobacteria. Thus, the rrs gene of any mycobacteria species is targeted by at least one primer pair of the set of primers of the present invention, resulting in an amplification product. However, the rrs gene of some mycobacteria species may be targeted by two primer pairs of the set of primers of the present invention. Thus, the rrs gene of any mycobacteria species is hybridized by at least one primer pair of the set of primers according to the present invention, but not necessarily by only one primer pair.

[0065] When using the set of three primer pairs of the present invention, under appropriate conditions, the absence of amplification in a sample containing DNA extracted from bacteria means that the sample does not contain sequences corresponding to the rrs genes of mycobacterial species and therefore does not contain any species of mycobacteria.

[0066] In a further aspect, the present invention relates to a kit for amplifying by PCR mycobacterial rrs gene sequences in a sample, such a kit comprising a set of primers as disclosed above, i.e. at least three primer pairs as disclosed above.

[0067] The kit of the present invention allows the use of different amplification methods based on polymerase chain reaction (PCR) in combination with an appropriate detection method. However, the conditions of the amplification cycle and the detection method may vary depending on the amplification method selected. Those skilled in the art will know how to adapt such conditions.

[0068] The amplification technique may inter alia be the LAMP (loop-mediated isothermal amplification) technique.

[0069] Another technique that is particularly suitable in the context of the present invention is amplification by qPCR (quantitative PCR, also known as real-time PCR). Quantitative PCR (or qPCR) is based on PCR and is suitable for amplifying and detecting short sequences in real time. This technique is particularly adapted to the present situation, inasmuch as the rrs sequences amplified by the primer pairs disclosed in this application are indeed short sequences. In the context of the present invention, this technique can be used to detect the amplification (or absence of amplification) of sequences or to quantify the amplification products.

[0070] For the detection of amplification products by qPCR, different techniques are available. One such technique is the use of DNA intercalating agents such as SYBRGreen, which non-specifically intercalate into the minor groove of double-stranded DNA structures and emit fluorescence. Thus, an increase in the fluorescent signal indicates an increase in the number of amplified sequences and thus the presence of the target sequence.

[0071] Another means for detecting amplification is a technique based on hydrolysis probes (such as TaqMan® probes) with a dual-labeled probe with a sequence complementary to the amplified target sequence. The probe is bound to a fluorophore and a quencher. As long as the fluorophore and the quencher are in close proximity, the fluorescent signal of the fluorophore is quenched. During the hybridization step of qPCR, the primer and the probe hybridize to the target sequence. During extension, due to the 5'-3' exonuclease activity of the polymerase, the probe is degraded, releasing the fluorophore, thus relieving the quenching effect of the quencher and allowing the fluorescence of the fluorophore.

[0072] The use of hydrolysis probes generally increases the specificity of quantitative PCR.

[0073] The sequences of probes suitable for the present invention are disclosed below.

[0074] Amplification can also be performed by digital PCR, or dPCR. dPCR is a technique that provides absolute quantification of nucleic acid target sequences, being an end-point measurement that allows quantifying nucleic acids without the use of a standard curve. The PCR reaction mixture is divided into a number of independent reactions. Each reaction is analyzed to determine whether it is positive or negative by specific software. Poisson's statistical law provides absolute quantification of the initial copy number / μL.

[0075] Detection can be achieved with hydrolysis probes or by intercalating agents such as EvaGreen.

[0076] The main advantage of this technique is that the quantification is absolute, so there is no reference scale.

[0077] The kit of the invention may advantageously contain further components, in particular those necessary for amplification by PCR, such as a polymerase, in particular a DNA polymerase and deoxyribonucleotide triphosphates. This list is not exhaustive. According to a preferred embodiment, the polymerase is Taq Polymerase. Many other suitable polymerases are known to those skilled in the art and will not be detailed here. They will depend on the amplification method chosen.

[0078] According to one embodiment, the kit according to the invention does not contain, in addition to the three primer pairs disclosed above, any further primers or any further primer pairs that hybridize to mycobacterial sequences.

[0079] According to another embodiment, the kit of the invention may contain additional primers. Advantageously, the kit may in fact contain a further pair of primers suitable for amplifying a nucleic acid sequence to be used as a control, namely a forward primer and a reverse primer. Such a nucleic acid sequence to be used as an exogenous control is added to the sample at a defined concentration or such a nucleic acid sequence to be used as an endogenous control is present in the sample at a defined concentration. The exogenous control may be any DNA sequence already extracted or present in a microorganism unrelated to the mycobacteria to be detected. The endogenous control may be any DNA sequence present in the sample and known to be unrelated to the mycobacteria to be detected, for example in the test of a cell bank, the endogenous control may be a DNA sequence from a gene of a cell not present in the mycobacteria genome.

[0080] According to a further embodiment, a nucleic acid control is part of the kit of the invention and will be added at a defined concentration to the sample to be tested.

[0081] Furthermore, the kit may also contain additional primers for the detection of contaminants other than mycobacteria, particularly for the detection of harmful or infectious agents such as viruses, bacteria, fungi, etc. It is therefore an important advantage of the present invention to design only three primer pairs for the amplification of the entire mycobacteria family, in order to incorporate these potential additional primers.

[0082] In certain embodiments, the kit according to the invention not only comprises a set of primers for amplifying a sequence of a Mycobacterium species, if present in a sample, but also comprises a probe for detecting the amplification product, if present.

[0083] According to such an embodiment, the kit of the invention advantageously comprises a probe S1 having the sequence 5'-CGGTGGGTACTAGGTGTG-3' (SEQ ID NO: 3) or a modification thereof, for detecting the sequence amplified by the forward primer F1 and the reverse primer R1 of the first primer pair disclosed above.

[0084] In a particular embodiment, the kit of the present invention also comprises a probe S3 having the sequence: 5'-TCGGTTCCCTTGTGGC-3' (SEQ ID NO: 6) or a modification thereof, for detecting the sequence amplified by the forward primer F3 and reverse primer R3 of the second primer pair disclosed above.

[0085] In a particular embodiment, the kit of the present invention also comprises a probe S4 having the sequence: 5'-ACATGCTACAATGGCCGG-3' (SEQ ID NO: 9) or a modification thereof, for detecting the sequence amplified by the forward primer F4 and reverse primer R4 of the third primer pair disclosed above.

[0086] The inventors have indeed shown in the Examples section that the probes having SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 9 are suitable for detecting the amplification products obtained by amplification with the first primer pair, the second primer pair and the third primer pair, respectively, either in the original design of the primers or with the modified sequences detailed above.

[0087] The inventors have further demonstrated that modifications of the sequences of these probes can nevertheless retain the ability to detect the amplification products obtained by amplification using the first primer pair, the second primer pair and the third primer pair according to the present invention.

[0088] The modification of the probe is, for example, the addition and / or deletion of 1 to 3 nucleotides, particularly 1 or 2 nucleotides, at the 3' end and / or 5' end of the probe.

[0089] The probes defined above have a length which perfectly matches the length of the amplification product expected when the primers of the invention are used to amplify a sequence corresponding to the rrs gene of a mycobacterial species.

[0090] Considering the modifications detailed above, according to a different embodiment of the invention, the first probe S1 has the following sequence: Probe S1-0: 5'-CGGTGGGTACTAGGTGTG-3' (SEQ ID NO: 3), corresponding to the original design without modifications; Probe S1-1: 5'-CGGTGGGTACTAGGTGT-3' (SEQ ID NO: 22), and Probe S1-2: 5'-CGGTGGGTACTAGGTG-3' (SEQ ID NO: 23) The present invention may have any one of the following features.

[0091] According to another independent embodiment, the probe S3 is Probe S3-0: 5'-TCGGTTCCCTTGTGGC-3' (SEQ ID NO: 6), corresponding to the original design without modifications; Probe S3-1: 5'-ATCGGTTCCCTTGTGGC-3' (SEQ ID NO: 24), and Probe S3-2: 5'-CGGTTCCCTTGTGGC-3' (SEQ ID NO: 25) is selected from the group:

[0092] According to yet another independent embodiment, the probe S4 is Probe S4-0: 5'-ACATGCTACAATGGCCGG-3' (SEQ ID NO: 9), corresponding to the original design without modifications, and Probe S4-1: 5'-ACATGCTACAATGGCCGGT-3' (SEQ ID NO: 26) is selected from the group:

[0093] According to a particular combination, probe S1-0 is used in combination with any one of the primer pairs F1-1 (SEQ ID NO: 1)&R1-1 (SEQ ID NO: 2); F1-1&R1-6 (SEQ ID NO: 13); F1-1&R1-5 (SEQ ID NO: 12); F1-2 (SEQ ID NO: 10)&R1-1; F1-2&R1-3 (SEQ ID NO: 27); F1-2&R1-5; F1-2&R1-6 and F1-3 (SEQ ID NO: 11)&R1-6.

[0094] According to another combination, probe S1-1 is used in combination with any one of the primer pairs F1-1&R1-1; F1-1&R1-6; F1-1&R1-5; F1-2&R1-1 and F1-3&R1-6.

[0095] According to yet another combination, probe S1-2 is used in combination with any one of the primer pairs F1-1&R1-1; F1-1&R1-6; F1-1&R1-5; F1-2&R1-1, F1-2&R1-5 and F1-3&R1-6.

[0096] According to a particular combination, probe S3-0 is used in combination with any one of the primer pairs F3-1 (SEQ ID NO: 4)&R3-1 (SEQ ID NO: 5); F3-3 (SEQ ID NO: 14)&R3-1; F3-1&R3-2 (SEQ ID NO: 15); F3-1&R3-3 (SEQ ID NO: 16); F3-1&R3-4 (SEQ ID NO: 17); F3-3&R3-2; F3-3&R3-3; and F3-3&R3-4.

[0097] According to another combination, probe S3-1 is used in combination with any one of the primer pairs F3-1&R3-1; F3-3&R3-1; F3-1&R3-2; F3-3&R3-2 and F3-3&R3-3.

[0098] According to yet another combination, probe S3-2 is used in combination with any one of primers F3-1&R3-1; F3-3&R3-1; F3-1&R3-2; F3-1&R3-3; F3-3&R3-3; and F3-3&R3-4.

[0099] According to a particular combination, probe S4-0 is used in combination with any one of the primer pairs F4-1 (SEQ ID NO: 7)&R4-1 (SEQ ID NO: 8); F4-3 (SEQ ID NO: 18)&R4-1; F4-5 (SEQ ID NO: 19)&R4-1; F4-1&R4-3 (SEQ ID NO: 20); F4-1&R4-5 (SEQ ID NO: 28); F4-3&R4-3; F4-5&R4-3 and F4-5&R4-6 (SEQ ID NO: 21).

[0100] According to other combinations, probe S4-1 is used in combination with any one of the primer pairs F4-1&R4-1; F4-3&R4-1; F4-5&R4-1; F4-1&R4-3; F4-3&R4-3; F4-5&R4-3 and F4-5&R4-6.

[0101] In one embodiment, the kit comprises forward primer F1-1 (SEQ ID NO:1), reverse primer R1-1 (SEQ ID NO:2) and probe S1-0 (SEQ ID NO:3).

[0102] In another embodiment, the kit comprises forward primer F3-1 (SEQ ID NO: 4), reverse primer R3-1 (SEQ ID NO: 5) and probe S3-0 (SEQ ID NO: 6).

[0103] In yet another embodiment, the kit comprises forward primer F4-1 (SEQ ID NO: 7), reverse primer R4-1 (SEQ ID NO: 8) and probe S4-0 (SEQ ID NO: 9).

[0104] According to certain embodiments, the kit comprises F1-1, R1-1, S1-0, F3-1, R3-1, S3-0, F4-1, R4-1 and S4-0.

[0105] The inventors have demonstrated that the combination of primer pairs and associated probes disclosed herein not only amplifies the sequence of the rrs gene of any mycobacterial species, but also makes it possible to detect only sequences of mycobacterial species, and not those of other bacterial origin. Thus, the kit of the present invention, by combining a specific primer pair with a corresponding probe, provides not only comprehensiveness of mycobacterial detection, but also specificity of such detection with respect to other bacteria. Thus, the kit of the present invention, together with the corresponding probe, is a detection kit for mycobacterial species, providing comprehensiveness and specificity.

[0106] The kits of the invention may also include probes designed to detect sequences amplified from the nucleic acid controls by the additional primer pairs disclosed above.

[0107] The kits of the invention, in which at least three primer pairs and associated probes are disclosed, are particularly suitable for amplification by qPCR or dPCR. Detection can be performed using probes to provide specificity to the mycobacteria detection.

[0108] The probes of the kit of the invention are advantageously linked to a label, either directly or indirectly. Such a label can be any kind of label, in particular any kind of fluorophore label, suitable for the detection of the probe and adapted to a thermocycler. Particular labels are dye or fluorophore markers. A particular marker shown in the examples is 6-carboxyfluorescein (6-FAM or FAM). Some other markers or fluorophores that are well known to those skilled in the art and can be used are, for example, Cy3 dyes, Cy5 dyes, VIC, YAK (Yakima Yellow), Red, HEX, TET, cyan dyes, etc.

[0109] In certain embodiments, the probe is also linked, directly or indirectly, to a quencher for use as a hydrolysis probe.

[0110] According to another embodiment, when the kit comprises probes S1, S3 and S4 as detailed above, the three probes are directly or indirectly linked to the same label, e.g. a fluorophore marker. Indeed, insofar as the kit allows the comprehensive and selective amplification of sequences of the rrs genes of any mycobacterial species, the detection of the amplification products by the probes indicates the presence of the mycobacterial sequence in the sample, and whether an amplification product is detected by probes S1, S3 and S4 is not necessarily informative. In this regard, it is noted that a given rrs gene of a mycobacterial species can be further amplified and detected by multiple combinations of primers and probes.

[0111] If the kit further comprises one primer pair for amplifying a nucleic acid control and a probe for detecting the corresponding amplification product, said probe for the nucleic acid control can also be directly or indirectly linked to a label, which is advantageously different from the labels of probes S1, S3 and S4. In this way, the amplification resulting from the control system can be readily distinguished from the amplification resulting from the presence of rrs gene sequences of mycobacterial origin. A quencher is also linked to the probe for the control.

[0112] The disclosed kits are intended for use with samples likely to contain mycobacterial DNA from mycobacteria, especially those obtained after DNA extraction. The mycobacterial DNA must be freely accessible in the sample, i.e. not trapped intracellularly, not fragmented, and must correspond to whole DNA, i.e. DNA extracted from living mycobacteria according to a protocol that does not damage the DNA. Controls for DNA extraction may be added as necessary and may be part of the kit.

[0113] Kits according to the invention comprising specific combinations of the disclosed sets of primers and corresponding probes are such that detection of an amplification product with any of probes S1, S3 and S4 indicates the specific presence in the sample of at least one mycobacterium, regardless of its classification, and more particularly the DNA of at least one of the 217 different species or subspecies or variants of mycobacteria known to date.

[0114] In contrast, due to the comprehensiveness provided by the set of primers of the kit, the absence of detection of an amplification product when using the kit of the invention indicates the absence of DNA of any species of mycobacteria in the sample, i.e. the absence of the target sequence to be amplified. According to certain embodiments, an internal control is added to the kit to ensure that the absence of amplification and detection is indeed due to the absence of the target sequence.

[0115] An internal control is added to the sample, for example, at the extraction step, which makes it possible to control for this extraction step, in the absence of mycobacterial amplification, and ensures that this is not linked to a faulty extraction step.

[0116] According to yet another embodiment, the kit according to the invention comprises polymerase chain reaction reagents and a set of at least three primer pairs according to the invention, with or without the presence of an associated probe, and possibly also a DNA extraction kit.

[0117] In another aspect, the present invention relates to different methods of amplifying and detecting the sequence of the rrs gene of any mycobacteria using the kit of the present invention described in the previous section. Indeed, taking into account the comprehensiveness of primer combinations and the sensitivity of PCR technology, the present invention provides a highly sensitive detection test for mycobacteria applicable to several domains, especially applicable for the detection of mycobacteria in samples with very low mycobacterial content, for example less than 100 viable mycobacteria per mL, even less than 10 viable mycobacteria per mL, even about 5 or 1 viable mycobacteria per mL.

[0118] The present invention relates inter alia to such a method for amplifying and detecting sequences of the rrs genes of any mycobacteria present in a sample, which comprises the following steps: a) performing in vitro PCR on nucleic acid extracted from the sample using at least the above three sets of primer pairs; b) detecting the presence or absence of an amplification product; Includes.

[0119] It is immediately clear that depending on the method used for amplification by PCR, the two steps can be performed simultaneously or successively. As explained above, techniques such as qPCR actually make it possible to carry out the amplification and detection of the amplification products simultaneously.

[0120] The amplification step can be carried out without additional primers hybridizing to the mycobacterial sequence, using only the three primer pairs, in particular the first, second and third pairs of the invention. Such an embodiment does not exclude the amplification of a nucleic acid control, using one primer pair specific for this control and unrelated to the mycobacterial sequence (internal control). According to such an embodiment, the amplification step a) is carried out using an additional control primer pair comprising a forward primer and a reverse primer designed for the amplification of a nucleic acid control present or added to the sample, such a nucleic acid control being unrelated to the mycobacterial sequence and not amplified by the three primer pairs of the invention.

[0121] According to a particular embodiment, the three primer pairs are the following primers: First pair: Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-1: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO: 2) Second pair: Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-1: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO: 5) Third pair: Forward primer F4-1: 5'-CCCTTATGTCCAGGGCTTCA-3' (SEQ ID NO: 7) Reverse primer R4-1: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8) It comprises or consists of:

[0122] Primers having modified sequences with respect to F1-1, F3-1, F4-1, R1-1, R3-1 and R4-1 are also suitable for use in the methods of the invention. Suitable modifications have been discussed extensively in relation to the primer sets of the invention and are equally applicable to this aspect of the invention.

[0123] As disclosed in the preceding embodiment in relation to the set of primers of the invention, the different primer pairs independently comprise: - for the first pair, a list of primer pairs consisting of F1-1 (SEQ ID NO: 1)&R1-1 (SEQ ID NO: 2); F1-1&R1-6 (SEQ ID NO: 13); F1-1&R1-5 (SEQ ID NO: 12); F1-2 (SEQ ID NO: 10)&R1-1; F1-2&R1-3 (SEQ ID NO: 27); F1-2&R1-5; F1-2&R1-6 and F1-3 (SEQ ID NO: 11)&R1-6; - for the second pair, a list of primer pairs consisting of F3-1 (SEQ ID NO: 4)&R3-1 (SEQ ID NO: 5); F3-3 (SEQ ID NO: 14)&R3-1; F3-1&R3-2 (SEQ ID NO: 15); F3-1&R3-3 (SEQ ID NO: 16); F3-3&R3-2; F3-3&R3-3; F3-1&R3-4 (SEQ ID NO: 17) and F3-3&R3-4; and - for the third pair, a list of primer pairs consisting of F4-1 (SEQ ID NO: 7)&R4-1 (SEQ ID NO: 8); F4-3 (SEQ ID NO: 18)&R4-1; F4-5 (SEQ ID NO: 19)&R4-1; F4-1&R4-3 (SEQ ID NO: 20); F4-1&R4-5 (SEQ ID NO: 28); F4-3&R4-3; F4-5&R4-3 and F4-5&R4-6 (SEQ ID NO: 21); It can also be selected.

[0124] According to the method of the present invention, the absence of detection of an amplification product indicates the absence of any species of mycobacteria or at least any of the 217 species, subspecies and variants of Table 4 in the sample, as long as the set of primers used in the method confers comprehensiveness to the method, i.e. regardless of the mycobacterial species that may be present in the sample, its rrs gene is amplified by at least one primer pair.

[0125] Furthermore, as explained in detail in connection with the kit of the invention, the amplification step a) can be carried out by any suitable PCR method, but highly preferred methods according to the invention are quantitative PCR and digital PCR, which are illustrated in the Examples section of the present application, and similar results are obtained by dPCR.

[0126] As is immediately clear from the above, the method of the invention does not require any step of culturing mycobacteria. The method can be performed directly on the sample to be tested without the need for culturing once the nucleic acid has been extracted from the sample. The method is therefore particularly rapid, especially for methods based on mycobacterial culture, and can actually reduce the time to result by more than 90%, especially 96%. The method is also more rapid than methods that require a step of pre-cultivation of mycobacteria before amplification. The claimed method does not require any culturing step at all. The method also presents the advantage of being able to detect any mycobacteria, including fastidious intracellular mycobacterial species.

[0127] According to a particular embodiment, the amplification by the three primer pairs is carried out simultaneously, i.e. in triplex, in the same reaction mixture.Furthermore, if an additional primer pair, designed for example for the amplification of a nucleic acid control present in or added to the sample, is also added, the amplification by the additional primer pair is advantageously carried out simultaneously, i.e. in quadruple.The inventors have actually demonstrated in Example 5 that such an amplification in quadruple is possible without negative interactions, thus improving the efficiency, while also maintaining the comprehensiveness of the detection method and its specificity when appropriate probes are used.

[0128] The amplification step a) of the method of the invention should be carried out in the presence of all the appropriate components required for the chosen amplification technique, i.e. with the appropriate deoxyribonucleotide triphosphates and polymerase, under conditions suitable for amplification.

[0129] Concerning the detection step b), this step may be carried out using the relevant probes corresponding to the primer pairs, as already disclosed in connection with the kits of the invention. In this respect, the detection is advantageously carried out using a probe S1 with the sequence 5'-CGGTGGGTACTAGGTGTG-3' (SEQ ID NO: 3) or a modification thereof for the detection of the sequence amplified by said primers F1 and R1; a probe S3 with the sequence 5'-TCGGTTCCCTTGTGGC-3' (SEQ ID NO: 6) or a modification thereof for the detection of the sequence amplified by said primers F3 and R3; and a probe S4 with the sequence 5'-ACATGCTACAATGGCCGG-3' (SEQ ID NO: 9) or a modification thereof for the detection of the sequence amplified by said primers F4 and R4. Suitable modifications have already been detailed in the previous section and are applicable to this aspect of the invention.

[0130] According to a particular embodiment, the probe comprises: - For probe S1, Probe S1-0: 5'-CGGTGGGTACTAGGTGTG-3' (SEQ ID NO: 3), Probe S1-1: 5'-CGGTGGGTACTAGGTGT-3' (SEQ ID NO: 22), and Probe S1-2: 5'-CGGTGGGTACTAGGTG-3' (SEQ ID NO: 23) The group consisting of When probe S1 is S1-0, the first primer pair is a) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-1: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO:2); b) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13); c) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-5: 5'-ACGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO: 12); d) Forward primer F1-2: 5'-CCTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-1: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO:2); e) Forward primer F1-2: 5'-CCTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-3: 5'-CACGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO:27); f) Forward primer F1-2: 5'-CCTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-5: 5'-ACGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO: 12); g) Forward primer F1-2: 5'-CCTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13); and h) Forward primer F1-3: 5'-CTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 11) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13) is selected from When probe S1 is S1-1, the first primer pair is a) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-1: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO:2); b) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13); c) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-5: 5'-ACGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO: 12); d) Forward primer F1-2: 5'-CCTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-1: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO: 2); and e) Forward primer F1-3: 5'-CTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 11) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13) is selected from When probe S1 is S1-2, the first primer pair is a) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-1: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO:2); b) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13); c) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-5: 5'-ACGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO: 12); d) Forward primer F1-2: 5'-CCTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-1: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO:2); e) Forward primer F1-2: 5'-CCTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-5: 5'-ACGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO: 12); and f) Forward primer F1-3: 5'-CTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 11) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13) The group selected from - Regarding probe S3, Probe S3-0: 5'-TCGGTTCCCTTGTGGC-3' (SEQ ID NO: 6), Probe S3-1: 5'-ATCGGTTCCCTTGTGGC-3' (SEQ ID NO: 24), and Probe S3-2: 5'-CGGTTCCCTTGTGGC-3' (SEQ ID NO: 25) The group consisting of When probe S3 is S3-0, the second primer pair is a) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-1: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO:5); b) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-1: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO:5); c) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-2: 5'-CATGCACCACCTGCACACAG-3' (SEQ ID NO: 15); d) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-3: 5'-ATGCACCACCTGCACACAG-3' (SEQ ID NO: 16); e) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-2: 5'-CATGCACCACCTGCACACAG-3' (SEQ ID NO: 15); f) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-3: 5'-ATGCACCACCTGCACACAG-3' (SEQ ID NO: 16); g) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-4: 5'-TGCACCACCTGCACACAG-3' (SEQ ID NO: 17); and h) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-4: 5'-TGCACCACCTGCACACAG-3' (SEQ ID NO: 17) is selected from When probe S3 is S3-1, the second primer pair is a) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-1: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO:5); b) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-1: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO:5); c) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-2: 5'-CATGCACCACCTGCACACAG-3' (SEQ ID NO: 15); d) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-2: 5'-CATGCACCACCTGCACACAG-3' (SEQ ID NO: 15); and e) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-3: 5'-ATGCACCACCTGCACACAG-3' (SEQ ID NO: 16) is selected from When probe S3 is S3-2, the second primer pair is a) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-1: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO:5); b) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-1: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO:5); c) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-2: 5'-CATGCACCACCTGCACACAG-3' (SEQ ID NO: 15); d) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-3: 5'-ATGCACCACCTGCACACAG-3' (SEQ ID NO: 16); e) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-4: 5'-TGCACCACCTGCACACAG-3' (SEQ ID NO: 17), and f) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-3: 5'-ATGCACCACCTGCACACAG-3' (SEQ ID NO: 16) The group selected from - Regarding the probe S4, Probe S4-0: 5'-ACATGCTACAATGGCCGG-3' (SEQ ID NO: 9), and Probe S4-1: 5'-ACATGCTACAATGGCCGGT-3' (SEQ ID NO: 26) The group consisting of When probe S4 is S4-0, the third primer pair is a) forward primer F4-1: 5'-CCCTTATGTCCAGGGCTTCA-3' (SEQ ID NO: 7); Reverse primer R4-1: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); b) forward primer F4-3: 5'-CCCCTTATGTCCAGGGCTTC-3' (SEQ ID NO: 18); Reverse primer R4-1: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); c) forward primer F4-5: 5'-GCCCCTTATGTCCAGGGC-3' (SEQ ID NO: 19); Reverse primer R4-1: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); d) forward primer F4-1: 5'-CCCTTATGTCCAGGGCTTCA-3' (SEQ ID NO: 7); Reverse primer R4-3: 5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); e) forward primer F4-1: 5'-CCCTTATGTCCAGGGCTTCA-3' (SEQ ID NO: 7); Reverse primer R4-5: 5'-GCATCGCAGCCCTTTGTA-3' (SEQ ID NO:28); f) forward primer F4-3: 5'-CCCCTTATGTCCAGGGCTTC-3' (SEQ ID NO: 18); Reverse primer R4-3: 5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); g) forward primer F4-5: 5'-GCCCCTTATGTCCAGGGC-3' (SEQ ID NO: 19); Reverse primer R4-3: 5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); and h) forward primer F4-5: 5'-GCCCCTTATGTCCAGGGC-3' (SEQ ID NO: 19); Reverse primer R4-6: 5'-GGCATCGCAGCCCTTTGTA-3' (SEQ ID NO: 21) is selected from When probe S4 is S4-1, the third primer pair is i. Forward primer F4-1: 5'-CCCTTATGTCCAGGGCTTCA-3' (SEQ ID NO: 7); Reverse primer R4-1: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); ii. Forward primer F4-3: 5'-CCCCTTATGTCCAGGGCTTC-3' (SEQ ID NO: 18); Reverse primer R4-1: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); iii. Forward primer F4-5: 5'-GCCCCTTATGTCCAGGGC-3' (SEQ ID NO: 19); Reverse primer R4-1: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); iv. Forward primer F4-1: 5'-CCCTTATGTCCAGGGCTTCA-3' (SEQ ID NO: 7); Reverse primer R4-3: 5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); v. forward primer F4-3: 5'-CCCCTTATGTCCAGGGCTTC-3' (SEQ ID NO: 18); Reverse primer R4-3: 5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); vi. Forward primer F4-5: 5'-GCCCCTTATGTCCAGGGC-3' (SEQ ID NO: 19); Reverse primer R4-3: 5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); and vii. forward primer F4-5: 5'-GCCCCTTATGTCCAGGGC-3' (SEQ ID NO: 19); Reverse primer R4-6: 5'-GGCATCGCAGCCCTTTGTA-3' (SEQ ID NO: 21) selected from the group is selected from.

[0131] In one embodiment, the method is carried out using forward primer F1-1 (SEQ ID NO:1), reverse primer R1-1 (SEQ ID NO:2) and probe S1-0 (SEQ ID NO:3).

[0132] In another embodiment, the method is carried out using forward primer F3-1 (SEQ ID NO: 4), reverse primer R3-1 (SEQ ID NO: 5) and probe S3-0 (SEQ ID NO: 6).

[0133] In yet another embodiment, the method is carried out using forward primer F4-1 (SEQ ID NO:7), reverse primer R4-1 (SEQ ID NO:8) and probe S4-0 (SEQ ID NO:9).

[0134] According to certain embodiments, the method is carried out using F1-1, R1-1, S1-0, F3-1, R3-1, S3-0, F4-1, R4-1 and S4-0.

[0135] Detection step b) of the method may also be performed by using a DNA intercalating agent such as SYBRGreen, as detailed above, however, to provide specificity, detection is in certain embodiments performed using a probe as disclosed above.

[0136] If probes are potentially used for the detection of the amplification products, according to a particular embodiment, the amplification step a) and the detection step b) are carried out in a single reaction mixture comprising different primer pairs, namely F1, R1, F3, R3, F4 and R4 and the associated probes S1, S3 and S4. The inventors have indeed demonstrated that simultaneous amplification and detection with three primer pairs and three associated probes gives satisfactory results.

[0137] The inventors have further demonstrated that amplification and detection can be carried out simultaneously using additional primer pairs and associated probes, particularly primers and probes specifically designed to amplify and detect nucleic acid controls. Thus, the methods of the present invention can also include additional primer pairs and associated probes designed for the detection of nucleic acid controls present or added to the sample.

[0138] As detailed in connection with the kits of the invention, the probes may be linked, either directly or indirectly, to a label, such as a dye or fluorophore marker. Suitable markers are given in the examples and include 6-FAM.

[0139] Moreover, all the specific embodiments already disclosed in relation to the kit of the invention are fully applicable to this aspect of the invention in relation to the method. In this respect, as already detailed, the probes S1, S3 and S4 are directly or indirectly linked to the same label, and the probes of the control system are directly or indirectly linked to a different label. The reasons behind this choice will be made clear.

[0140] As mentioned above, the amplification and detection steps can be performed simultaneously or sequentially. Step a) can also include at least several cycles of amplification by PCR before step b) is performed or before the reading corresponding to step b). In particular, there are at least 30 cycles of PCR, such as at least 35 cycles, for example about 37 cycles, in particular at least or about 40 cycles. Depending on the experimental conditions, the skilled person can easily determine the appropriate number of cycles to distinguish between specific amplification of mycobacterial nucleic acids and non-specific amplification of non-mycobacterial nucleic acids or noise. The determination of the appropriate threshold value of the number of PCR cycles is illustrated, inter alia, in Example 8.

[0141] The method according to the invention can advantageously be used with different kinds of samples. The sample may in fact be a biopharmaceutical sample that is likely to be contaminated by mycobacteria, for example due to the use of eukaryotic cells in its manufacturing process, or the sample may also be an environmental sample, for example a water sample from an installation, the sample may also be a biological sample obtained from an animal or human that is likely to be contaminated by mycobacteria. The sample may also be a medium for cell culture or a matrix used in the manufacture of a pharmaceutical product, a virus seed, a starting material for cell culture, a cell culture or a supernatant, and thus an intermediate product, such as a crude harvest of drug substance, a formulated drug product or a final product. The sample may be from any step of any pharmaceutical product manufacturing, such as, for example, CHO cells used for recombinant protein production, crude harvest of drug substance from, for example, MRC5 cells used for virus production, such as, for example, cytomegalovirus protein antigens, for example, HAV (hepatitis A virus), crude harvest of drug substance from, for example, Vero cells used for virus production, such as, for example, yellow fever virus or rabies virus. The inventors have indeed demonstrated that specific and comprehensive amplification of any mycobacteria potentially present in these matrices can be performed according to the method of the invention.

[0142] In a particular embodiment, the method is performed on a biopharmaceutical sample from a drug or vaccine at any stage of pharmaceutical production, in particular on the final product, any intermediate matrix, a virus seed or starting material such as a cell culture or medium.

[0143] According to the method of the present invention, detection of an amplification product by any of probes S1, S3 and S4 indicates the specific presence of at least one species of mycobacteria in the sample among at least 200 different mycobacterial species, more particularly among 217 mycobacterial species, subspecies and variants.

[0144] Depending on the expected mycobacterial DNA concentration and the recommendations of the PCR mix provider's user manual, the skilled person will adapt the concentrations of the different primers F1, R1, F3, R3, F4 and R4. For biopharmaceutical samples and detection by qPCR, the concentrations used are generally in the range of 100 nM to 400 nM for the primers in the reaction mixture.

[0145] Likewise, the concentrations of the probes are adapted depending, inter alia, on the expected concentration of mycobacterial DNA, the number of PCR amplification cycles before detection and the type of detection technique. For biopharmaceutical samples and detection by qPCR, probes S1, S3 and S4 should generally be added at concentrations in the range of 50 nM to 250 nM.

[0146] According to a particular embodiment, steps a) and b) are carried out simultaneously in the same reaction mixture. Thus, the DNA extract of the sample is contacted simultaneously with the three primer pairs and the three associated probes in the same reaction mixture. As detailed above, a fourth primer pair for amplifying a control sequence is advantageously also added to the same reaction mixture and associated probe. Thus, three or four different primers and associated probes are added to the DNA extract, and amplification and detection are carried out in this single reaction mixture. The inventors have actually demonstrated that even in the presence of associated probes, a quadruple reaction is possible without losing the comprehensiveness and specificity of detection. In the case of triplex or quadruple amplification and detection, the PCR technique is, for example, qPCR or dPCR, in particular qPCR.

[0147] Performing amplification and detection in triplex or quadruple is a major time gain and also allows saving of materials, reagents, machine time and costs.

[0148] The inventors have also demonstrated that the method disclosed above is capable of detecting very low amounts of mycobacteria species and is therefore a sensitive method suitable for use in the pharmaceutical field. The method can indeed detect mycobacterial DNA at concentrations of less than 10 fg / μL, less than 1 fg / μL, even less than 0.3 fg / μL, and generally less than 0.03 fg / μL for most strains. Thus, by adjusting the DNA extraction step, it is possible to detect mycobacteria with a sensitivity essentially similar to or better than that of the official method that meets the Ph Eur.2.6.2 requirements for mycobacteria detection. The present invention provides a sensitive detection test for mycobacteria applicable to several domains, and is particularly applicable to the detection of mycobacteria in samples with very low mycobacteria content, for example less than 100 viable mycobacteria per mL, even less than 10 viable mycobacteria per mL, even about 5 or 1 viable mycobacteria per mL.

[0149] Therefore, this method is an alternative to the compendial method that can advantageously be performed in less than 48 hours, and even less than 24 hours.

[0150] The method is therefore highly valuable for biopharmaceutical testing, especially vaccine testing, where it can replace compendium methods at the same or even increased sensitivity and comparable or increased specificity (see Example 8.1) and with a broader detection range.

[0151] According to yet another aspect, the present invention also relates to the use of the set of primers as detailed according to the previous aspect or the use of the kit as detailed in the same way, for amplifying by PCR mycobacterial rrs gene sequences present in a sample. It is immediately clear that all potential or specific embodiments disclosed for the set of primers and the kit of the present invention are also applicable to this further aspect. In this regard, suitable primer pairs have already been disclosed and are also suitable for this aspect, the same being true for the associated probes. Similarly, the different PCR techniques that can be used have already been described in detail in relation to the previous aspect, particular techniques being qPCR and dPCR, in particular qPCR. The use of the kit of the present invention can be for amplifying and detecting mycobacterial rrs gene sequences present in a sample, if suitable probes for detection are present in the kit.

[0152] The use according to the invention results in the comprehensive amplification of any mycobacterial rrs genes potentially present in a sample. Moreover, by using the kit of the invention together with the associated probe, the use results in the specific detection of mycobacterial rrs genes, while other bacterial rrs genes potentially present in the sample are not detected.

[0153] This use according to the invention may be either in vivo, ex vivo or in vitro, in particular in vitro. The use may further be carried out on the sample.

[0154] According to a particular embodiment, the present invention provides a method for the detection of genomic DNA comprising the steps of: System 1: Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-1: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO: 2) Probe S1-0: 5'-CGGTGGGTACTAGGTGTG-3' (SEQ ID NO: 3) System 3: Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-1: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO: 5) Probe S3-0: 5'-TCGGTTCCCTTGTGGC-3' (SEQ ID NO: 6) System 4: Forward primer F4-1: 5'-CCCTTATGTCCAGGGCTTCA-3' (SEQ ID NO: 7) Reverse primer R4-1: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8) Probe S4-0: 5'-ACATGCTACAATGGCCGG-3' (SEQ ID NO: 9) for the in vitro use of the method for specifically amplifying and detecting by PCR mycobacterial nucleic acid present in a sample without amplifying other bacterial nucleic acid.

[0155] In certain embodiments, only the three primer pairs and associated probes disclosed are used in conjunction with other probes or primers potentially not associated with Mycobacteria species.

[0156] In the different uses according to the invention, the primers and probes of the three systems are advantageously used simultaneously, i.e. in a single amplification step. A fourth system relating to the amplification and detection of control nucleic acid molecules may also be combined with the first three systems in these uses.

[0157] As regards other aspects of the invention, the PCR technique may be any suitable technique, such as qPCR or dPCR, in particular qPCR.

[0158] According to another specific embodiment, the present invention also relates to a method for detecting the presence of mycobacterial contamination in a medicinal product, such as a vaccine, without any culture step of mycobacteria, which comprises the following steps: a) a step of in vitro amplification by quantitative PCR (qPCR) or dPCR, carried out on nucleic acids extracted from said product sample, using the first, second and third primer pairs described above that hybridize to the rrs gene; b) The following probes: i) a probe S1, Probe S1-0: 5'-CGGTGGGTACTAGGTGTG-3' (SEQ ID NO: 3), Probe S1-1: 5'-CGGTGGGTACTAGGTGT-3' (SEQ ID NO: 22), and Probe S1-2: 5'-CGGTGGGTACTAGGTG-3' (SEQ ID NO: 23) is selected from the group consisting of When probe S1 is S1-1, the first primer pair is 1) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-1: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO:2); 2) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13); 3) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-5: 5'-ACGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO: 12); 4) Forward primer F1-2: 5'-CCTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-1: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO: 2); and 5) Forward primer F1-3: 5'-CTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 11) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13) is selected from When probe S1 is S1-2, the first primer pair is 1) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-1: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO:2); 2) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13); 3) Forward primer F1-1: 5'-CCTGGTAGTCCACGCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-5: 5'-ACGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO: 12); 4) Forward primer F1-2: 5'-CCTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-1: 5'-CGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO:2); 5) Forward primer F1-2: 5'-CCTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-5: 5'-ACGGATCCCAAGGAAGGAAAC-3' (SEQ ID NO: 12); and 6) Forward primer F1-3: 5'-CTGGTAGTCCACGCCGTAAA-3' (SEQ ID NO: 11) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13) Selected from: probe S1; ii) a probe S3, Probe S3-0: 5'-TCGGTTCCCTTGTGGC-3' (SEQ ID NO: 6), Probe S3-1: 5'-ATCGGTTCCCTTGTGGC-3' (SEQ ID NO: 24), and Probe S3-2: 5'-CGGTTCCCTTGTGGC-3' (SEQ ID NO: 25) is selected from the group consisting of When probe S3 is S3-1, the second primer pair is 1) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-1: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO:5); 2) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-1: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO:5); 3) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-2: 5'-CATGCACCACCTGCACACAG-3' (SEQ ID NO: 15); 4) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-2: 5'-CATGCACCACCTGCACACAG-3' (SEQ ID NO: 15); and 5) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-3: 5'-ATGCACCACCTGCACACAG-3' (SEQ ID NO: 16) is selected from When probe S3 is S3-2, the second primer pair is 1) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-1: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO:5); 2) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-1: 5'-CATGCACCACCTGCACACA-3' (SEQ ID NO:5); 3) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-2: 5'-CATGCACCACCTGCACACAG-3' (SEQ ID NO: 15); 4) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-3: 5'-ATGCACCACCTGCACACAG-3' (SEQ ID NO: 16); 5) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-4: 5'-TGCACCACCTGCACACAG-3' (SEQ ID NO: 17); and 6) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-3: 5'-ATGCACCACCTGCACACAG-3' (SEQ ID NO: 16) Selected from: probe S3; iii) a probe S4, Probe S4-0: 5'-ACATGCTACAATGGCCGG-3' (SEQ ID NO: 9), and Probe S4-1: 5'-ACATGCTACAATGGCCGGT-3' (SEQ ID NO: 26) is selected from the group consisting of When probe S4 is S4-1, the third primer pair is 1) Forward primer F4-1: 5'-CCCTTATGTCCAGGGCTTCA-3' (SEQ ID NO: 7); Reverse primer R4-1: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); 2) forward primer F4-3: 5'-CCCCTTATGTCCAGGGCTTC-3' (SEQ ID NO: 18); Reverse primer R4-1: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); 3) Forward primer F4-5: 5'-GCCCCTTATGTCCAGGGC-3' (SEQ ID NO: 19); Reverse primer R4-1: 5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); 4) Forward primer F4-1: 5'-CCCTTATGTCCAGGGCTTCA-3' (SEQ ID NO: 7); Reverse primer R4-3: 5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); 5) Forward primer F4-3: 5'-CCCCTTATGTCCAGGGCTTC-3' (SEQ ID NO: 18); Reverse primer R4-3: 5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); 6) Forward primer F4-5: 5'-GCCCCTTATGTCCAGGGC-3' (SEQ ID NO: 19); Reverse primer R4-3: 5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); and 7) Forward primer F4-5: 5'-GCCCCTTATGTCCAGGGC-3' (SEQ ID NO: 19); Reverse primer R4-6: 5'-GGCATCGCAGCCCTTTGTA-3' (SEQ ID NO: 21) Selected from the probe S4 Detection of the presence or absence of an amplification product by Includes.

[0159] The steps may be performed sequentially or simultaneously, eg with partial concurrence.

[0160] The first primer pair is, for example, F1-1 and R1-1. In such a case, the first probe can be any of S1-0, S1-1 and S1-2.

[0161] The second primer pair is, for example, F3-1 and R3-1, in which case the second probe can be any of S3-0, S3-1, and S3-2.

[0162] The third primer pair is, for example, F4-1 and R4-1. In such a case, the third probe can be either S4-0 or S4-1.

[0163] Probe S1 may be S1-0, in which case the first primer pair is selected from F1-1&R1-1; F1-1&R1-6; F1-1&R1-5, F1-2&R1-1; F1-2&R1-3; F1-2&R1-5; F1-2&R1-6 and F1-3&R1-6.

[0164] Probe S3 may be S3-0, in which case the second primer pair is selected from F3-1&R3-1; F3-3&R3-1; F3-1&R3-2; F3-1&R3-3; F3-1&R3-4; F3-3&R3-2; F3-3&R3-3; and F3-3&R3-4.

[0165] Probe S4 may be S4-0, in which case the third primer pair is selected from F4-1&R4-1; F4-3&R4-1; F4-5&R4-1; F4-1&R4-3; F4-1&R4-5; F4-3&R4-3; F4-5&R4-3 and F4-5&R4-6.

[0166] In a particular embodiment, the first primer pair is F1-1 and R1-1 and associated probe S1-0, the second primer pair is F3-1 and R3-1 and associated probe S3-0, and the third primer pair is F4-1 and R4-1 and associated probe S4-0.

[0167] In the experimental section, the inventors have indeed demonstrated that the specificity and sensitivity of the method, primers and probes is not lost in the presence of biopharmaceutical matrices such as cell supernatants, crude harvests of drug substance or culture media (see especially Examples 8 and 9).

[0168] According to a particular embodiment of this particular method, a fourth primer pair is added along with an associated probe that amplifies an exogenous nucleic acid molecule used as a control. This fourth primer pair and associated probe is thus a control system that is used in combination with the three systems that provide for amplification and detection of mycobacterial rrs genes.

[0169] Three primer pairs and associated probes that hybridize to the rrs gene and a fourth pair and associated probe that hybridize to a control molecule are used in a quadruple in the same reaction mixture, e.g., as already described in detail in connection with other aspects of the invention.

[0170] The method according to the invention is therefore such that detection of an amplification product indicates the specific presence in the medicinal product of at least one species of mycobacteria out of at least 217 different mycobacterial species, subspecies or variants, and lack of detection of an amplification product indicates the absence of any species of mycobacteria in the medicinal product.

[0171] Such methods are therefore suitable as alternative safety release tests and can be used directly after product validation in accordance with regulatory requirements.

[0172] Regarding the three primer pairs and associated probes that hybridize to the rrs genes, different alternative systems have been described in relation to other aspects of the invention and are entirely applicable to this method.

[0173] The present invention also relates to different processes, in particular the process of releasing pharmaceutical products that have passed the above-mentioned safety release test, based on the absence of detection of any amplification products by using the three primer pairs and associated probes of the present invention.

[0174] The present invention also relates to a process for the production of a biopharmaceutical, which comprises a step of testing for mycobacterial contamination by carrying out the method for detecting any mycobacterial rrs gene sequence according to the present invention.

[0175] The present invention also relates to a process for producing a biopharmaceutical product free of mycobacterial contamination, which comprises: - Providing biopharmaceutical products; - subjecting the product to the method according to the invention for detecting the sequence of the rrs gene of any mycobacterium; Includes.

[0176] According to this method, if no amplification product is detected by the method of the invention, it is concluded that the product is devoid of mycobacterial contamination.

[0177] A biopharmaceutical may be a biological product or a pharmaceutical; a preferred product is a vaccine.

[0178] Also in this embodiment, all the specific features already disclosed in relation to the previous embodiments of the invention are applicable. EXAMPLES

[0179] Mycobacteria detection is a safety release test that may be required for biopharmaceuticals expressed in or based on eukaryotic cells. Current methods described in the pharmacopoeia are based on microbial culture (see European Pharmacopoeia. Chapter 2.6.2 and Technical Report Series-TRS 978 section B.11.3.3 of WHO). The samples to be tested must be inoculated in triplicate on two different suitable solid media and one suitable liquid medium. All media are incubated for 56 days.

[0180] The main problems and limitations of the above official culture-based assays are, inter alia, the availability and potential shortage of specific media, the very long test turnaround time of 56 days and the lack of detection of an important human origin species, namely M. leprae, an obligate intracellular parasite, as well as the lack of detection of other intracellular mycobacteria or fastidious species with specific growth requirements, and hence the lack of comprehensiveness of the assay.

[0181] Therefore, the inventors developed an alternative method, which is as follows. - specific and comprehensive, i.e. capable of detecting low levels of all mycobacterial species but not non-mycobacterial species; - rapid, meaning results are obtained within a few days or even less; and - Sensitive enough to replace official methods.

[0182] Such a method is more in line with Good Manufacturing Practice.

[0183] The various mycobacteria currently known and contemplated by the present inventors are listed in Table 4.

[0184] Example 1: Materials and Methods KK: The mycobacterial strains used to validate this method include slow- and fast-growing strains and are detailed in Table 1. The rationale underlying the selection of these strains is explained in Example 5.

[0185] The growth rate is defined as fast (also defined as rapid) if the reproductive cycle is 7 days or less. The growth rate is defined as slow for mycobacteria that have a reproductive cycle of more than 7 days.

[0186] [Table 1]

[0187] Production and characterization of mycobacterial banks. The bank's mycobacteria have been characterized and calibrated by flow cytometry and digital PCR (dPCR).

[0188] DNA extraction and purification For DNA purification, the QIAmp DNA Micro Kit (for the preliminary steps of primer design and LOQ determination) and the QIAamp UCP DNA Micro Kit (Qiagen) are used according to the manufacturer's recommendations.

[0189] Alternative commercially available kits such as the EliGene® MTB Isolation kit (Elisabeth Pharmacon) have also been tested.

[0190] amplification: PCR amplification The amplification is carried out using a commercially available ready to use Master Mix for probe detection, containing DNA Taq polymerase and oligonucleotides suitable for the PCR technique used, e.g. qPCR.

[0191] Extraction / amplification controls: Exogenous internal controls for extraction and amplification should be used. The following two internal controls were tested: - DiaControlDNA™: Diagenode, reference number DICD-CY-L100 (with detection with Cy5)

[0192] The kit contains primers and probes as well as a mycobacterial gene and a seal herpes virus at a concentration of 1000 TCID50 / mL that is unrelated to the product being evaluated. - The second internal control is a plasmid containing a mycobacterial gene and a gene encoding GFP, unrelated to the product being evaluated.

[0193] To detect this control, appropriate primers and corresponding probes are designed.

[0194] Primers and probes The primers and probes used to amplify and detect the rrs gene (16S rRNA) amplification products are detailed in Table 2.

[0195] [Table 2]

[0196] The probe used for detection of the internal control is linked at the 5' end to the fluorophore Cy5 and the quencher at the 3' end is BHQ1.

[0197] Example 2: Selection of targets, primers and amplification methods. In order to develop an alternative to the official method for detecting mycobacteria, one that would be quicker, more comprehensive and easier to perform, we tested different methods based on molecular biology rather than culture, and more specifically based on sequence amplification.

[0198] Therefore, we first designed potentially suitable primers in silico, tested and compared different amplification methods, and constructed a calibrated bank of mycobacteria to validate the specificity and comprehensiveness of the primers, detection methods, and evaluate the sensitivity.

[0199] 2.1. Selection of the target to be amplified. Genome-wide approach One approach to define a suitable target for the purpose of defining an alternative approach to the official method is to target a single gene or a single sequence that is strictly specific to a mycobacteria species.For this purpose, a sequence alignment should be performed on the whole genome of all species.This approach therefore requires that the whole sequence of all mycobacteria species is available with sufficient quality.However, this is not the case for all species, which compromises the comprehensiveness of the method.This approach has therefore been ignored by the inventors.

[0200] Gene or region-specific approaches Various genes or regions have been reported in the art as primary targets for detecting mycobacteria (Bhatnagar, et al 2017). However, after studying these genes or regions, the inventors have concluded that in most cases, these genes are present only in tuberculous mycobacteria or only in nontuberculous mycobacteria (NTM). Furthermore, the sequences of these genes are not available or reported for all species of the genus, which does not meet the required comprehensiveness.

[0201] The intergenic sequence between the rrs and rrl genes encoding the 23S RNA (ITS1 region) has also been investigated and initially retained. However, the corresponding sequences are not available for all mycobacteria, which compromises the comprehensiveness of the detection, and furthermore, the ITS1 target does not appear to be specific enough for the required specificity.

[0202] The rrs gene is a gene encoding 16S rRNA and contains about 1400 base pairs. It is a major target for detecting specific microorganisms because it contains conserved regions shared by several bacterial species and variable regions depending on the species. Moreover, the rrs gene appears to be well documented in different species of mycobacteria and was therefore retained by the present inventors.

[0203] A file containing the sequences of the rrs genes in FASTA format was generated from the Ezbiocloud database, which contains only quality-checked sequences, and contains 195 sequences corresponding to all the different species and subspecies referred to as mycobacteria.

[0204] These 195 sequences, completed to cover the 217 sequences known so far, were then aligned with the Clustal omega program adapted for multiple sequence alignment in order to determine suitable primers.

[0205] 2.2. Primer and probe design: Single primer pair approach: All available sequences of mycobacteria rrs genes were aligned. Once the sequences were aligned, they were used in Primer express software (Thermo Fisher Scientific) to design primer pairs with the associated MGB probes (Minor Groove Binder). Different potential pairs were proposed by the software, and the different sequences were then compared with Primer Blast (NCBI) and de Silva test Prime (German Network for Bioinformatics).

[0206] This step is essential not only to check the mycobacterial sequence recognized by the pair or primers, but also to ensure specificity, ie lack of amplification and detection of other bacterial species.

[0207] Primer mismatches are not tolerated in the database.

[0208] Insofar as the targeted region of the 195 species or subspecies is a conserved region in bacteria, the results obtained with the selected primers are disappointing since species other than mycobacteria are amplified.

[0209] Therefore, it was not possible to design a single primer pair to amplify exclusively all sequences from mycobacteria.

[0210] Multiplexed Approach: Since it was not possible to design a single primer pair to amplify only rrs sequences from mycobacteria, but all of them could be designed, we decided to design different groups of mycobacteria according to their phylogenetic evolution before searching for suitable primer pairs. The rationale underlying this approach is to identify target regions of rrs genes that are more variable but more specific to mycobacteria.

[0211] Furthermore, we decided to design the different groups to take advantage of the phylogenetic tree detailed according to the previously defined genus Mycobacterium, since phylogenetically closer species are more likely to present common conserved regions specific to those groups (Gupta et al., 2018).

[0212] In a first step, two primer pairs were designed covering 175 different species: the first pair F1-1 and R1-1 allows the detection of 151 species of mycobacteria; the second pair allows the detection of 24 additional species, although about 100 species are amplified by both pairs.

[0213] The remaining 20 sequences in FASTA format are realigned with another alignment software, namely Seaview. Primer Express (version 3) software generates new primer proposals, which are then compared with different databases. In this way, two additional primer pairs covering 17 additional species and specific for the genus Mycobacterium are obtained (F3-1&R3-1 and F4-1&R4-1).

[0214] Thus, four different primer pairs were generated, targeting the same rrs gene but on different regions.

[0215] In addition to updated taxonomy and software revisions defining primers and probes, the inventors determined that since systems 1, 3 and 4 cover all species mentioned to date, the second primer pair does not need to cover all mycobacteria.

[0216] The list of aligned mycobacterial species, including accession numbers and primer pairs leading to amplification, is detailed in Table 4. The table is based on the List of Prokaryotic names with Standing in Nomenclature (LPSN) and the publications Parte et al., 2018, Parte et al. 2020, Parte et al. 2014, and Euzeby 1997.

[0217] [Table 3]

[0218] [Table 4]

[0219] [Table 5]

[0220] [Table 6]

[0221] [Table 7]

[0222] [Table 8]

[0223] As can be seen in Table 4, for five species, namely M. genavense, M. stomatepiae, M. hassiacum, M. thermoresistible and M. heraklionense, there are mismatches between one of the primers and the rrs gene sequence of these species. Given the location of these mismatches on the primers and the fact that the mismatches are with respect to the primers and not the probe, it is presumed that such mismatches should not prevent amplification. This has been confirmed by the inventors (see Example 3).

[0224] Conclusion: The three primer pairs F1-1&R1-1, F3-1&R3-1 and F4-1&R4-1 allow to detect the rrs genes of mycobacteria and cover all mycobacterial species.

[0225] Probe and specificity verification: Using the same software, probes specific to the sequences amplified by the three primer pairs F1-1&R1-1, F3-1&R3-1 and F4-1&R4-1, respectively, namely S1-0, S3-0 and S4-0, are designed. A perfect match between these probes and the amplified sequences is required.

[0226] Furthermore, we performed an analysis using Primer-BLAST software developed at NCBI to help users create primers specific to the intended PCR target. This program uses BLAST and global alignment algorithms to screen primers against a database selected by the user to avoid primer pairs (all combinations including forward primer-reverse primer pair, forward-forward pair, and reverse-reverse pair) that may cause non-specific amplification. In this case, the database selected was the RefSeq Representative Genome Database (Organism limited to Bacteria).

[0227] The primer BLAST parameters were set as follows:

[0228] [Table 9]

[0229] The numbers of Blast hits analyzed were 36,477 for F1-1&R1-1, 8,072 for F3-1&R3-1, and 8,971 for F4-1&R4-1.

[0230] The results are as follows: F1-1&R1-1: If two mismatches are allowed, the specificity is 98.72%. Two of the 156 amplified sequences are not actually of mycobacterial origin. These two sequences are derived from: - Lujinxingia litoralis strain B210 B210. However, the amplified fragment contains more than 1000 base pairs and is therefore not compatible with qPCR. In any case, this amplified fragment does not hybridize with probe S1-0. - Virgibacillus indicus strain P2-C2, however no amplification is detected with probe S1-0.

[0231] The specificity is therefore 100% for the selected probe S1-0.

[0232] For F3-1&R3-1: If two mismatches are permitted, the specificity is 100%. Thirteen sequences, all of mycobacterial origin, are amplified.

[0233] For F4-1&R4-1: If two mismatches are allowed, the specificity is 95.65%. Eight of the 184 amplified sequences, which are not actually of mycobacterial origin, do not hybridize with probe S4-0.

[0234] Conclusion: The three primer pairs F1-1&R1-1, F3-1&R3-1 and F4-1&R4-1 with the corresponding probes S1-0, S3-0 and S4-0 allow the specific detection of all mycobacteria species without detecting other bacteria.

[0235] 2.3. Creation of a calibrated mycobacterial bank The production of mycobacterial cell banks is carried out during the exponential growth phase. The viable mycobacterial concentration is characterized by flow cytometry and the number of genome copies (GC) (live and dead bacteria) is determined by classical methods known to those skilled in the art (qPCR, dPCR, fluorescence measurement or UV detection, etc.) on a single copy reference gene.

[0236] The ratio of GC / mL to viable mycobacteria / mL is calculated and should be as close to 1 (mostly viable organisms) as possible to disfavor genome amplification-based methods over culture-based methods. This calibration method provides a mycobacterial cell bank suitable for PCR use.

[0237] 2.4. Comparison of the sensitivities obtained with different amplification methods. Quantitative PCR (qPCR), also known as real-time PCR, and digital PCR (dPCR) were evaluated in parallel.

[0238] For primer pairs F1-1&R1-1 and F4-1&R4-1 (similar to F2-R2, but this pair was not ultimately retained), these pairs amplify sequences present in the M. bovis genome (now further classified as M. tuberculosis var BCG due to the last evolution of the nomenclature). Therefore, this mycobacterium was used to determine the detection limit of these primer pairs according to the amplification method, i.e. qPCR or dPCR, based on the concentrations evaluated as detailed in section 2.3. Mycobacterial DNA was extracted with the appropriate commercially available kit and used according to the manufacturer's recommendations.

[0239] For pairs F3-1 and R3-1, the corresponding DNA of M. hiberniae was used.

[0240] Amplification by dPCR This technique was initially tested by the present inventors using primers targeting the ITS1 region.

[0241] We then tested this technique using primer pairs targeting the rrs genes (F1-1&R1-1, F2&R2, and F4-1&R4-1) on M. bovis DNA to assess the detection limit of the technique.

[0242] Dilutions of the samples are carried out until the disappearance of the signal, i.e. no positive droplets are detectable. The limits of detection are comparable for the three primer pairs tested (including but not retained pair F2-R2), regardless of the detection system, i.e. probe or Evagreen.

[0243] The limit of detection is up to 10 (for M. tuberculosis var BCG) based on initial studies performed by the inventors. -4 of dilution (on a bank roughly calibrated by optical density).

[0244] It is expected that sensitivity can be improved by optimizing the DNA extraction process and PCR conditions, including the number of cycles.

[0245] Amplification by qPCR To assess the detection limit of this technique, an initial reference range was prepared, allowing to estimate the amount of DNA present from the amplification detected.

[0246] To this end, for each selected target, a corresponding synthetic reference nucleotide sequence was synthesized, introduced into a plasmid and quantified.

[0247] Then, using the primer pairs F1-1&R1-1, F2-R2 and F4-1&R4-1, the associated probes S1-0, S2 and S4-0, respectively, and synthetic sequences with known amounts in ng / mL, we were able to set up a reference range for the detection of the target sequence by qPCR using the probes.

[0248] The limit of detection is up to 10 (for M. tuberculosis var BCG) based on early studies performed by the inventors. -5 of dilution (on a bank roughly calibrated by optical density).

[0249] Considering the sensitivity and rapidity of qPCR, which is also cheaper than -dPCR, qPCR was selected by the inventors for the next step. Moreover, this technique meets the requirements of Good Manufacturing Practice (GMP).

[0250] Example 3: Verification of comprehensiveness. For five species, namely M. genavense, M. stomatepiae, M. hassiacum, M. thermoresistible and M. heraklionense, there are mismatches between one of the primers and the rrs gene sequence of these species. Given the location of these mismatches on the primers and the fact that the mismatches relate to the primers and not the probe, it is presumed that such mismatches should not prevent amplification.

[0251] We confirmed this by using synthetic rrs genes corresponding to each of these five species to test three sets of primers.

[0252] The results obtained by the inventors confirmed that F1-1&R1-1 and / or F4-1&R4-1 are able to amplify these five rrs genes. These results are reported in Table 4, where "NO*" denotes in vitro amplification, not amplification based on in silico studies.

[0253] Thus, the combination of the three primer pairs disclosed in Example 2 allows the amplification of the rrs gene of any mycobacterium.

[0254] Example 4: Validation of multiplex amplification. In a first step, we performed duplex amplification with or without an internal control (IC), i.e. the internal control of the Diagenode kit (spiked with 10 TCID50) or the internal control GFP plasmid (10 per 5 μL). 6 We prepared samples of M. tuberculosis var BCG at different concentrations, either spiked with GFP or with 100 copies of the plasmid. Both internal controls tested performed well (data not shown), but for further work we used the GFP IC.

[0255] The different samples (including different concentrations of DNA extracted from M. tuberculosis var BCG as an internal control) were analyzed by qPCR under the following conditions: - simplex: only the primer pair F1-1&R1-1 for amplifying the rrs gene or only the primer pair for amplifying the internal control with the appropriate probe, and - Duplex: primer pair F1-1&R1-1 and primer pair IC simultaneously with the appropriate probe.

[0256] The results obtained show that it is possible to simultaneously detect both the target rrs gene and the internal control. As shown in Figure 1A and Figure 1B, there is no difference in the detection of the target rrs gene or the internal control between the simplex and duplex setups.

[0257] As can be seen from these figures, there is no fluorescence interference between the probe used for the rrs genes (fluorophore FAM) and the probe used for the internal control (fluorophore Cy5) because the wavelengths are sufficiently different, i.e., ≤ 100 Hz. FAM: Excitation at 490 nm / Emission at 520 nm Cy5: excitation at 650 nm / emission at 670 nm.

[0258] To confirm the above results, the experiment was replicated using three different primer pairs and associated probes.

[0259] Mycobacterium tuberculosis (M. tuberculosis) var BCG 10 5 , 10 4 and 10 3 Mycobacteria extract / mL, GFP plasmid at 10 5 The same was done with M. hiberniae DNA at concentrations of 5.3 pg / μL and 5.3 fg / μL.

[0260] Using qPCR, the three systems disclosed in Example 2 (primers and corresponding probes) as well as the primers and probes of the GFP plasmid were tested first in simplex reactions and then in duplex.

[0261] We then concluded that simplex or duplex amplification does not alter the results obtained by qPCR.

[0262] Multiplex detection We first tested duplex qPCR amplification with targeted M. tuberculosis var BCG and both the first system (F1-1, R1-1 and S1-0) and the fourth system (F4-1, R4-1 and S4-0).

[0263] The results show that duplex qPCR is possible and allows for better detection of mycobacteria. Indeed, these detection systems, namely the first and fourth systems, amplify separate regions of the rrs gene so that there is no competition between the primers for the target DNA. The other components of the master mix (e.g. dNTPs, polymerase, etc.) are in excess, which also ensures that there is no competition between the primers for other resources. As long as the probes of both systems are detected by the same fluorophore, the signal is even amplified, since the amplified sequence increases by two-fold. Signal amplification is expected for all species likely to be amplified by at least two of the three systems disclosed in Example 2.

[0264] We then added an internal control, i.e., GFP internal control, together with the relevant primers and probes and performed qPCR in triplex. The results obtained show that the triplex reaction does not adversely affect the detection of the internal control. Similarly, the detection of the targeted rrs gene is uniform.

[0265] Therefore, we tested quadrupleplex qPCR. In this regard, four different reactions were performed: - 3 duplex reactions, corresponding to one of the three systems (System 1, System 3 or System 4) and an internal control (GFP plasmid) and the associated primers and probes; - 1 Quadruplex with 3 systems and internal control.

[0266] By comparing side-by-side the results obtained for each system, either in combination with the internal control alone or with the internal control and two additional systems, it can be concluded that quadruple amplification gives satisfactory results with sufficient sensitivity and specificity. Indeed, as can be seen in Figure 2, in each system (first system: Figure 2A; third system Figure 2B and fourth system Figure 2C), the detection of the target sequence is either earlier with respect to the duplex detection with the control alone or amplified in quadrupleplex.

[0267] Example 5: Validation of multiplex amplification for representative species. The detection method disclosed in Example 2 was then experimentally validated by the inventors for mycobacteria in quadruplex conditions as detailed in Example 4.

[0268] The 12 species / strains selected for the development and validation of the detection method are listed in Example 1, Table 1. They represent samples of different genera included in mycobacteria (according to previous classification, the single genus Mycobacterium is now reconstructed according to the latest classification, but without changing the bacteria considered as mycobacteria), in fact, four out of five different mycobacterial genera are represented, namely Mycobacteroides, Mycolicibacterium, Mycolicibacter and Mycobacterium. Mycobacillus is not represented, since it includes only three unconventional mycobacteria with very low occurrence.

[0269] M. xenopi and M. chelonae were available in insufficient quantities for this experiment, and we used synthetic rrs genes encoding the 16S rRNA from these species. For M. hiberniae, we worked with DNA already extracted from this mycobacterium for safety reasons. All strains, including those represented only by DNA, had been extracted with one of the kits described in Example 1 and then samples were spiked with the internal control (10 per 5 μL). 5 Spike in 10 copies of the GFP plasmid.

[0270] The samples are then analyzed by qPCR using commercially available kits in quadrupleplex.

[0271] For each test strain, a standard range is obtained for quantifying the extracted samples to define the detection limit of the test. This standard range is calibrated in pg / μL of DNA and is obtained with 6 points and 3 replicates. The margin of error must be less than 0.1 and the linear regression coefficient must be as close as possible to 1.

[0272] To estimate the detection limit for each strain, we first analyzed each strain extract by qPCR undiluted and then diluted it at 10 -3 Dilute to .

[0273] The results are shown in FIG. 3 (FIG. 3A: slow growing mycobacteria; FIG. 3B: fast growing mycobacteria).

[0274] For a given dilution to be considered "quantifiable" in quadrupleplex conditions, both replicates must be similar (less than 0.8 Cp difference) and the result in terms of Cp must match the previous dilution, i.e., 3.3 Cp away from the previous dilution.

[0275] Cp means "crossover point", also called Cq, and is the number of amplification cycles above which amplification is considered significant, i.e. above a threshold level, which is indicated by a line in Figures 1, 2 and 3.

[0276] Table 5 summarizes the minimal amount of DNA amplified for each strain by the Quadruplex detection system. This is not the limit of detection of the method, but rather evidence that the method is suitable for detecting very small amounts of DNA for each species.

[0277] [Table 10]

[0278] In conclusion, we have shown that using standard extraction conditions, the detection test detailed in Example 2 is able to detect targeted mycobacteria as well as the internal control within quadrupleplex pools.

[0279] Sensitivity is included between 0.03 and 0.005 fg / μL for all strains except M. hiberniae (0.26 fg / μL).

[0280] Conclusions of Examples 2 to 5: The inventors have demonstrated that a qPCR-based technique provides a suitable test for detecting the presence of mycobacteria without culture using the primers, probes and conditions disclosed in these examples. This technique provides results faster than culture-based compendial methods, as it provides results in 2 days or even fewer days. Thus, the time to result can be reduced by up to 96% versus compendial methods.

[0281] qPCR is also a technology that is compatible with GMP environments.

[0282] The above results further demonstrate that the detection of mycobacteria is comprehensive, as demonstrated in silico and with a proof of concept having a representative number of mycobacteria.

[0283] The test detailed in the Examples is particularly efficient as it allows the simultaneous detection of the three systems and an internal control.

[0284] The specificity of detection is also demonstrated by alignments with other bacterial sequences, this specificity being provided by the probe.

[0285] The sensitivity of this method can be further improved by optimizing the extraction process and qPCR conditions.

[0286] Finally, although the proof of concept has been established with qPCR, we have started to test with other techniques such as dPCR, which may also be applicable for detection, especially since the comprehensiveness and specificity depend on the primer and probe design and not on the amplification method itself.

[0287] Example 6: Mycobacteria detection test Taking into account the results obtained in Examples 1 to 5, the inventors have therefore defined a suitable method for the detection of mycobacteria in a sample by qPCR, which is both comprehensive and specific.

[0288] This method is capable of detecting indeed any mycobacteria from any of the following five genera: - Mycolicidbacterium - Mycobacteroides - Mycolicibacter - Mycobacterium - Mycobacterium This represents a total of 200 and 217 species, subspecies or variants (see Table 4). The new classification does not affect the number of mycobacteria.

[0289] This method relies on three different primer pairs and associated probes designed as detailed in Example 2 to amplify regions of the sequence of the rrs gene of any mycobacterium. The three different primer pairs or systems target different regions of the rrs gene. The three associated probes are coupled to a FAM fluorophore.

[0290] A fourth primer pair and associated probe is used to detect an internal exogenous control, the control probe being coupled to another fluorophore different from Cy5 or FAM.

[0291] The internal exogenous control can be any DNA sequence already extracted or present in a microorganism unrelated to the mycobacteria to be detected. The control is added to the sample specifically at the extraction step. It makes it possible to control this extraction step in the absence of mycobacteria amplification and ensures that this is not linked to a faulty extraction step.

[0292] All primers, i.e., all four primer pairs, and all probes, i.e., all four probes, are mixed together in a single reaction mixture for PCR. Thus, a single PCR reaction is sufficient to detect all mycobacteria potentially present in the sample, corresponding to quadruple-plex detection.

[0293] Primers F1-1, R1-1, F3-1, R3-1, F4-1 and R4-1 are those disclosed in Table 2 of Example 1.

[0294] Primers and probes appropriate for the internal control are also added; the fluorophore attached to the probe detecting the internal control must be different from the FAM fluorophore in order to distinguish between amplification from systems 1, 3 and 4, which indicate the presence of mycobacteria, and amplification from the control system, which indicates the presence of control.

[0295] For qPCR, the reaction mixture is prepared according to the following, depending on the number of samples to be analyzed and the volume required for each well (20 µL - 50 µL): - Commercially available master mix qPCR with probes: TaqPolymerase, dNTPs, Mg2+, buffers (non-exhaustive list), - Primers at a concentration of 100nM to 400nM - Probe at a final concentration of 50nM to 250nM - primers and probes adapted for internal control of extraction - Nuclease-free water qsp

[0296] The composition of the master mix and the concentrations of primers and probes can be adjusted to improve the sensitivity or specificity of detection.

[0297] The reaction mixture is then placed into the wells of a 96-well plate, and then the samples are added. The plate is photographed, centrifuged, and inserted into a thermocycler.

[0298] The number of cycles should be adapted depending on the enzymes present in the mixture. The hybridization temperature is defined according to the fusion temperature of the primer and the probe.

[0299] Positive and negative controls for extraction and qPCR are advantageously added.

[0300] Example 7: Variations in primer and probe design In addition to demonstrating in the previous examples that a defined set of primers and probes can specifically and comprehensively detect all mycobacterial rrs genes, the inventors have demonstrated that variations of the primers and probes can be designed without losing the specificity and comprehensiveness of the initial primers and probes.

[0301] Therefore, the inventors have studied in silico various variations, more specifically the addition or suppression of 1 to 4 bases at each end. This study aims to define the influence of these modifications on the specificity and coverage of the initial primers and probes. In this context, primer design software such as Primer Expresss and international databases such as PrimerBlast have been utilized. The inventors have further tested combinations of primer variants and probes.

[0302] Materials and Methods As an initial step, 217 rrs gene sequences of mycobacterial origin, corresponding to a total of 200 different species and 217 species, subspecies or variants, were aligned with the software Geneious (10.2.6) and the program ClustalW, positioning the three primer pairs identified in Example 2, namely F1-1&R1-1, F3-1&R3-1 and F4-1&R4-1.

[0303] The modifications that may be introduced into the primers without compromising the comprehensiveness are defined based on this alignment diagram. For the probe, the permissible modifications are much less extensive, insofar as the probe is necessarily based on the sequence amplified by the primers and is therefore limited to the sequence between the primer sequences (one or two bases apart). This emphasizes that the sequence of the primer pair is closely linked to the sequence of the associated probe.

[0304] Primer Variation Design: Primer variations are first designed on the Primer Blast data using the program Primer3. They are named F1-2, F1-3, etc. for primer F1, with F1-1, R1-1, F3-1, R3-1, F4-1 and R4-1 corresponding to the initial design. For each variation, the GC content (percentage) and Tm (temperature melting) are calculated. These variations are then analyzed using the software Primer Express and set the probe MGB (Minor Groove Binder), i.e. quantification using the same software as the initial primers for consistency.

[0305] For each primer variation tested, the following parameters / criteria set by the program should be met in order for the variation to be retained: - Primer Tm (melting temperature) must be between 58°C and 60°C; - The probe Tm (melting temperature) must be between 68°C and 71°C; - The GC content should be between 40% and 70%, in particular around 50%.

[0306] Furthermore, the difference in Tm within one primer pair should not exceed 2°C, and the difference in Tm between the probe and primer should be approximately 10°C.

[0307] In a first step, based on this criterion, different primer pairs were tested using the relevant initial probes, namely S1-0, S3-0 and S4-0 without variations.

[0308] In a second step, the probe variations are then tested in combination with the primer (variation) pairs retained and selected in the first step.

[0309] Two different types of results ultimately held: - Primer and probe combinations that fully fulfill all criteria as the initial design of primers and probes does. These combinations should be adapted for selective and exhaustive amplification as demonstrated for the initial design. - combinations in which one requirement is not fully met, e.g. primer Tm comprised between 57°C and 58°C or between 60°C and 61°C, i.e. primer Tm differing from the set criteria by less than 1°C; other criteria are fully met. These combinations are also expected to be fully suitable for the intended purpose.

[0310] Specificity analysis: Once the different combinations are retained, based on the criteria and selections above, the specificity of the different primer pairs and associated probes is tested to ensure that specificity is not lost.

[0311] result: 1. Primer combination Various variations on the primer sequences are detailed in Table 6 below, specifying modifications at the 5' and 3' ends with respect to the initial design.

[0312] [Table 11]

[0313] For each system amplifying a different region of the rrs gene, namely System 1, System 3 and System 4, each variation of the forward primer is tested with each mutation of the reverse primer including the original design using PrimerExpress software.

[0314] Therefore, F1-1 is tested independently of R1-1 (initial pair, no variation), R1-2, R1-3, R1-4, R1-5 and R1-6. Similarly for F1-2, F1-3 and F1-4.

[0315] The same combination strategy applies to systems 3 and 4.

[0316] In this regard, it is emphasized that a given variation of the forward primer may indeed be suitable in combination with a particular variation of the reverse primer, and inappropriate in combination with another variation of the reverse primer, which is why the forward and reverse primer variations are tested in pairs.

[0317] The tables below (Tables 7, 8 and 9) report for each system the combinations where all criteria are fulfilled (++), the combinations where only one parameter does not fully fulfill one of the criteria but the difference remains acceptable (+), the combinations where the Tm typically differs by 1°C from the target Tm and the combinations where one or more of the criteria are not fulfilled (-), representing less preferred primer sequences.

[0318] [Table 12]

[0319] [Table 13]

[0320] [Table 14]

[0321] 2. Probe combination The variations of probes S1-0, S3-0 and S4-0 tested are reported in Table 10 and contain modifications at the 5' and 3' ends.

[0322] [Table 15]

[0323] Different associations have been made between the primer combinations detailed in Tables 7-9 with results of (++) or (+) and the associated probes and variations in Table 10.

[0324] The following tables (Tables 11 to 13) report for each system the associations where all criteria are met (++), the associations where one parameter does not fully meet one of the criteria but the difference remains acceptable (+) and the associations where one or more of the criteria are not met (-), representing a less favorable association.

[0325] [Table 16]

[0326] [Table 17]

[0327] [Table 18]

[0328] 3. Specificity We used the tool Primer BLAST to check whether the retained primer combinations could amplify sequences other than mycobacterial sequences in the NCBI database.

[0329] System 1 For combinations 6, 5, 7, 11 and 18 detailed above, primer BLAST results show the same specificity as combination 1 (corresponding to the original primers), therefore these variations do not impair specificity.

[0330] System 3: For combinations 9, 2, 3, 10, 11 and 12 detailed above, primer BLAST results show the same specificity as combination 1 (corresponding to the original primers), therefore these variations do not impair specificity.

[0331] System 4: In this system, it has already been shown that specificity is provided by probe S4-0 for the primer pair disclosed in Example 2. In order to maintain this specificity provided by the probe, we have come to the conclusion that the 5' end of the probe cannot be shortened, i.e. S4-2 and S4-3 are not acceptable, and therefore only variations corresponding to S4-1 are retained.

[0332] For the combinations 13, 25, 3, 15, 27 and 30 detailed above, primer BLAST results show the same specificity as combination 1 (corresponding to the original primers), therefore these variations do not impair specificity.

[0333] Combination 5 amplifies 13 additional species not of mycobacterial origin, but relative to probe S4-0 (initial design), specificity can be preserved.

[0334] Therefore, these results are F1-1&R1-1 with probe S1-0 F3-1&R3-1 with probe S3-0 F4-1&R4-1 with probe S4-0 Not only have three primer pairs corresponding to and associated probes demonstrated their ability to specifically and comprehensively detect mycobacteria by PCR-based amplification methods with probe detection, but the same ability is shared by slight variations of the primers and probes.

[0335] Example 8: Further validation of specificity. The specificity of the methods disclosed in Examples 4 and 5 demonstrated in Example 2, especially with respect to other non-mycobacterial bacteria and in the presence of matrix, has been verified by the inventors.

[0336] 8.1. Testing on other species. Four other species are selected to evaluate the specificity of qPCR using primers and probes F1-1, R1-1, S1-0, F3-1, R3-1, S3-0, F4-1, R4-1 and S4-0 in the conditions detailed in Examples 4 and 5. The species selected by the inventors in this regard are from the genus Mycobacterium (class Actinomyces), namely: Nocardia asteroides ·Cutibacterium acnes Corynebacterium diphteriae Dietzia papillomatosis It is a species that is thought to be very close phylogenetically to.

[0337] Each strain was characterized at a viable concentration of 100 CFU / mL.

[0338] The mycobacteria qPCR test is performed simultaneously and under the same experimental conditions against the mycobacterial strains M. tuberculosis var BCG strain (positive control) and different Actinomyces strains.

[0339] The results obtained for N. asteroides at 100 CFU / mL and D. papillomatosis at 100 CFU / mL are shown in Figures 4A and 4B, respectively. Comparable results were obtained with C. acnes at 100 CFU / mL and C. diphteriae at 100 CFU / mL.

[0340] These results show specific detection of the Mycobacteria strain, while no detection was found for the other four Actinomycetes strains at cycle numbers between 37 and 40, depending on the strain. Thus, in the experimental conditions of this test, the cycle threshold is approximately 37 cycles.

[0341] These results demonstrate that qPCR using the primers and probes designed by the inventors is specific for the detection of mycobacterial strains.

[0342] Comparison with official assays: To compare the specificity of the qPCR method of the present invention with the culture-based standard assay, the standard assay is performed with the same strains and concentrations. Positive results are obtained in spiked liquid medium for all four Actinomycetales strains.

[0343] N. asteroides, C. acnes, C. diphteriae, and D. papillomatosis are detected by the mycobacteria test in culture (corresponding to the official assay), but are not detected by the qPCR of the present invention ("qPCR Mycobacteria"), which specifically detects mycobacteria.

[0344] The method according to the invention therefore allows for a specific detection, more specific than the classical methods showing culture.

[0345] 8.2. Testing on the matrix. The qPCR mycobacteria according to the present invention can be used for pharmaceutical testing.The inventors have actually verified that this technique can be carried out in different matrices, and have confirmed both the absence of non-specific cross-detection and the absence of PCR inhibition by matrix components.Therefore, the qPCR mycobacteria technique can be carried out for pharmaceutical testing.Therefore, the qPCR mycobacteria technique can be carried out in the process of producing biopharmaceuticals as a step of testing for mycobacteria contamination.

[0346] The matrices tested by the inventors are as follows: - Supernatant of CHO cells producing recombinant cytomegalovirus protein antigen; - HAV Hepatitis A virus crude harvest produced on MRC5 cells; - Yellow fever virus crude harvest produced in Vero cells; - Crude rabies virus produced on Vero cells.

[0347] The mycobacteria qPCR test was performed at 10 5 ~10 viable mycobacteria / mL or two concentrations of 10 5 (to simulate a high level of contamination) and 10 (to simulate a low level of contamination) viable mycobacteria / mL using primers and probes F1-1, R1-1, S1-0, F3-1, R3-1, S3-0, F4-1, R4-1 and S4-0.

[0348] The results obtained were similar for the matrix and PBS samples, some of which are shown in Figures 5A, 5B and 5C.

[0349] Conclusion: The qPCR Mycobacteria according to the invention allows the detection of M. tuberculosis var BCG on different matrices. This ensures that the matrix does not induce PCR inhibition and there is no non-specific amplification on non-spiked matrices. Therefore, the qPCR according to the invention is suitable for pharmaceutical testing at any stage of pharmaceutical production, especially on the final product, any intermediate matrix, virus seeds and starting materials such as cell cultures or media.

[0350] Example 9: Demonstration of qPCR sensitivity.

[0351] [Table 19]

[0352] 9.2. Matrices tested For M. tuberculosis var BCG, M. avium, M. hiberniae, M. xenopi, and M. phlei: >10 in media containing serum and antibiotics, molecules known to interfere with nucleic acid-based assays. 6 The high concentration of Vero cell suspensions in cells / mL represents the "worst case scenario" likely to be encountered in pharmaceutical manufacturing.

[0353] For M. flavescens, M. fortuitum, M. abscessus, and M. kansasii: Vero yellow fever virus crude harvest matrix

[0354] 9.3. Method. The dilution range for each strain detailed in Section 9.1 is performed in PBS with a maximum of 10 or 1 viable mycobacteria per mL (depending on the strain tested). Spike the appropriate dilution of mycobacterial strain into the sample containing matrix (see 9.2) according to Table 14.

[0355] [Table 20]

[0356] The volume of the sample analyzed is 1.5 mL. Considering the extraction volume and the fact that the entire extraction product is analyzed by qPCR, there are 6 determinations for one sample. If one of the 6 determinations is positive (significant amplification), the sample is considered positive for the presence of a mycobacterial strain.

[0357] 9.4.Results M. tuberculosis var BCG with 1 viable mycobacteria / mL: 4 / 6 measurements show positive and significant PCR amplification (i.e. above the threshold at approximately 37-40 PCR cycles, in contrast to the negative (non-spiked) control), see Figure 6A. The test is therefore positive for this concentration, with 1 viable mycobacteria / mL constituting the limit of detection.

[0358] 1 viable mycobacteria / mL of M. avium: 4 / 6 measurements show positive and significant PCR amplification (i.e. above the threshold at approximately 37-40 PCR cycles, in contrast to the negative (non-spiked) control). The test is positive for this concentration, with 1 viable mycobacteria / mL constituting the limit of detection.

[0359] 1 viable mycobacteria / mL of M. hiberniae: 6 / 6 measurements show positive and significant PCR amplification (i.e., above the threshold at approximately 37-40 PCR cycles, in contrast to the negative (unspiked) control). See Figure 6B. The test is positive for this concentration, with 1 viable mycobacteria / mL constituting the limit of detection.

[0360] 0.1 viable mycobacteria / mL of M. xenopi: 6 / 6 measurements show positive and significant PCR amplification (i.e. above the threshold at approximately 37-40 PCR cycles, in contrast to the negative (unspiked) control). The test is positive for this concentration, with 0.1 viable mycobacteria / mL constituting the limit of detection.

[0361] 0.1 viable mycobacteria / mL of M. phlei: 6 / 6 measurements show positive and significant PCR amplification (i.e., above the threshold at approximately 37-40 PCR cycles, in contrast to the negative (non-spiked) control), see Figure 6C. The test is positive for this concentration, with 0.1 viable mycobacteria / mL constituting the limit of detection.

[0362] 10 viable mycobacteria / mL of M. flavescens: 4 / 6 measurements have positive and significant PCR amplification (i.e. above the threshold at approximately 37-40 PCR cycles, in contrast to the negative (non-spiked) control). The test is positive for this concentration, with 10 viable mycobacteria / mL constituting the limit of detection.

[0363] 10 viable mycobacteria / mL of M. fortuitum: 3 / 6 measurements have positive and significant PCR amplification (i.e., above the threshold at approximately 37-40 PCR cycles, in contrast to the negative (non-spiked) control). The test is positive for this concentration, with 10 viable mycobacteria / mL constituting the limit of detection.

[0364] 10 viable mycobacteria / mL of M. abscessus: 2 / 6 measurements have positive and significant PCR amplification (i.e., above the threshold at approximately 37-40 PCR cycles, in contrast to the negative (unspiked) control). The test is positive for this concentration, with 10 viable mycobacteria / mL constituting the limit of detection.

[0365] 10 viable mycobacteria / mL of M. kansasii: 5 / 6 measurements have positive and significant PCR amplification (i.e., above the threshold at approximately 37-40 PCR cycles, in contrast to the negative (unspiked) control). The test is positive for this concentration, with 10 viable mycobacteria / mL constituting the limit of detection.

[0366] 9.5. Conclusion The detection limit is 10 viable mycobacteria / mL for M. flavescens, M. fortuitum, M. abscessus and M. kansasii in complex matrices (crude harvest of Vero yellow fever virus).

[0367] The detection limit is 1 viable mycobacteria / mL for M. tuberculosis var BCG, M. avium and M. hiberniae in complex matrices (highly enriched Vero cell cultures).

[0368] The detection limit is 0.1 viable mycobacteria / mL for M. xenopi and M. phlei in complex matrices (highly enriched Vero cell cultures).

[0369] This confirms the very high sensitivity of the method, which allows the detection of representative mycobacterial strains at concentrations as low as 10 or 1 per mL, and even lower for some strains, even in complex matrices such as highly concentrated Vero cell cultures or viral crude harvest vaccines. Thus, the qPCR mycobacteria technique can be implemented in biopharmaceutical manufacturing processes as a step to test for mycobacterial contamination.

[0370] List of references: Bhatnagar, J. et al. (2017).Improved Detection and Accuracy of Mycobacterium Species Identification from Paraffin Embedded Tissues of Patients by Using Multigene Targeted PCR and Sequencing.Open Forum Infectious Diseases 4,S620-S621. Euzeby,J.P.(1997).List of Bacterial Names with Standing in Nomenclature:a Folder Available on the Internet.International Journal of Systematic Bacteriology,47,590-592. Gupta,R.S.,Lo,B.,and Son,J.(2018).Phylogenomics and Comparative Genomic Studies Robustly Support Division of the Genus Mycobacterium into an Emended Genus Mycobacterium and Four Novel Genera.Frontiers in Microbiology 9,67. Haig,S.-J.,Kotlarz,N.,LiPuma,J.J.,and Raskin,L.(2018).A High-Throughput Approach for Identification of Nontuberculous Mycobacteria in Drinking Water Reveals Relationship between Water Age and Mycobacterium avium.MBio 9.9:e02354-17. Meehan,C.J.,Barco,R.A.,Loh,Y.-H.E.,Cogneau,S.,and Rigouts,L.(2021).Reconstituting the genus Mycobacterium.International Journal of Systematic and Evolutionary Microbiology 71:004922. Parte,A.C.(2014).LPSN-List of Prokaryotic names with Standing in Nomenclature.Nucleic Acids Research,42,Issue D1,D613-D616 Parte,A.C.(2018).LPSN-List of Prokaryotic names with Standing in Nomenclature(bacterio.net),20 years on.International Journal of Systematic and Evolutionary Microbiology,68,1825-1829. Parte,A.C.,et al.(2020).List of Prokaryotic names with Standing in Nomenclature(LPSN)moves to the DSMZ.International Journal of Systematic and Evolutionary Microbiology,70,5607-5612. Riojas,M.A.et al.(2018).Phylogenomic analysis of the species of the Mycobacterium tuberculosis complex demonstrates that Mycobacterium africanum,Mycobacterium bovis,Mycobacterium caprae,Mycobacterium microti and Mycobacterium pinnipedii are later heterotypic synonyms of Mycobacterium tuberculosis.Int J Syst Evol Microbiol.68(1):324-332. Tortoli E,Brown-Elliott BA,Chalmers JD,et al.(2019)Same meat,different gravv:ignore the new names of mycobacteria.Eur Respir J 2019;54.

Claims

1. A set of primers for amplifying the sequence of any mycobacterial rrs gene present in a sample by polymerase chain reaction (PCR), comprising at least three of the following primer pairs: The first pair: Sequence: 5'-CCTGGTAGTCCAGCCCGTAA-3' (Sequence ID 1) or a forward primer F1 having a modification thereof. Sequence: 5'-CGGATCCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 2) or a reverse primer R1 having a modification thereof; The second pair: Sequence: 5'-CACAGGACGCCGGTAGAT-3' (SEQ ID NO: 4) or forward primer F3 having a modification thereof. Sequence: 5'-CATGCACCACCCTGCACACA-3' (SEQ ID NO: 5) or a modified version thereof, reverse primer R3; The third pair: Sequence: 5'-CCCTTAGTTCCAGGGGCTTTCA-3' (Sequence ID 7) or forward primer F4 having a modification thereof. Sequence: 5'-GGCATCGCAGCCCTTTG-3' (Sequence ID 8) or a modified version thereof Reverse primer R4 Includes, The modifications include the addition and / or deletion of 1 to 4 nucleotides at the 3' and / or 5' ends, and / or the substitution of 1 or 2 nucleotides. The three primer pairs described above are a set of primers that enable amplification of the sequence from the rrs gene of any mycobacterial species.

2. The first primer pair is, 1) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-1: 5'-CGGATCCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 2); 2) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-6:5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13); 3) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-5:5'-ACGGATCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 12); 4) Forward primer F1-2: 5'-CCTGGTAGTCCAGCCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-1: 5'-CGGATCCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 2); 5) Forward primer F1-2: 5'-CCTGGTAGTCCAGCCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-3: 5'-CACGGATCCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 27); 6) Forward primer F1-2: 5'-CCTGGTAGTCCAGCCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-5:5'-ACGGATCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 12); 7) Forward primer F1-2: 5'-CCTGGTAGTCCAGCCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-6:5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13); and 8) Forward primer F1-3: 5'-CTGGTAGTCCACGCCGTAAAA-3' (SEQ ID NO: 11) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13) Selected from, The aforementioned second primer pair is 1) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAT-3' (SEQ ID NO: 4) Reverse primer R3-1:5'-CATGCACCACCCTGCACACA-3' (SEQ ID NO: 5); 2) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-1:5'-CATGCACCACCCTGCACACA-3' (SEQ ID NO: 5); 3) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAT-3' (SEQ ID NO: 4) Reverse primer R3-2:5'-CATGCACCACCCTGCACACAG-3' (SEQ ID NO: 15); 4) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAT-3' (SEQ ID NO: 4) Reverse primer R3-3:5'-ATGCACCACCTGGCACACAG-3' (SEQ ID NO: 16); 5) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-2:5'-CATGCACCACCCTGCACACAG-3' (SEQ ID NO: 15); 6) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-3:5'-ATGCACCACCTGGCACACAG-3' (SEQ ID NO: 16); 7) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) Reverse primer R3-4:5'-TGCAACCACCTGCAACACAG-3' (SEQ ID NO: 17), and 8) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-4:5'-TGCAACCACCTGCAACACAG-3' (SEQ ID NO: 17) Selected from, and / or The third primer pair described above is 1) Forward primer F4-1: 5'-CCCTTAGTTCCAGGGGCTTCCA-3' (SEQ ID NO: 7), Reverse primer R4-1:5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); 2) Forward primer F4-3: 5'-CCCCTTATGTCCCAGGGGCTTC-3' (SEQ ID NO: 18), Reverse primer R4-1:5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); 3) Forward primer F4-5: 5'-GCCCCTTAGTCCCAGGGC-3' (SEQ ID NO: 19), Reverse primer R4-1:5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); 4) Forward primer F4-1: 5'-CCCTTAGTTCCAGGGCTTCA-3' (SEQ ID NO: 7), Reverse primer R4-3:5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); 5) Forward primer F4-1: 5'-CCCTTAGTTCCAGGGCTTCA-3' (SEQ ID NO: 7), Reverse primer R4-5:5'-GCATGCCAGCCCTTTTGTA-3' (SEQ ID NO: 28); 6) Forward primer F4-3: 5'-CCCCTTATGTCCCAGGGGCTTC-3' (SEQ ID NO: 18), Reverse primer R4-3:5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); 7) Forward primer F4-5: 5'-GCCCCTTAGTCCCAGGGC-3' (SEQ ID NO: 19), Reverse primer R4-3:5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); and 8) Forward primer F4-5: 5'-GCCCCTTAGTCCCAGGGC-3' (SEQ ID NO: 19), Reverse primer R4-6: 5'-GGCATCGCAGCCCTTTTGTA-3' (SEQ ID NO: 21) A set of primers according to claim 1, selected from the following.

3. A set of primers, - The first primer pair is, Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) and Reverse primer R1-1: 5'-CGGATCCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 2) And, - The second primer pair described above is Forward primer F3-1: 5'-CACAGGACGCCGGTAGAGAT-3' (SEQ ID NO: 4) and Reverse primer R3-1:5'-CATGCACCACCCTGCACACA-3' (SEQ ID NO: 5) And, - The third primer pair described above is Forward primer F4-1:5'-CCCTTAGTTCCAGGGGCTTCCA-3' (SEQ ID NO: 7), and Reverse primer R4-1:5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8) The set of primers according to claim 1.

4. A kit for amplifying the mycobacterial rrs gene sequence in a sample by PCR, comprising a set of primers according to any one of claims 1 to 3.

5. This is for detecting the mycobacterial rrs gene sequence amplified by PCR in a sample, and the following related probes are used to detect the amplification product: - A probe S1 having the sequence 5'-CGGTGGGTACTAGGTGTG-3' (SEQ ID NO: 3) or a modification thereof, for detecting the sequence amplified by the primers F1 and R1; - A probe S3 having the sequence: 5'-TCGGTTCCCCTTGTGGGC-3' (SEQ ID NO: 6) or a modification thereof, for detecting the sequence amplified by the primers F3 and R3; - A probe S4 having the sequence: 5'-ACATGCTACAATGGCCGG-3' (SEQ ID NO: 9) or a modification thereof, for detecting the sequence amplified by the primers F4 and R4. The kit according to claim 4, further comprising, wherein the modification is the addition and / or deletion of 1-2 nucleotides at the 3' and / or 5' ends.

6. The aforementioned probe a) Regarding the probe S1, Probe S1-0: 5'-CGGTGGGTACTAGGTGTG-3' (Sequence ID 3), Probe S1-1: 5'-CGGTGGGTACTAGGTGT-3' (SEQ ID NO: 22), and Probe S1-2: 5'-CGGTGGGTACTAGGTG-3' (Sequence ID 23) A group consisting of, When the probe S1 is S1-1, the first primer pair is 1) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-1: 5'-CGGATCCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 2); 2) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-6:5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13); 3) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-5:5'-ACGGATCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 12); 4) Forward primer F1-2: 5'-CCTGGTAGTCCAGCCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-1:5'-CGGATCCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 2); and 5) Forward primer F1-3: 5'-CTGGTAGTCCACGCCGTAAAA-3' (SEQ ID NO: 11) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13) Selected from, When the probe S1 is S1-2, the first primer pair is 1) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-1: 5'-CGGATCCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 2); 2) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-6:5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13); 3) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-5:5'-ACGGATCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 12); 4) Forward primer F1-2: 5'-CCTGGTAGTCCAGCCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-1: 5'-CGGATCCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 2); 5) Forward primer F1-2: 5'-CCTGGTAGTCCAGCCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-5:5'-ACGGATCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 12); and 6) Forward primer F1-3: 5'-CTGGTAGTCCACGCCGTAAAA-3' (SEQ ID NO: 11) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13) A group selected from; b) Regarding the probe S3, Probe S3-0: 5'-TCGGTTCCCCTTGTGGGC-3' (Sequence ID 6), Probe S3-1: 5'-ATCGGTTCCCTTGTGGGC-3' (SEQ ID NO: 24), and Probe S3-2: 5'-CGGTTCCCTTGTGGGC-3' (Sequence ID 25) A group consisting of, When the probe S3 is S3-1, the second primer pair is 1) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAT-3' (SEQ ID NO: 4) Reverse primer R3-1:5'-CATGCACCACCCTGCACACA-3' (SEQ ID NO: 5); 2) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-1:5'-CATGCACCACCCTGCACACA-3' (SEQ ID NO: 5); 3) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAT-3' (SEQ ID NO: 4) Reverse primer R3-2:5'-CATGCACCACCCTGCACACAG-3' (SEQ ID NO: 15); 4) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-2:5'-CATGCACCACCTGGCACACAG-3' (SEQ ID NO: 15); and 5) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-3: 5'-ATGCACCACCTGGCACACAG-3' (SEQ ID NO: 16) Selected from, When the probe S3 is S3-2, the second primer pair is 1) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAT-3' (SEQ ID NO: 4) Reverse primer R3-1:5'-CATGCACCACCCTGCACACA-3' (SEQ ID NO: 5); 2) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-1:5'-CATGCACCACCCTGCACACA-3' (SEQ ID NO: 5); 3) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAT-3' (SEQ ID NO: 4) Reverse primer R3-2:5'-CATGCACCACCCTGCACACAG-3' (SEQ ID NO: 15); 4) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAT-3' (SEQ ID NO: 4) Reverse primer R3-3:5'-ATGCACCACCTGGCACACAG-3' (SEQ ID NO: 16); 5) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-4:5'-TGCAACCACCTGCAACACAG-3' (SEQ ID NO: 17); and 6) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-3: 5'-ATGCACCACCTGGCACACAG-3' (SEQ ID NO: 16) A group selected from; c) Regarding the probe S4, Probe S4-0:5'-ACATGCTACAATGGCCGG-3' (SEQ ID NO: 9), and Probe S4-1: 5'-ACATGCTACAATGGCCCGGT-3' (Sequence ID 26) A group consisting of, When the probe S4 is S4-1, the third primer pair is 1) Forward primer F4-1: 5'-CCCTTAGTTCCAGGGGCTTCCA-3' (SEQ ID NO: 7), Reverse primer R4-1:5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); 2) Forward primer F4-3: 5'-CCCCTTATGTCCCAGGGGCTTC-3' (SEQ ID NO: 18), Reverse primer R4-1:5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); 3) Forward primer F4-5: 5'-GCCCCTTAGTCCCAGGGC-3' (SEQ ID NO: 19), Reverse primer R4-1:5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); 4) Forward primer F4-1: 5'-CCCTTAGTTCCAGGGCTTCA-3' (SEQ ID NO: 7), Reverse primer R4-3:5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); 5) Forward primer F4-3: 5'-CCCCTTATGTCCAGGGCTTC-3' (SEQ ID NO: 18), Reverse primer R4-3:5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); 6) Forward primer F4-5: 5'-GCCCCTTAGTCCCAGGGC-3' (SEQ ID NO: 19), Reverse primer R4-3:5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); and 7) Forward primer F4-5: 5'-GCCCCTTAGTCCCAGGGC-3' (SEQ ID NO: 19), Reverse primer R4-6: 5'-GGCATCGCAGCCCTTTTGTA-3' (SEQ ID NO: 21) A group selected from A kit according to claim 5, selected from the following.

7. The kit according to claim 4 for amplification by quantitative polymerase chain reaction (qPCR) or digital polymerase chain reaction (dPCR).

8. The kit according to claim 5 for amplification by quantitative polymerase chain reaction (qPCR) or digital polymerase chain reaction (dPCR).

9. The kit according to claim 6 for amplification by quantitative polymerase chain reaction (qPCR) or digital polymerase chain reaction (dPCR).

10. A method for amplifying and detecting the rrs gene sequence of any mycobacterium present in a sample, comprising the following steps performed simultaneously or sequentially: a) A step of performing in vitro PCR on nucleic acids extracted from the sample using at least a set of three primer pairs as described in any one of claims 1 to 3; b) A step to detect the presence or absence of amplification products. A method that includes this.

11. Claim 10, wherein the amplification step a) is performed by quantitative PCR or digital PCR. Methods used.

12. The detection step b) involves the following related probes: i) Probe S1, Probe S1-0: 5'-CGGTGGGTACTAGGTGTG-3' (Sequence ID 3), Probe S1-1: 5'-CGGTGGGTACTAGGTGT-3' (SEQ ID NO: 22), and Probe S1-2: 5'-CGGTGGGTACTAGGTG-3' (Sequence ID 23) Selected from the group consisting of, When the probe S1 is S1-1, the first primer pair is 1) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-1: 5'-CGGATCCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 2); 2) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-6:5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13); 3) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-5:5'-ACGGATCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 12); 4) Forward primer F1-2: 5'-CCTGGTAGTCCAGCCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-1:5'-CGGATCCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 2); and 5) Forward primer F1-3: 5'-CTGGTAGTCCACGCCGTAAAA-3' (SEQ ID NO: 11) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13) Selected from, When the probe S1 is S1-2, the first primer pair is 1) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-1: 5'-CGGATCCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 2); 2) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-6:5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13); 3) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-5:5'-ACGGATCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 12); 4) Forward primer F1-2: 5'-CCTGGTAGTCCAGCCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-1: 5'-CGGATCCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 2); 5) Forward Primer F1-2: 5'-CCTGGTAGTCCAGCCCGTAA A-3' (Sequence No. 10) Reverse primer R1-5:5'-ACGGATCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 12); and 6) Forward primer F1-3: 5'-CTGGTAGTCCACGCCGTAAAA-3' (SEQ ID NO: 11) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13) Probe S1 is selected from; ii) Probe S3, Probe S3-0: 5'-TCGGTTCCCCTTGTGGGC-3' (Sequence ID 6), Probe S3-1: 5'-ATCGGTTCCCTTGTGGGC-3' (SEQ ID NO: 24), and Probe S3-2: 5'-CGGTTCCCTTGTGGGC-3' (Sequence ID 25) Selected from the group consisting of, When the probe S3 is S3-1, the second primer pair is 1) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAT-3' (SEQ ID NO: 4) Reverse primer R3-1:5'-CATGCACCACCCTGCACACA-3' (SEQ ID NO: 5); 2) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-1:5'-CATGCACCACCCTGCACACA-3' (SEQ ID NO: 5); 3) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAT-3' (SEQ ID NO: 4) Reverse primer R3-2:5'-CATGCACCACCCTGCACACAG-3' (SEQ ID NO: 15); 4) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-2:5'-CATGCACCACCTGGCACACAG-3' (SEQ ID NO: 15); and 5) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-3: 5'-ATGCACCACCTGGCACACAG-3' (SEQ ID NO: 16) Selected from, When the probe S3 is S3-2, the second primer pair is 1) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAT-3' (SEQ ID NO: 4) Reverse primer R3-1:5'-CATGCACCACCCTGCACACA-3' (SEQ ID NO: 5); 2) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-1:5'-CATGCACCACCCTGCACACA-3' (SEQ ID NO: 5); 3) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAT-3' (SEQ ID NO: 4) Reverse primer R3-2:5'-CATGCACCACCCTGCACACAG-3' (SEQ ID NO: 15); 4) Forward Primer F3-1: 5'-CACAGGACGCCGGTAGAT -3' (Sequence No. 4) Reverse primer R3-3:5'-ATGCACCACCTGGCACACAG-3' (SEQ ID NO: 16); 5) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-4:5'-TGCAACCACCTGCAACACAG-3' (SEQ ID NO: 17); and 6) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-3: 5'-ATGCACCACCTGGCACACAG-3' (SEQ ID NO: 16) Selected from, probe S3; iii) Probe S4, Probe S4-0:5'-ACATGCTACAATGGCCGG-3' (SEQ ID NO: 9), and Probe S4-1: 5'-ACATGCTACAATGGCCCGGT-3' (Sequence ID 26) Selected from the group consisting of, When the probe S4 is S4-1, the third primer pair is 1) Forward primer F4-1: 5'-CCCTTAGTTCCAGGGGCTTCCA-3' (SEQ ID NO: 7), Reverse primer R4-1:5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); 2) Forward primer F4-3: 5'-CCCCTTATGTCCCAGGGGCTTC-3' (SEQ ID NO: 18), Reverse primer R4-1:5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); 3) Forward primer F4-5: 5'-GCCCCTTAGTCCCAGGGC-3' (SEQ ID NO: 19), Reverse primer R4-1:5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); 4) Forward primer F4-1: 5'-CCCTTAGTTCCAGGGCTTCA-3' (SEQ ID NO: 7), Reverse primer R4-3:5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); 5) Forward primer F4-3: 5'-CCCCTTATGTCCAGGGCTTC-3' (SEQ ID NO: 18), Reverse primer R4-3:5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); 6) Forward primer F4-5: 5'-GCCCCTTAGTCCCAGGGC-3' (SEQ ID NO: 19), Reverse primer R4-3:5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); and 7) Forward primer F4-5: 5'-GCCCCTTAGTCCCAGGGC-3' (SEQ ID NO: 19), Reverse primer R4-6: 5'-GGCATCGCAGCCCTTTTGTA-3' (SEQ ID NO: 21) Selected from, probe S4 The method according to claim 10, which is carried out using

13. The method according to claim 12, wherein the amplification step a) is carried out simultaneously in a single reaction mixture using the three primer pairs and the associated probe, and / or the amplification step a) is carried out by quantitative PCR or digital PCR.

14. The method according to claim 10, comprising an initial step of extracting the nucleic acid from the sample before performing step a).

15. A method for detecting the presence of mycobacterial contamination in pharmaceuticals without any culture step, comprising the following simultaneous steps: a) A step of in vitro amplification by quantitative PCR (qPCR) or dPCR performed on nucleic acids extracted from a sample of the product using at least three primer pairs according to any one of claims 1 to 3, wherein the primers hybridize to the rrs gene. b) The following probes: i. Probe S1, Probe S1-0: 5'-CGGTGGGTACTAGGTGTG-3' (Sequence ID 3), Probe S1-1: 5'-CGGTGGGTACTAGGTGT-3' (SEQ ID NO: 22), and Probe S1-2: 5'-CGGTGGGTACTAGGTG-3' (Sequence ID 23) Selected from the group consisting of, When the probe S1 is S1-1, the first primer pair is 1) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-1: 5'-CGGATCCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 2); 2) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-6:5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13); 3) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-5:5'-ACGGATCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 12); 4) Forward primer F1-2: 5'-CCTGGTAGTCCAGCCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-1:5'-CGGATCCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 2); and 5) Forward primer F1-3: 5'-CTGGTAGTCCACGCCGTAAAA-3' (SEQ ID NO: 11) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13) Selected from, When the probe S1 is S1-2, the first primer pair is 1) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-1: 5'-CGGATCCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 2); 2) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-6:5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13); 3) Forward primer F1-1: 5'-CCTGGTAGTCCAGCCCGTAA-3' (SEQ ID NO: 1) Reverse primer R1-5:5'-ACGGATCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 12); 4) Forward primer F1-2: 5'-CCTGGTAGTCCAGCCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-1: 5'-CGGATCCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 2); 5) Forward primer F1-2: 5'-CCTGGTAGTCCAGCCCGTAAA-3' (SEQ ID NO: 10) Reverse primer R1-5:5'-ACGGATCCCAAGGAAGGAAAAC-3' (SEQ ID NO: 12); and 6) Forward primer F1-3: 5'-CTGGTAGTCCACGCCGTAAAA-3' (SEQ ID NO: 11) Reverse primer R1-6: 5'-GGATCCCAAGGAAGGAAACC-3' (SEQ ID NO: 13) Probe S1 is selected from; ii. Probe S3, Probe S3-0: 5'-TCGGTTCCCCTTGTGGGC-3' (Sequence ID 6), Probe S3-1: 5'-ATCGGTTCCCTTGTGGGC-3' (SEQ ID NO: 24), and Probe S3-2: 5'-CGGTTCCCTTGTGGGC-3' (Sequence ID 25) Selected from the group consisting of, When the probe S3 is S3-1, the second primer pair is 1) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAT-3' (SEQ ID NO: 4) Reverse primer R3-1:5'-CATGCACCACCCTGCACACA-3' (SEQ ID NO: 5); 2) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-1:5'-CATGCACCACCCTGCACACA-3' (SEQ ID NO: 5); 3) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAT-3' (SEQ ID NO: 4) Reverse primer R3-2:5'-CATGCACCACCCTGCACACAG-3' (SEQ ID NO: 15); 4) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-2:5'-CATGCACCACCTGGCACACAG-3' (SEQ ID NO: 15); and 5) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-3: 5'-ATGCACCACCTGGCACACAG-3' (SEQ ID NO: 16) Selected from, When the probe S3 is S3-2, the second primer pair is 1) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAT-3' (SEQ ID NO: 4) Reverse primer R3-1:5'-CATGCACCACCCTGCACACA-3' (SEQ ID NO: 5); 2) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-1:5'-CATGCACCACCCTGCACACA-3' (SEQ ID NO: 5); 3) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAT-3' (SEQ ID NO: 4) Reverse primer R3-2:5'-CATGCACCACCCTGCACACAG-3' (SEQ ID NO: 15); 4) Forward primer F3-1: 5'-CACAGGACGCCGGTAGAT-3' (SEQ ID NO: 4) Reverse primer R3-3:5'-ATGCACCACCTGGCACACAG-3' (SEQ ID NO: 16); 5) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-4:5'-TGCAACCACCTGCAACACAG-3' (SEQ ID NO: 17); and 6) Forward primer F3-3: 5'-CACAGGACGCCGGTAGAGATA-3' (SEQ ID NO: 14) Reverse primer R3-3: 5'-ATGCACCACCTGGCACACAG-3' (SEQ ID NO: 16) Selected from, probe S3; iii. Probe S4, Probe S4-0:5'-ACATGCTACAATGGCCGG-3' (SEQ ID NO: 9), and Probe S4-1: 5'-ACATGCTACAATGGCCCGGT-3' (Sequence ID 26) Selected from the group consisting of, When the probe S4 is S4-1, the third primer pair is 1) Forward primer F4-1: 5'-CCCTTAGTTCCAGGGGCTTCCA-3' (SEQ ID NO: 7), Reverse primer R4-1:5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); 2) Forward primer F4-3: 5'-CCCCTTATGTCCCAGGGGCTTC-3' (SEQ ID NO: 18), Reverse primer R4-1:5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); 3) Forward primer F4-5: 5'-GCCCCTTAGTCCCAGGGC-3' (SEQ ID NO: 19), Reverse primer R4-1:5'-GGCATCGCAGCCCTTTG-3' (SEQ ID NO: 8); 4) Forward primer F4-1: 5'-CCCTTAGTTCCAGGGCTTCA-3' (SEQ ID NO: 7), Reverse primer R4-3:5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); 5) Forward primer F4-3: 5'-CCCCTTATGTCCAGGGCTTC-3' (SEQ ID NO: 18), Reverse primer R4-3:5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); 6) Forward primer F4-5: 5'-GCCCCTTAGTCCCAGGGC-3' (SEQ ID NO: 19), Reverse primer R4-3:5'-CGGCATCGCAGCCCTT-3' (SEQ ID NO: 20); and 7) Forward primer F4-5: 5'-GCCCCTTAGTCCCAGGGC-3' (SEQ ID NO: 19), Reverse primer R4-6: 5'-GGCATCGCAGCCCTTTTGTA-3' (SEQ ID NO: 21) Selected from, probe S4 Steps for detecting the presence or absence of amplification products A method that includes this.

16. A process for manufacturing a biopharmaceutical, comprising the step of testing for mycobacterial contamination by performing the method according to claim 10.

17. A process for manufacturing a biopharmaceutical, comprising the step of testing for mycobacterial contamination by performing the method according to claim 12.

18. A process for manufacturing a biopharmaceutical, comprising the step of testing for mycobacterial contamination by performing the method according to claim 15.

Images