Method for detecting microorganisms

Abstract

The present invention relates to a method for detecting a microorganism, such as Mycoplasma or mouse minute virus (MMV), in a sample, the method comprising: (i) performing a PCR on a sample suspected of containing Mycoplasma DNA with a PCR reaction mixture comprising (1) a first primer (P1) that anneals to the reverse complement of the nucleic acid sequence of SEQ ID NO: 1 and a second primer (P2) that anneals to the nucleic acid sequence of SEQ ID NO: 2, or (2) a third primer (P3) that anneals to the reverse complement of the nucleic acid sequence of SEQ ID NO: 109 and a fourth primer (P4) that anneals to the nucleic acid sequence of SEQ ID NO: 110; and (ii) detecting the PCR product, wherein detection of the PCR product indicates that the sample contains the microorganism, and absence of the PCR product indicates that the sample does not contain the microorganism. The present invention further relates to primer pairs for detecting Mycoplasma or a virus in a sample, and kits for detecting Mycoplasma or a virus in a sample.

Description

[0001] This invention relates to a method for detecting microorganisms, such as mycoplasma or mouse parvovirus (MMV), in a sample by PCR using specific primers and optional additional probes. The invention also provides primer pairs and kits for performing this method. Background Technology

[0002] Cell lines used to manufacture active pharmaceutical ingredients (APIs) intended for clinical trials or commercial use must be tested and cleaned of microbial contamination (e.g., mycoplasma or parvovirus) (see European Pharmacopoeia and ICH Guideline Q5 for viral safety). In addition to mandatory testing required by health authorities, the industry requires regular testing to protect facilities from contamination before introducing samples into production facilities, and to detect contamination early and protect downstream equipment. Several methods can be used to document the status of cell substrates in terms of microbial contamination (such as mycoplasma or viral contamination). These methods primarily include sample culture in media suitable for microbial growth and colony identification, and nucleic acid techniques (NATs) such as quantitative PCR (qPCR) and droplet digital PCR (ddPCR) for detecting bacterial or viral genetic material, for example, derived from mycoplasma or parvovirus. Due to the diversity of mycoplasma strains, for example, health authorities expect various assays to detect pathogen subgroups based on the incidence of the pathogen and its impact on human health.

[0003] Mouse minute virus (MMV), alternatively known as the Minute Virus of Mice, is a member of the Parvoviridae family and is a non-enveloped virus with a diameter of approximately 20 nm. MMV contains approximately 5 kb of single-stranded DNA and two genes: ns1 (non-structural protein 1) and vp1 (viral protein 1). Four MMV strains (i, p, m, and c) and seven genome sequences have been reported in the NCBI database. MMV is known to have contaminated the production processes of several companies through external sources. While MMV can infect humans, it is not considered a significant risk to operator and patient safety. However, the risks to the production process are significant, as MMV contamination could lead to supply disruptions to patients, product loss, costs of cleaning facilities and equipment, and potential disposal of expensive chromatography resins.

[0004] Cell culture-based methods are difficult to implement because they require optimal cell growth media in which all mycoplasma strains mentioned in the guidelines, as well as parvoviruses such as MMV, can potentially grow. Furthermore, due to the slow replication of mycoplasma, these assays can take up to several weeks to complete, during which time contamination from external sources can occur, leading to false positives and lengthy investigations. Therefore, these assays are outsourced to contract manufacturing organizations, but their long turnaround times are incompatible with the short timelines required for development and manufacturing, where products should be released without delay.

[0005] NAT represents an interesting alternative to cell culture-based technologies. Since PCR equipment is commonplace in all laboratories, numerous kits have been developed to ensure the detection of microorganisms such as mycoplasma or MMV contamination, with limits of detection (LODs) meeting guideline requirements. However, existing solutions rely on complex and suboptimal designs. For example, because the assay must be able to detect different mycoplasma strains, some kits require the use of several primer sets specific to each strain. The use of SYBR green in kits (which allows for the detection of amplicon) is also problematic, as it may detect nonspecific DNA amplification, leading to false positives and triggering lengthy investigations. This was recently demonstrated in the applicant's own laboratory, where the MycoTOOL kit (Roche) yielded positive results in the analysis of samples contaminated with bacteria but without mycoplasma.

[0006] Therefore, this invention addresses the need for a simple, rapid, and cost-effective method for detecting microorganisms (such as mycoplasma or viral contamination) in samples. This method allows for highly specific and sensitive detection of microorganisms (such as mycoplasma or viruses) by using novel primers that target previously untargeted mycoplasma regions. Summary of the Invention

[0007] The object of the present invention is achieved by providing a method for detecting microorganisms in a sample, the method comprising: (i) performing PCR on a sample suspected of containing mycoplasma DNA using a PCR reaction mixture comprising (1) a first primer (P1) annealed to the reverse complementary sequence of the nucleic acid sequence of SEQ ID NO: 1 and a second primer (P2) annealed to the nucleic acid sequence of SEQ ID NO: 2; or (2) a third primer (P3) annealed to the reverse complementary sequence of the nucleic acid sequence of SEQ ID NO: 109 and a fourth primer (P4) annealed to the nucleic acid sequence of SEQ ID NO: 110; and (ii) detecting PCR products, wherein the detection of PCR products indicates that the sample contains microorganisms, and wherein the absence of PCR products indicates that the sample does not contain microorganisms.

[0008] According to one aspect, the present invention provides a method for detecting mycoplasma in a sample, the method comprising: (i) performing PCR on a sample suspected of containing mycoplasma DNA using a PCR reaction mixture comprising a first primer (P1) annealed to the reverse complementary sequence of a nucleic acid sequence of SEQ ID NO: 1 and a second primer (P2) annealed to a nucleic acid sequence of SEQ ID NO: 2; and (ii) detecting PCR products, wherein the detection of PCR products indicates that the sample contains mycoplasma, and the absence of PCR products indicates that the sample does not contain mycoplasma.

[0009] According to one embodiment, (i) P1 has a length between 18 and 24 nucleotides, preferably between 19 and 21 nucleotides, and most preferably 19 nucleotides, and / or P2 has a length between 17 and 22 nucleotides, preferably between 18 and 20 nucleotides, and most preferably 20 nucleotides; and / or (ii) P1 has a Tm between 60°C and 70°C, preferably between 63°C and 69°C, more preferably between 66°C and 68°C, and / or P2 has a Tm between 60°C and 67°C, preferably between 62°C and 66°C, and most preferably between 64°C and 65°C; and / or (iii) P1 comprises or is composed of the nucleic acid sequence of SEQ ID NO: 3 to 49 and / or P2 comprises or is composed of the nucleic acid sequence of SEQ ID NO: 50 to 52.

[0010] According to another embodiment, the reaction mixture further comprises at least a first nucleic acid probe (probe 1) that is annealed or bound to the nucleic acid sequence of SEQ ID NO: 53 or its reverse complementary sequence.

[0011] According to one embodiment, (i) the reaction mixture further comprises at least a second nucleic acid probe (probe 2) annealed with SEQ ID NO: 53; and / or (ii) probe 1 and / or probe 2 each have a length between 20 and 42 nucleotides, preferably between 21 and 30 nucleotides, and most preferably between 22 and 24 nucleotides; and / or (iii) probe 1 and / or probe 2 each have a Tm between 55°C and 70°C, preferably between 58°C and 69°C, more preferably between 59°C and 68°C; and / or (iv) probe 1 comprises or is composed of the nucleic acid sequence of SEQ ID NO: 54 to 65 and / or probe 2 comprises or is composed of the nucleic acid sequence of SEQ ID NO: 66; and / or (v) probe 1 and / or probe 2 each comprise one or more locked nucleic acid (LNA) and / or minor groove binding (MGB) moieties; and / or (vi) Probe 1 and / or probe 2 each contain a detectable marker, wherein preferably probe 1 and probe 2 contain the same detectable marker.

[0012] According to one embodiment, (i) the PCR is qPCR, dPCR, ddPCR, or cdPCR; and / or (ii) the PCR uses an annealing temperature between 55°C and 65°C, preferably between 58°C and 60°C; and / or (iii) the PCR includes an activation step and / or a degradation step; and / or (iv) annealing and elongation are performed at the same temperature; and / or (v) the PCR includes 40 to 50 denaturation and annealing / elongation cycles, preferably 45 cycles.

[0013] According to a preferred embodiment, the reaction mixture further comprises an internal positive control nucleic acid sequence comprising at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 1 and at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 2, and an intercalation nucleic acid sequence of at least 18 nucleotides in length between the two nucleic acid sequences, wherein the intercalation nucleic acid sequence is preferably different from SEQ ID NO: 53 in that neither probe 1 nor probe 2 is annealed to the intercalation nucleic acid sequence, and wherein the reaction mixture preferably further comprises at least a third nucleic acid probe (probe 3) annealed to the intercalation nucleic acid sequence or its reverse complementary sequence.

[0014] According to another embodiment, i) probe 3 has a length between 20 and 42 nucleotides, preferably between 21 and 30 nucleotides, and most preferably between 22 and 24 nucleotides; and / or (ii) probe 3 has a Tm between 55°C and 70°C, preferably between 58°C and 69°C, more preferably between 59°C and 68°C; and / or (iii) probe 3 comprises the nucleic acid sequence of SEQ ID NO: 69 or its reverse complementary sequence or is composed of the nucleic acid sequence of SEQ ID NO: 69 or its reverse complementary sequence; and / or (iv) probe 3 comprises one or more locked nucleic acids (LNA) and / or minor groove binding (MGB) portions; and / or (v) probe 3 comprises a detectable label different from the labels of probe 1 and probe 2.

[0015] According to a preferred embodiment, the internal positive control nucleic acid sequence comprises or consists of the following: a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 68 or its reverse complementary sequence.

[0016] According to yet another embodiment, the reaction mixture further comprises a differential positive control nucleic acid sequence comprising at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 1 and at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 2, and an intercalation nucleic acid sequence of at least 18 nucleotides in length between the two nucleic acid sequences, wherein the intercalation nucleic acid sequence is preferably identical to SEQ ID NO: 53, such that probe 1 and probe 2 can anneal with the intercalation nucleic acid sequence, wherein the reaction mixture preferably further comprises at least a fourth nucleic acid probe (probe 4) which anneals with the intercalation nucleic acid sequence or its reverse complementary sequence at a sequence different from the sequence annealed with probe 1 and / or probe 2.

[0017] According to the embodiments, i) probe 4 has a length between 20 and 42 nucleotides, preferably between 21 and 30 nucleotides, and most preferably between 22 and 24 nucleotides; and / or (ii) probe 4 has a Tm between 55°C and 70°C, preferably between 58°C and 69°C, more preferably between 59°C and 68°C; and / or (iii) probe 4 comprises the nucleic acid sequence of SEQ ID NO: 71 or its reverse complementary sequence or is composed of the nucleic acid sequence of SEQ ID NO: 71 or its reverse complementary sequence; and / or (iv) probe 4 comprises one or more locked nucleic acids (LNA) and / or minor groove binding (MGB) portions; and / or (v) probe 4 comprises a detectable label different from the labels of probe 1, probe 2 and probe 3.

[0018] According to a preferred embodiment, the distinguishing positive control nucleic acid sequence comprises or consists of the following: a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 70 or its reverse complementary sequence.

[0019] According to yet another embodiment, the sample is a biological sample, preferably selected from the group consisting of: cell banks, cell cultures, culture media, cell culture supernatants, pharmaceutical products, vaccine preparations, blood, saliva, and sputum.

[0020] According to yet another aspect, the present invention provides a primer pair for detecting mycoplasma in a sample, the primer pair comprising primer P1 annealed to the reverse complementary sequence of the nucleic acid sequence of SEQ ID NO: 1 and primer P2 annealed to the nucleic acid sequence of SEQ ID NO: 2.

[0021] According to another aspect, the present invention provides a kit for detecting mycoplasma in a sample, wherein the kit comprises the primer pair of the present invention and a first nucleic acid probe (probe 1) annealed to the nucleic acid sequence of SEQ ID NO: 53 or its reverse complementary sequence, and optionally a second nucleic acid probe (probe 2) annealed to SEQ ID NO: 53 or its reverse complementary sequence. The kit preferably further comprises one or more of the following: (i) a PCR reaction mixture; (ii) an internal positive control nucleic acid sequence comprising at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 1 and at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 2, and an intercalation nucleic acid sequence of at least 18 nucleotides in length between the two nucleic acid sequences, wherein the intercalation nucleic acid sequence is preferably different from SEQ ID NO: 53 in that neither probe 1 nor probe 2 anneals to the intercalation nucleic acid sequence, wherein the kit preferably further comprises at least a third nucleic acid probe (probe 3) annealed to the intercalation nucleic acid sequence or its reverse complementary sequence; (iii) a differential positive control nucleic acid sequence comprising at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 1 and at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 2, and an intercalation nucleic acid sequence of at least 60 nucleotides in length between the two nucleic acid sequences, wherein the intercalation nucleic acid sequence is preferably different from SEQ ID NO: 53 in that neither probe 1 nor probe 2 anneals to the intercalation nucleic acid sequence, wherein the kit preferably further comprises at least a third nucleic acid probe (probe 3) annealed to the intercalation nucleic acid sequence or its reverse complementary sequence; 53. No difference, which allows probe 1 and probe 2 to anneal with intercalated nucleic acid sequences, wherein the kit preferably further comprises at least a fourth nucleic acid probe (probe 4) which anneal with an intercalated nucleic acid sequence or its reverse complementary sequence that is different from the sequence annealed with probe 1 and / or probe 2; (iv) tools for extracting and / or purifying DNA from a sample; (v) instructions for PCR using primers.

[0022] According to one embodiment, in the method of the present invention, in the primer pair of the present invention, or in the kit of the present invention, the mycoplasma is one or more mycoplasma species selected from the group consisting of: Mycoplasma arginini, Mycoplasma buccale, Mycoplasma hominis, Mycoplasma orale, Mycoplasma salivarium, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma synoviae, Mycoplasma pneumoniae, Acholeplasma laidlawii, Mycoplasma gallisepticum, and Spiroplasma citri.

[0023] According to another aspect, the present invention provides a method for detecting a virus in a sample, the method comprising: (i) performing PCR on a sample suspected of containing viral DNA using a PCR reaction mixture comprising a third primer (P3) annealed to the reverse complementary sequence of the nucleic acid sequence of SEQ ID NO: 109 and a fourth primer (P4) annealed to the nucleic acid sequence of SEQ ID NO: 110; and (ii) detecting PCR products, wherein the detection of PCR products indicates that the sample contains a virus, and the absence of PCR products indicates that the sample does not contain a virus.

[0024] According to one embodiment, (i) P3 has a length between 20 and 25 nucleotides, preferably between 21 and 24 nucleotides, and most preferably 23 nucleotides, and / or P4 has a length between 15 and 21 nucleotides, preferably between 16 and 20 nucleotides, and most preferably 23 nucleotides; and / or (ii) P3 has a Tm between 60°C and 70°C, preferably between 63°C and 69°C, more preferably between 66°C and 68°C, and / or P4 has a Tm between 60°C and 67°C, preferably between 62°C and 66°C, and most preferably between 64°C and 65°C; and / or (iii) P3 comprises or is composed of the nucleic acid sequence of SEQ ID NO: 104 and / or P4 comprises or is composed of the nucleic acid sequence of SEQ ID NO: 105.

[0025] According to one embodiment, the reaction mixture further comprises at least one nucleic acid probe (probe 5) annealed to the nucleic acid sequence of SEQ ID NO: 111 or its reverse complementary sequence.

[0026] According to another embodiment, (i) probe 5 has a length between 15 and 30 nucleotides, preferably between 18 and 22 nucleotides, and most preferably between 19 and 21 nucleotides; and / or (ii) probe 5 has a Tm between 59°C and 71°C, preferably between 60°C and 70°C, and more preferably between 63°C and 67°C; and / or (iii) probe 5 comprises or is composed of the nucleic acid sequence of SEQ ID NO: 106; and / or (iv) probe 5 comprises one or more locked nucleic acids (LNA) and / or minor groove binding (MGB) moieties; and / or (v) probe 5 comprises a detectable marker.

[0027] According to yet another embodiment, (i) the PCR is qPCR, dPCR, ddPCR, or cdPCR; and / or (ii) the PCR uses an annealing temperature between 55°C and 65°C, preferably between 58°C and 60°C; and / or (iii) the PCR includes an activation step and / or a degradation step; and / or (iv) annealing and elongation are performed at the same temperature; and / or (v) the PCR includes 40 to 50 denaturation and annealing / elongation cycles, preferably 45 cycles.

[0028] According to another embodiment, the reaction mixture further comprises an internal positive control sequence comprising at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 68 or 112 or 115.

[0029] According to another embodiment, the internal positive control nucleic acid sequence comprises or consists of the following: a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 68 or 112 or 115 or its reverse complementary sequence.

[0030] According to another embodiment, the reaction mixture further comprises a differential positive control nucleic acid sequence comprising at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 109 and at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 110, and an intercalation nucleic acid sequence of at least 18 nucleotides in length between the two nucleic acid sequences.

[0031] According to an embodiment, the reaction mixture further comprises at least a third nucleic acid probe (probe 3) and / or a fourth nucleic acid probe (probe 4), which anneal to the intercalated nucleic acid sequence or its reverse complementary sequence at a sequence different from the sequence annealed with probe 5.

[0032] According to a preferred embodiment, the probe 3 has a length between 20 and 42 nucleotides, preferably between 21 and 30 nucleotides, and most preferably between 22 and 24 nucleotides, and / or a Tm between 55°C and 70°C, preferably between 58°C and 69°C, more preferably between 59°C and 68°C, and / or contains the nucleic acid sequence of SEQ ID NO: 69 or its reverse complementary sequence or is composed of the nucleic acid sequence of SEQ ID NO: 69 or its reverse complementary sequence, and / or contains one or more locked nucleic acids (LNA) and / or minor groove binding (MGB) portions. According to a further preferred embodiment, probe 4 has a length between 20 and 42 nucleotides, preferably between 21 and 30 nucleotides, and most preferably between 22 and 24 nucleotides, and / or a Tm between 55°C and 70°C, preferably between 58°C and 69°C, more preferably between 59°C and 68°C, and / or contains the nucleic acid sequence of SEQ ID NO: 71 or its reverse complementary sequence or is composed of the nucleic acid sequence of SEQ ID NO: 71 or its reverse complementary sequence, and / or contains one or more locked nucleic acids (LNA) and / or minor groove binding (MGB) portions, and / or contains a detectable label different from the labels of probes 3 and 5. According to one embodiment, the differential positive control nucleic acid sequence contains or is composed of a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 113 or its reverse complementary sequence.

[0033] According to one embodiment, the sample is a biological sample, preferably selected from the group consisting of: cell banks, culture media, cell cultures, cell culture supernatants, pharmaceutical products, vaccine preparations, blood, saliva, and sputum.

[0034] According to another aspect, the present invention provides a primer pair for detecting MMV in a sample, the primer pair comprising: primer P3 annealed to the reverse complementary sequence of the nucleic acid sequence of SEQ ID NO: 109 and primer P4 annealed to the nucleic acid sequence of SEQ ID NO: 110. Primer P3 preferably comprises or is composed of the nucleic acid sequence of SEQ ID NO: 104. Primer P4 preferably comprises or is composed of the nucleic acid sequence of SEQ ID NO: 105.

[0035] According to another aspect, the present invention provides a kit for detecting MMV in a sample, wherein the kit comprises the primer pair of the present invention and a nucleic acid probe (probe 5) annealed to the nucleic acid sequence of SEQ ID NO: 111 or its reverse complementary sequence, preferably wherein the kit further comprises one or more of the following: (i) a PCR reaction mixture; (ii) an internal positive control nucleic acid sequence comprising at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 109 and at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 110, and an intercalation nucleic acid sequence of at least 18 nucleotides in length between the two nucleic acid sequences; (iii) a differential positive control nucleic acid sequence comprising at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 109 and at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 110, and an intercalation nucleic acid sequence of at least 29 nucleotides in length between the two nucleic acid sequences, wherein the intercalation nucleic acid sequence preferably annealed to the nucleic acid sequence of SEQ ID NO: 111. 111 shows no difference, allowing probe 5 to anneal with intercalated nucleic acid sequences; (iv) tools for extracting and / or purifying DNA from samples; (v) instructions for PCR using primers.

[0036] According to one embodiment, in the method according to the invention, in the primer pair according to the invention, or in the kit according to the invention, the virus is selected from the group consisting of: Anelloviridae, Inoviridae, Parvoviridae, preferably Parvovirus, Erythovirus, Dependovirus, Amdovirus, and Bocavirus, more preferably Parvovirus, and most preferably Mouse parvovirus.

[0037] Further aspects and embodiments of the invention will become apparent from the appended claims and the following detailed description. Attached Figure Description

[0038] The present invention is further illustrated by the following figures and embodiments, but is not limited thereto.

[0039] Figure 1This graph displays search results for 100 bp fragments with 80% homology in mycoplasma 16S rRNA sequences across species, where primers and probes can be aligned and bound separately. The graph shows the similarity of RNA from all tested species, with +1 indicating 100% similarity. Identified sequence segments meeting the criteria are highlighted: approximately 500-600 bp in the top row, approximately 800 bp in the middle row, and approximately 1100 bp in the bottom row.

[0040] Figure 2 shows the sequence details of the regions where the forward primer, reverse primer, and probe were designed. For clarity, the sequences are aligned to the corresponding species by subgroup. A: All mycoplasma species. B: Bacterial and mycoplasma species. C: Mycoplasma hyoides, prokaryotic and eukaryotic species.

[0041] Figure 3 Fwd7 / Rev1 was shown on the mycoplasma DNA sample. Analysis of PCR products. A: PAGE 15% analysis of PCR performed on reference DNA from a specified organism. B: Melting curves (left) and peaks (right) of the PCR products.

[0042] Figure 4 The schematic localization of primers and probes on the control construct is shown. A: Location of primers and probes on the differential positive control (DPC). B: Location of primers and probes on the internal positive control (IPC).

[0043] Figure 5 The comparison of MMV serotype genomes is shown. Sequences from MMVp, MMVi, MMVc, and MMVm listed in Table 24 were compared and similarities were calculated.

[0044] Figure 6 Sequence details of the regions where the forward primer, reverse primer, and probe were designed are shown. For clarity, sequences are aligned by subgroup. A) All MMV serotypes; B) MMV and parvovirus of interest; C) MMV and parvovirus with higher sequence homology in the target region.

[0045] Figure 7 The locations of primers and probes on the control constructs are schematically shown. A) Location of primers and probes on the MMV differential positive control (DPC). B) Location of primers and probes on the internal positive control v2 (IPC MMV). C) Location of primers and probes on the internal positive control v3 (IPC Myco / MV).

[0046] Figure 8The results show the amplification of various gDNAs using a first PCR mixture containing all reagents for specific MMV detection, with an MMV primer set. A) MMV and DPC amplification on the FAM channel; B) DPC amplification on the HEX channel only; C) IPC amplification of all samples on the Cy5 channel. The X-axis shows fluorescence intensity, and the Y-axis shows the PCR cycle number.

[0047] Figure 9 The results show the amplification of various gDNAs using their own primer sets, employing a second PCR mixture containing all reagents for specific MMV detection. A) Human amplification on the FAM channel; B) E. coli amplification on the HEX channel; C) CHO amplification on the Cy5 channel. The X-axis represents fluorescence intensity, and the Y-axis represents the PCR cycle number.

[0048] Figure 10 Visualizations of amplification products in 15% PAGE are shown using two different PCR mixtures targeting MMV and other gDNA matrices, respectively. A) Amplification from MMV using PCR mixture 1; B) Amplification from gDNA using PCR mixture 2.

[0049] Figure 11 This represents the calibration curves for two different data units. After data reformatting, a 10 Cq drift was observed, which implies an absolute difference of approximately 3 Log10. sequence list

[0050] The sequences mentioned herein are disclosed in detail in the attached sequence listing. The sequences of the present invention are also listed in Table 1 below.

[0051] Table 1: All examples of nucleic acid sequences of the present invention exhibit a 5'→3' orientation.

[0052]

[0053]

[0054]

[0055] The SEQ ID NOs 72 to 103 shown in the figure are listed in the sequence listing, as are SEQ ID NOs 117 to 192. Detailed Implementation

[0056] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0057] Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, edited by Leuenberger, HGW, Nagel, B. and Klbl, H. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0058] Throughout this specification and the claims thereafter, unless the context otherwise requires, the words “comprise” and variations such as “comprises” and “comprising” should be understood to imply inclusion of a whole or a group of steps of a statement, but do not exclude any other whole or a group of steps. In the following paragraphs, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any one or more other aspects unless expressly stated to the contrary. Any feature indicated as optional, preferred, or advantageous may be combined with any one or more other features indicated as optional, preferred, or advantageous.

[0059] The elements of the invention will be described below. These elements are all illustrated with specific embodiments; however, it should be understood that they can be combined in any manner and in any number to form other embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context otherwise indicates, it should be considered that any permutation and combination of all the elements described in this application is disclosed through the description of this application.

[0060] This instruction manual contains numerous references. Each reference cited herein (including all scientific publications, manufacturer's specifications, instructions, etc.) is hereby incorporated in its entirety by way of citation, both above and below.

[0061] definition

[0062] The following sections provide definitions for some terms commonly used in this specification. In the remainder of this specification, these terms will have their respective defined meanings and preferred meanings each time they are used.

[0063] As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” include plural indicators.

[0064] The terms “nucleic acid” or “polynucleotide” as used in this specification include polymeric or oligomeric macromolecules or large biomolecules essential for all known forms of life. Nucleic acids, including DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are made of monomers called nucleotides. Most naturally occurring DNA molecules consist of two complementary biopolymer chains that are intertwined to form a double helix. DNA chains are also referred to as polynucleotides, which are composed of nucleotides. Each nucleotide consists of a nitrogenous nucleotide base and a monosaccharide called deoxyribose or ribose, and a phosphate group. Naturally occurring nucleotide bases include guanine (G), adenine (A), thymine (T), uracil (U), or cytosine (C). Nucleotides are linked together in the chain by covalent bonds between the sugar of one nucleotide and the phosphate of the next, creating an alternating sugar-phosphate backbone. If the sugar is deoxyribose, the polymer is DNA. If the sugar is ribose, the polymer is RNA. Typically, polynucleotides are formed by phosphodiester bonds between individual nucleotide monomers. In the context of this invention, the term "nucleic acid" includes, but is not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof, such as RNA-DNA hybrids (within one strand), as well as cDNA, genomic DNA, recombinant DNA, cRNA, and mRNA. Nucleic acids can also be artificial nucleic acids. Artificial nucleic acids include polyamide or peptide nucleic acids (PNA), morpholino and locked nucleic acids (LNA), ethylene glycol nucleic acids (GNA), and threonine nucleic acids (TNA). Each of these is distinguished from naturally occurring DNA or RNA by changes in the molecular backbone. Nucleic acids can be chemically synthesized, for example, according to the phosphotriester method (see, for example, Uhlmann, E. and Peyman, A. (1990) Chemical Reviews, 90, 543-584).

[0065] The terms “nucleic acid” and “nucleic acid molecule” are used synonymously herein and are understood in the art to refer to single- or double-stranded oligomers or polymers of deoxyribonucleotide bases or ribonucleotide bases, or both. As used herein, the term “nucleic acid” includes not only deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), but also all other linear polymers in which the bases adenine (A), cytosine (C), guanine (G), and thymine (T) or uracil (U) are arranged in the corresponding sequence (nucleic acid sequence). The invention also includes the corresponding RNA sequence (in which thymine is replaced by uracil), complementary sequence, and sequence having a modified nucleic acid backbone or 3' or 5' end. However, nucleic acids in the form of DNA are preferred.

[0066] The nucleotide base symbols used throughout this disclosure are consistent with those in accordance with WIPO ST.26, as outlined in Table 2 below.

[0067] Table 2: Standard Abbreviations for Nucleotides

[0068]

[0069] The term "primer" has the meaning understood by those of ordinary technical skill in the field of genetics. It refers to a short, single-stranded nucleic acid molecule, typically between 18 and 24 nucleotides in length, that is able to bind to a target nucleic acid (also known as a template) via annealing (hybridization with a template through Watson-Crick base pairing).

[0070] The "sequence identity percentage" is determined by comparing two best-aligned sequences within a comparison window, where the sequence portion in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which contains no additions or deletions). The percentage can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears to generate a matching position number, dividing this matching position number by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage.

[0071] The term "identical" in this document, in the context of two or more nucleic acids, refers to two or more identical sequences or subsequences, i.e., sequences containing the same nucleotide or amino acid sequence. Sequences are considered "identical" to each other if they have a specified percentage of identical nucleotide or amino acid residues. According to the invention, at least 90% identity, measured by manual alignment and visual inspection, when comparing and aligning maximum correspondences over a comparison window or specified region, includes at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity on the specified sequence. These definitions also refer to complementary sequences of the test sequence. Therefore, the term "at least XY% sequence identity" is used throughout the specification with respect to polynucleotide sequence comparisons. In the context of this invention, a nucleic acid sequence having at least 90% sequence identity with a given SEQ ID NO or a nucleic acid sequence that is inversely complementary to it preferably means that the nucleic acid has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a given SEQ ID NO or a nucleic acid sequence that is inversely complementary to it.

[0072] The term "sequence comparison" is used herein to refer to the process of comparing a test sequence to a reference sequence, with one of the sequences serving as the reference sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and subsequence coordinates are specified if necessary, along with the sequence algorithm program parameters. Default program parameters are typically used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence relative to the reference sequence based on the program parameters. When comparing two sequences and no reference sequence is specified for calculating the percentage of sequence identity, the longer of the two sequences being compared is used to calculate the sequence identity unless otherwise explicitly stated. If a reference sequence is specified, the sequence identity is determined based on the full length of the reference sequence indicated by one of the SEQ ID NOs of this invention, unless otherwise explicitly stated.

[0073] Sequence alignment methods for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by: Smith and Waterman’s local homology algorithm (Adv. Appl. Math. 2:482, 1970), Needleman and Wunsch’s homology alignment algorithm (1970), Pearson and Lipman’s similarity search method (1988), computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA from the Wisconsin Genetics Software Package, 575 Ph.D. Genetics Computer Group, Madison, Wisconsin, 575), or by manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology (Supplement 1995)). Suitable algorithms for determining sequence identity and sequence similarity percentages are the BLAST algorithm and the BLAST 2.0 algorithm, described by Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977) and Altschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence. These short words match or satisfy a positive threshold score T when compared to words of the same length in a database sequence. T is called the neighborhood word score threshold (Altschul et al., ibid.). These initial neighborhood word hits act as seeds to initiate a search for longer HSPs containing these initial neighborhood word hits. Word hits expand in both directions along each sequence, as far as possible to increase the cumulative alignment score. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a score matrix is ​​used to calculate the cumulative score. Word hit expansion in each direction is halted if the cumulative alignment score drops by an amount X from its maximum gain; the cumulative score moves towards zero or lower due to the accumulation of one or more negatively scored residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the alignment sensitivity and speed.The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expected value (E) of 10, M = 5, N = -4, and a comparison of the two strands as default values. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expected value (E) of 10, a BLOSUM62 score matrix (see Henikoff and Henikoff, 1989) alignment (B) of 50, an expected value (E) of 10, M = 5, N = -4, and a comparison of the two strands as default values. The BLAST algorithm also performs statistical analysis on the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 90:5873-87, 1993). One similarity metric provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability of a chance match between two nucleotide or amino acid sequences. For example, if the minimum sum probability in a comparison of the test nucleic acid with a reference nucleic acid is less than about 0.2, typically less than about 0.01, and more typically less than about 0.001, then the nucleic acid is considered to be similar to the reference sequence.

[0074] As used herein, the term “annealing with” refers to the preferred pairing of complementary sequences of a single-stranded nucleic acid under stringent conditions to form a double-stranded polynucleotide. As used herein, the term “subject” refers to an animal, preferably a mammal, and most preferably a human.

[0075] As used herein, the term "melting temperature" or "Tm" refers to the temperature at which half of the DNA strand is in a random coil or single-stranded (ssDNA) state. Tm depends on the length of the DNA molecule and its specific nucleotide sequence. In this invention, the Tm for a given nucleotide sequence is calculated using the software PrimerExpress. ®3.0 (Life Technologies Corporation) using standard settings; Geneious Prime 2023.2 software (Dotmatics) using the following settings: 50 mM monovalent ions, 3 mM divalent ions, 200 nM primers, 0.8 mM dNTPs; or primer design tools for PCR and qPCR (Integrated DNA Technologies) using the parameter set for qPCR: 50 mM monovalent ions, 3 mM divalent ions, 200 nM primers, 0.8 mM dNTPs, further using the Santa Lucia 1998 thermodynamic model and salinity correction (Santa Lucia, ProcNatl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America], 1998, Vol. 95(4): 1460-1465) or the Santa Lucia 1998 thermodynamic model and Owczarzy Salinity correction in 2004 (Owczarzy et al., Biochemistry 2004, Vol. 43(12): 3537-3554).

[0076] Description of the Implementation Examples

[0077] This invention belongs to the field of microbial detection and provides methods, specific primers and probes, and kits for detecting microorganisms such as mycoplasma or mouse parvovirus.

[0078] In the experiments that led to this invention, it was surprisingly found that, although three regions with sufficient homology between mycoplasma species were identified for primer and probe design ( Figure 1 However, only one region proved suitable for primer and probe design, and this region needed to be further widened to accommodate forward primers, probes, and reverse primers. Surprisingly, it was further discovered that only one genomic region proved suitable for primer and probe design for the detection of mouse parvovirus (MMV). This invention describes the identification of these specific regions for primer and probe design, which can be used for mycoplasma detection or MMV detection, optionally in conjunction with other probes for positive controls and internal controls. The primers and probes of this invention guarantee the high specificity (no detection of bacterial DNA, no detection of CHO DNA, no detection of human DNA) and sensitivity required by established guidelines, for example, for the detection of mycoplasma in samples as low as 10 CFU / mL.

[0079] Therefore, the present invention provides a method for detecting microorganisms in a sample, the method comprising the steps of: (i) performing PCR on a sample suspected of containing microbial DNA using a PCR reaction mixture comprising: (1) a first primer (P1) annealed to the reverse complementary sequence of the nucleic acid sequence of SEQ ID NO: 1 and a second primer (P2) annealed to the nucleic acid sequence of SEQ ID NO: 2, or (2) a third primer (P3) annealed to the reverse complementary sequence of the nucleic acid sequence of SEQ ID NO: 109 and a fourth primer (P4) annealed to the nucleic acid sequence of SEQ ID NO: 110; and (ii) detecting the PCR product. Since this method is intended to detect microorganisms in a sample, the detection of PCR products generated in the PCR reaction using both primers (1) and / or (2) indicates that the sample contains microorganisms. Similarly, the absence of such PCR products indicates that the sample does not contain microorganisms such as mycoplasma and MMV.

[0080] The samples used in the context of this invention are preferably biological samples. According to preferred embodiments, the samples are selected from, but are not limited to, samples from cell banks, culture media (such as, but not limited to, cell culture media), cell cultures, cell culture supernatants, pharmaceutical products, vaccine formulations, and bodily fluids (such as blood, saliva, or sputum). The samples can be used in the methods of this invention in a substantially untreated form. For example, the samples can be derived from or obtained from articles, objects, items, or subjects. The samples can be added directly to the reaction mixture without any prior purification steps. Methods for obtaining such samples are well known to those skilled in the art. Alternatively, the samples can be pretreated before being added to the reaction mixture. Such pretreatment can include methods for purifying or isolating DNA from the sample, which can then be used in the methods of this invention.

[0081] The method of the present invention allows for the detection of microorganisms, such as bacteria or viruses, in samples.

[0082] According to one aspect, the present invention provides a method for detecting mycoplasma in a sample, the method comprising: (i) performing PCR on a sample suspected of containing mycoplasma DNA using a PCR reaction mixture comprising a first primer (P1) annealed to the reverse complementary sequence of a nucleic acid sequence of SEQ ID NO: 1 and a second primer (P2) annealed to a nucleic acid sequence of SEQ ID NO: 2; and (ii) detecting PCR products, wherein the detection of PCR products indicates that the sample contains mycoplasma, and the absence of PCR products indicates that the sample does not contain mycoplasma.

[0083] This method allows for the detection of bacteria, particularly mycoplasma, in samples selected from the group consisting of the following mycoplasma species: Mycoplasma argininae, Mycoplasma buccalis, Mycoplasma hominis, Mycoplasma oralis, Mycoplasma salivariae, Mycoplasma fermentans, Mycoplasma hyoidosaurus, Mycoplasma synovitis, Mycoplasma pneumoniae, Acholestylasma reesei, Mycoplasma gallisepticum, and Mycoplasma citrinum.

[0084] According to another aspect, the present invention provides a method for detecting a virus in a sample, the method comprising: (i) performing PCR on a sample suspected of containing viral DNA using a PCR reaction mixture comprising a third primer (P3) annealed to the reverse complementary sequence of the nucleic acid sequence of SEQ ID NO: 109 and a fourth primer (P4) annealed to the nucleic acid sequence of SEQ ID NO: 110; and (ii) detecting PCR products, wherein the detection of PCR products indicates that the sample contains a virus, and the absence of PCR products indicates that the sample does not contain a virus.

[0085] This method allows the detection of viruses in samples selected from the group consisting of single-stranded DNA viruses, such as those from the families Circaviridae, Fibroviridae, and Parvoviridae. The Parvoviridae family can be further subdivided into the subfamilies Parvovirus, including parvoviruses, erythroviruses, dependent viruses, Aleutian viruses, and Bocaviruses. Preferably, the method of the present invention allows the detection of parvoviruses, particularly mouse parvovirus (MMV).

[0086] The reaction mixture used for PCR is not particularly limited to a specific type and can be selected by those skilled in the art or by following the manufacturer's instructions regarding the components of the PCR reaction mixture. In addition to primers and a DNA template, the PCR reaction mixture typically contains DNA polymerase, deoxynucleoside triphosphates (dNTPs), a buffer solution, and divalent cations (such as Mg²⁺). 2+ or Mn 2+ ).

[0087] The preferred characteristics of primers P1 and P2 used in the context of this invention are disclosed below.

[0088] Primer P1 preferably has a length between 18 and 24 nucleotides, such as 18, 19, 20, 21, 22, 23 and 24 nucleotides, preferably between 19 and 23, 19 and 22, 19 and 21 or 19 and 20 nucleotides, and most preferably 19 nucleotides.

[0089] Primer P1 preferably has a melting temperature (Tm) between 60°C and 70°C, such as 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C and 70°C, more preferably between 63°C and 69°C, more preferably between 66°C and 68°C, and most preferably 67°C.

[0090] Primer P1 preferably comprises or is composed of the nucleic acid sequence of SEQ ID NO: 3 to 49. More preferably, primer P1 comprises or is composed of the nucleic acid sequence according to SEQ ID NO: 9. Most preferably, P1 is composed of the nucleic acid sequence according to SEQ ID NO: 9.

[0091] Primer P2 preferably has a length between 17 and 22 nucleotides, such as 17, 18, 19, 20, 21 and 22 nucleotides, preferably between 18 and 20 nucleotides, or between 19 and 20 nucleotides, and most preferably 20 nucleotides.

[0092] Primer P2 preferably has a Tm between 60°C and 67°C, such as 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C and 67°C, more preferably between 62°C and 66°C, and most preferably between 64°C and 65°C.

[0093] Primer P2 preferably comprises or is composed of the nucleic acid sequence of SEQ ID NO: 50 to 52. More preferably, primer P1 comprises or is composed of the nucleic acid sequence according to SEQ ID NO: 50. Most preferably, P1 is composed of the nucleic acid sequence according to SEQ ID NO: 50.

[0094] The preferred characteristics of primers P3 and P4 used in the context of this invention are disclosed below.

[0095] Primer P3 preferably has a length between 20 and 25 nucleotides, such as 20, 21, 22, 23, 24 and 25 nucleotides, preferably between 21 and 24, 22 and 23, and most preferably 23 nucleotides.

[0096] Primer P3 preferably has a melting temperature (Tm) between 60°C and 70°C, such as 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C and 70°C, more preferably between 63°C and 69°C, more preferably between 66°C and 68°C, and most preferably 67°C.

[0097] Primer P3 preferably contains or is composed of the nucleic acid sequence of SEQ ID NO: 104. Most preferably, P3 is composed of the nucleic acid sequence according to SEQ ID NO: 104.

[0098] Primer P4 preferably has a length between 15 and 21 nucleotides, such as 15, 16, 17, 18, 19, 20 and 21 nucleotides, preferably between 16 and 20 nucleotides, or between 17 and 19 nucleotides, and most preferably 17 nucleotides.

[0099] Primer P4 preferably has a Tm between 60°C and 67°C, such as 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C and 67°C, more preferably between 62°C and 66°C, and most preferably between 64°C and 65°C.

[0100] Primer P4 preferably contains or is composed of the nucleic acid sequence of SEQ ID NO: 105. Most preferably, P4 is composed of the nucleic acid sequence according to SEQ ID NO: 105.

[0101] The Tm of primers, and especially the Tm of primers P1 and P2 or primers P3 and P4, is preferably calculated using the software and settings described in the "Definitions" section above.

[0102] The method of the present invention involves performing PCR on samples suspected of containing mycoplasma DNA and / or viral DNA (such as MMV DNA) using a PCR reaction mixture. The type of PCR is preferably real-time quantitative PCR (qPCR) or digital PCR (dPCR). dPCR can be, for example, droplet digital PCR (ddPCR) or microarray digital PCR (cdPCR). A particularly preferred PCR method according to the present invention is qPCR.

[0103] PCR is preferably performed by annealing P1 and P2 at a temperature between 55°C and 65°C, such as 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, and 65°C, more preferably between 58°C and 60°C. Preferably, the annealing temperatures of P1 and P2, or P3 and P4, are substantially the same, such that the difference between the two primers is in the range of about 2°C to 3°C, more preferably in the range of ±1°C. For example, if P1 has an annealing temperature of about 59°C, then it is preferred that P2 has an annealing temperature of 59°C ±1°C. Although it is preferred that the annealing temperatures of P1 and P2, or P3 and P4, are substantially the same (and more preferably the same), this is not mandatory.

[0104] The elongation temperature can be readily selected by those skilled in the art and can depend on the specific type of polymerase used in the reaction and the expected length of the PCR product generated in the reaction. Typical elongation temperatures are between 60°C and 80°C, typically around 72°C, but may vary depending on the type of enzyme used.

[0105] According to a preferred embodiment of the invention, the annealing and elongation steps are performed at the same temperature and therefore can be performed in the same step. For example, annealing and elongation can be performed at temperatures between about 57°C and 65°C, such as about 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, or 65°C. According to a particularly preferred embodiment, annealing and elongation can be performed at a temperature of about 60°C. Such a combined annealing and elongation step is also simply referred to as an amplification step and can accelerate the time required to run PCR. The duration of the amplification step depends on the size of the target to be amplified and the DNA polymerase used. For example, amplification may take about 20 to 60 seconds, about 30 to 50 seconds, or about 40 seconds. According to a preferred embodiment of the invention, the duration of amplification (i.e., annealing and elongation) is about 30 seconds.

[0106] Typical cycle numbers, including denaturation, annealing, and elongation, are between about 20 and 50 cycles, such as 20, 25, 30, 35, 40, 45, and 50 cycles, preferably between about 30 and 45 cycles. According to a particularly preferred embodiment, PCR is run for about 45 cycles.

[0107] In addition to the standard procedure of multiple cycles of denaturation, annealing, and elongation, PCR may also include one or more additional steps, preferably prior to said cycles, for degrading contaminant DNA, such as contaminant DNA from residual PCR products. Such degradation is preferably carried out by adding an enzyme, such as uracil-DNA glycosylase (UNGase, e.g., from ArcticZymes Technologies, Norway), to the reaction mixture and applying the degradation step at about 40°C for a period of 60 to 180 seconds (preferably 120 seconds). The temperature and duration of such additional steps may depend on the enzyme and its concentration used in the reaction mixture for degrading the contaminant DNA, and can be readily selected by those skilled in the art or by following the manufacturer's instructions for use with the enzyme for this purpose.

[0108] As a supplement to or alternative to the degradation step, PCR may include a step of activating the reaction prior to the start of the actual cycle. Such a start or initiation step may include applying a temperature between about 88°C and 98°C, preferably about 90°C, to the reaction mixture containing the sample and primers for about 45 seconds to 10 minutes, preferably about 60 seconds. The specific parameters of such an activation step can be readily selected by those skilled in the art or by following the instructions of the manufacturer of the polymerase or any other reaction mixture component used in PCR.

[0109] The particularly preferred PCR protocol used in the method of the present invention is disclosed below:

[0110]

[0111] Detection of PCR products can be performed by any appropriate method known in the art. For example, the generated PCR products can be detected during PCR (also known as real-time techniques) or after PCR (also known as endpoint techniques). Real-time techniques are performed within the PCR reaction vessel and during PCR thermal cycling using, for example, DNA-binding fluorescent dyes (e.g., non-specific fluorescent dyes with any double-stranded DNA inserted), DNA hybridization (e.g., sequence-specific DNA probes consisting of, for example, oligonucleotides labeled with fluorescent dyes), or dNTP nucleotides with fluorescent dyes. Endpoint techniques include, but are not limited to, DNA gel electrophoresis and the use of DNA insertion dyes. A particularly preferred detection method is the use of labeled sequence-specific DNA probes directly in the reaction mixture and therefore during PCR.

[0112] According to such a particularly preferred embodiment of the invention, the reaction mixture for detecting mycoplasma may further contain at least a first nucleic acid probe (hereinafter referred to as probe 1). Probe 1 is annealed with a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 53, or with the reverse complementary sequence of a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 53. Therefore, the reaction mixture for detecting MMV may further contain at least one nucleic acid probe (hereinafter referred to as probe 5). Probe 5 is annealed with a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 111, or with the reverse complementary sequence of a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 111.

[0113] According to another particularly preferred embodiment of the invention relating to mycoplasma detection, the reaction mixture comprises a second nucleic acid probe (probe 2) annealed to a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 53, or to the reverse complementary sequence of a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 53. Compared to probe 1, probe 2 anneals to or binds to different segments of a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 53 or its reverse complementary sequence. Preferably, probe 2 anneals to a segment of a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 53 or its reverse complementary sequence, which is at least two nucleotides apart from the segment of a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 53 or its reverse complementary sequence annealed to or bound by probe 1.

[0114] Probe 1 and / or probe 2 or probe 5 may be present in the premix of the reaction mixture or may be added before PCR begins.

[0115] The preferred characteristics of probe 1, probe 2 and probe 5 are disclosed below.

[0116] Probe 1 and / or probe 2 preferably have a length between about 20 and 42 nucleotides, such as about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42 nucleotides, more preferably between about 21 and 30 nucleotides, and most preferably between about 22 and 24 nucleotides. The most preferred length of probe 1 and / or probe 2 is 23 nucleotides. Probe 5 preferably has a length between about 15 and 30 nucleotides, such as about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 nucleotides, more preferably between about 18 and 22 nucleotides, and most preferably between about 19 and 21 nucleotides. The most preferred length of probe 5 is 20 nucleotides.

[0117] Probe 1 and / or probe 2 preferably have a Tm between about 55°C and 70°C, such as about 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C and 70°C, more preferably between about 58°C and 69°C, and most preferably between about 59°C and 68°C, such as between about 59°C and 67°C, between about 59°C and 66°C, between about 59°C and 65°C, between about 59°C and 64°C, between about 59°C and 63°C, between about 59°C and 62°C, between about 59°C and 61°C.

[0118] The probe 5 preferably has a Tm between about 59°C and 71°C, such as about 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C and 71°C, more preferably between about 60°C and 70°C, and most preferably between about 62°C and 68°C, such as between about 63°C and 67°C, between about 64°C and 66°C.

[0119] Probe 1 preferably comprises or is composed of the nucleic acid sequence of SEQ ID NO: 54 to 65. More preferably, probe 1 comprises or is composed of the nucleic acid sequence according to SEQ ID NO: 62, and most preferably, probe 1 is composed of the nucleic acid sequence according to SEQ ID NO: 62. Probe 1 may contain one or more locked nucleic acids (LNAs). As a supplement to or alternative to LNAs, probe 1 may contain one or more minor groove binding (MGB) moieties. According to a particularly preferred embodiment, probe 1 is composed of the nucleic acid sequence according to SEQ ID NO: 62 and contains LNAs, preferably five LNAs, as shown in SEQ ID NO: 60 (underlined nucleotides represent LNA modifications).

[0120] Probe 2 preferably comprises or is composed of the nucleic acid sequence of SEQ ID NO: 66. More preferably, probe 2 is composed of the nucleic acid sequence according to SEQ ID NO: 66. Probe 2 may contain one or more LNAs. As a supplement to or alternative to LNAs, probe 2 may contain one or more minor groove binding (MGB) moieties. According to a particularly preferred embodiment, probe 2 is composed of the nucleic acid sequence according to SEQ ID NO: 66 and contains LNAs, preferably three LNAs, as shown in SEQ ID NO: 67 (underlined nucleotides represent LNA modifications).

[0121] Probe 5 preferably comprises or is composed of the nucleic acid sequence of SEQ ID NO: 106. More preferably, probe 5 is composed of the nucleic acid sequence according to SEQ ID NO: 106. Probe 5 may contain one or more locked nucleic acids (LNAs). As a supplement to or alternative to LNAs, probe 5 may contain one or more minor groove binding (MGB) moieties.

[0122] If probe 1 and / or probe 2 are longer than approximately 30 nucleotides, an internal quencher can be used instead of LNA and / or MEG modification. The internal quencher can be, for example, ZEN and / or TAO modified. Such internal quenchers are particularly useful in long probes where one end contains a fluorescent dye and the other end contains a quencher, to increase assay efficiency by reducing the number of cycles required to meet the signal threshold compared to a single quenched probe, and by providing greater overall dye quenching, resulting in reduced background fluorescence and improved signal-to-noise ratio.

[0123] Probe 1 and / or probe 2 and / or probe 5 contain one or more detectable markers. According to one embodiment, probe 1 and probe 2 contain the same detectable marker. The detectable marker can be any marker that allows identification of the probe. Preferred detectable markers are fluorescent dyes. Preferred fluorescent dyes include, but are not limited to, FAM, HEX, NED, TET, VIC, Cy3, Cy5, Texas Red, or Tide Fluor™ dyes, with FAM being more preferred. The fluorescent dye is preferably attached to the 5' end of the nucleic acid probe. In such cases, the probe may further contain a quencher dye attached to its 3' end. Preferred quencher dyes include, but are not limited to, DABCYL, TAMRA, BXQ-1, BXQ-2, IABkFQ, IABkRQ, or Tide Quencher™ dyes, with IABkFQ being more preferred. IABkFQ and IABkRQ are Iowa Black... ®FQ and RQ are obtained from Integrated DNA Technologies, Inc., USA. IABkFQ is preferably used in combination with FAM, HEX, NED, TET, and VIC, and IABkRQ is preferably used in combination with Cy5 or Texas Red. Probe 1 and / or probe 2 and / or probe 5 are preferably hydrolyzing probes, which contain a fluorescent dye at their 5' end and a quencher dye at their 3' end. A particularly preferred combination of probe 1, probe 2, and probe 5 is FAM / IABkFQ. When the hydrolyzing probe is intact, the fluorescent dye and quencher dye remain in close proximity to each other, FRET occurs, and the fluorescent dye is quenched. Any probe that binds to the correct target sequence is hydrolyzed as DNA polymerase binds to and extends the primer upstream of the probe during PCR cycling. The release of the fluorescent dye fragment results in a fluorescence signal proportional to the amount of amplicon produced, thus allowing detection of PCR products during the reaction.

[0124] According to a particularly preferred embodiment, probe 1 consists of SEQ ID NO: 60 and contains the fluorescent dye / quencher pair FAM / IABkFQ. According to another particularly preferred embodiment, probe 2 consists of SEQ ID NO: 67 and contains the fluorescent dye / quencher pair FAM / IABkFQ. According to another particularly preferred embodiment, probe 5 consists of SEQ ID NO: 106 and contains the fluorescent dye / quencher pair FAM / IABkFQ.

[0125] According to a particularly preferred embodiment of the present invention for mycoplasma detection, the reaction mixture comprises primer P1, primer P2, probe 1 and probe 2 as described herein.

[0126] According to a particularly preferred embodiment of the present invention for MMV detection, the reaction mixture comprises primer P3, primer P4 and probe 5 as described herein.

[0127] According to one embodiment, in the method of the present invention, the reaction mixture further comprises a positive control nucleic acid sequence, also referred to as an internal positive control (IPC). The IPC may be present in a premix of the reaction mixture, or it may be added to the reaction mixture before PCR begins.

[0128] In embodiments used for detecting mycoplasma, the positive control nucleic acid sequence or myco-IPC comprises at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 1, at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 2, and an intercalation nucleic acid sequence of at least 18 nucleotides in length between the two nucleic acid sequences. This intercalation sequence of at least 18 nucleotides is preferably different from SEQ ID NO: 53, such that neither probe 1 (if present) nor probe 2 (if present) anneals to the intercalation nucleic acid sequence. According to a particularly preferred embodiment, a third probe (probe 3) anneals to or binds to the intercalation sequence or its reverse complementary sequence. The intercalation nucleic acid sequence preferably has a length of about 20 to 150 nucleotides, about 30 to 140 nucleotides, or about 40 to 120 nucleotides, such as 50, 60, 70, 80, 90, 100, 110, or about 120 nucleotides. According to a particularly preferred embodiment, the intercalation nucleic acid sequence has a length of about 120 nucleotides.

[0129] In embodiments used for detecting mycoplasma, the internal positive control nucleic acid sequence or myco-IPC preferably comprises or consists of the following: a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 68, or the reverse complementary sequence of a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 68. The myco-IPC is preferably in the form of a double-stranded DNA molecule, more preferably in the form of a gBlock. According to a particularly preferred embodiment, the myco-IPC is a gBlock comprising SEQ ID NO: 68 and its reverse complementary sequence or consisting of SEQ ID NO: 68 and its reverse complementary sequence.

[0130] Probe 3 can be used to detect the myco-IPC amplicons. Therefore, according to one embodiment, the reaction mixture further comprises probe 3, which may be present in the premix of the reaction mixture or added before PCR begins. Probe 3 preferably has one or more details described above in the context of probes 1 and 2 regarding its length, Tm, and labeling, provided that the labeling of probe 3 is different from the labeling chosen for probes 1 and 2, thereby distinguishing probe 3 from probes 1 and 2. Probe 3 may also contain one or more of LNA and / or MGB modifications. Probe 3 preferably has a length between 20 and 42 nucleotides, more preferably between 21 and 30 nucleotides, and most preferably between 22 and 24 nucleotides. According to one embodiment, probe 3 has a Tm between about 55°C and 70°C, preferably between about 58°C and 69°C, more preferably between about 59°C and 68°C, and most preferably about 60°C. Probe 3 preferably comprises the nucleic acid sequence of SEQ ID NO: 69 or its reverse complementary sequence, or is composed of the nucleic acid sequence of SEQ ID NO: 69 or its reverse complementary sequence. More preferably, probe 3 is composed of the nucleic acid sequence of SEQ ID NO: 69 or its reverse complementary sequence. According to a preferred embodiment, probe 3 comprises the fluorescent dye / quencher pair Cy5 / IABkRQ. According to a particularly preferred embodiment, probe 3 is composed of the nucleic acid sequence of SEQ ID NO: 69 and comprises the fluorescent dye / quencher pair Cy5 / IABkRQ.

[0131] If myco-IPC is present during PCR, primers 1 and 2 will also bind to the IPC and allow its amplification. The presence of the IPC amplicon can be detected using, for example, probe 3. Alternatively, the presence of the IPC amplicon can be detected using the real-time or endpoint techniques described herein. If IPC amplification products are detected during and / or at the end of PCR, the selected reaction parameters are sufficient to enable the amplification of the potential target in the sample. If only the myco-IPC amplicon is detected and no further PCR products are found, this means that the sample does not contain any mycoplasma.

[0132] In embodiments used for detecting MMV, the positive control nucleic acid sequence or mmv-IPC comprises at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 109, at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 110, and an intercalation nucleic acid sequence of at least 18 nucleotides in length between the two nucleic acid sequences. The intercalation sequence preferably disallows probe 3 (if present) or probe 5 (if present) from binding to the intercalation nucleic acid sequence. According to a particularly preferred embodiment, probe 4 anneals or binds to the intercalation sequence or its reverse complementary sequence. The intercalation nucleic acid sequence preferably has a length of about 20 to 150 nucleotides, about 30 to 140 nucleotides, or about 40 to 120 nucleotides, such as 50, 60, 70, 80, 90, 100, 110, or about 120 nucleotides. According to a particularly preferred embodiment, the intercalation nucleic acid sequence has a length of about 120 nucleotides.

[0133] In embodiments used for detecting MMV, the internal positive control nucleic acid sequence or mmv-IPC preferably comprises or consists of: a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 112 or 116, or an inverse complementary sequence of a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 112 or 116. According to alternative embodiments, the mmv-IPC comprises or consists of: a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 68, 112, or 115, or an inverse complementary sequence of a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 68, 112, or 115. The myco-IPC is preferably in the form of a double-stranded DNA molecule, more preferably in the form of a gBlock. According to a particularly preferred embodiment, the myco-IPC is a gBlock comprising or consisting of SEQ ID NO: 68 and its inverse complementary sequence.

[0134] Probe 4 can be used to detect the mmv-IPC amplicons. Therefore, according to one embodiment, the reaction mixture further comprises probe 4, which may be present in the premix of the reaction mixture or added before PCR begins. Probe 4 is as described above. Probe 4 may also contain one or more of LNA and / or MGB modifications. Probe 4 preferably has a length between 20 and 42 nucleotides, more preferably between 21 and 30 nucleotides, and most preferably between 22 and 24 nucleotides. According to one embodiment, probe 4 has a Tm between about 55°C and 70°C, preferably between about 58°C and 69°C, more preferably between about 59°C and 68°C, and most preferably about 60°C. Probe 4 preferably comprises the nucleic acid sequence of SEQ ID NO: 69 or its reverse complementary sequence, or is composed of the nucleic acid sequence of SEQ ID NO: 69 or its reverse complementary sequence. More preferably, probe 4 is composed of the nucleic acid sequence of SEQ ID NO: 69 or its reverse complementary sequence. According to a preferred embodiment, probe 4 comprises a fluorescent dye / quencher pair Cy5 / IABkRQ. According to a particularly preferred embodiment, probe 4 consists of the nucleic acid sequence of SEQ ID NO: 69 and comprises a fluorescent dye / quencher pair Cy5 / IABkRQ.

[0135] If mmv-IPC is present during PCR, primers 3 and 4 also bind to the IPC and allow its amplification. The presence of the IPC amplicon can be detected using, for example, probe 4. Alternatively, the presence of the IPC amplicon can be detected using the real-time or endpoint techniques described herein. If IPC amplification products are detected during and / or at the end of PCR, the selected reaction parameters are sufficient to enable the amplification of the potential target in the sample. If only mmv-IPC amplicons are detected and no further PCR products are found, this means that the sample does not contain any mouse parvovirus.

[0136] According to one embodiment, in the method of the present invention, the reaction mixture or sample additionally contains a differential positive control nucleic acid sequence, also referred to as DPC. Alternatively, DPC can be used in a separate control reaction using the same parameters as PCR for mycoplasma or MMV detection, but containing DPC instead of the sample.

[0137] In embodiments used for detecting mycoplasma, the differential positive control nucleic acid sequence or myco-DPC comprises at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 1, at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 2, and an intercalation nucleic acid sequence of at least 18 nucleotides in length between the two nucleic acid sequences. This intercalation sequence of at least 18 nucleotides preferably does not differ from SEQ ID NO: 53 or a portion thereof, but rather comprises SEQ ID NO: 53 or a portion thereof. The length of the intercalation nucleic acid sequence preferably allows for annealing or binding of probe 1 (if present) and preferably probe 2 (if present). According to a preferred embodiment, the intercalation sequence has a length that allows for binding or annealing of a fourth probe (probe 4). Therefore, the intercalation sequence of DPC preferably has a length between 18 and about 200 nucleotides, such as between about 25 and 190, about 30 and 180, about 35 and 170, about 40 and 160, about 50 and 150, about 60 and 140, about 70 and 130, about 80 and 120, or about 90 and 110 nucleotides. Preferably, the sequence of the intercalation nucleic acid sequence annealed or bound by probe 4, or its reverse complementary sequence, is different from the sequence of the intercalation nucleic acid sequence annealed or bound by probe 1 and / or probe 2.

[0138] In embodiments used for detecting mycoplasma, the differential positive control nucleic acid sequence or myco-DPC preferably comprises or consists of the following: a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 70, or the reverse complementary sequence of a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 70. The myco-DPC is preferably in the form of a double-stranded DNA molecule, more preferably in the form of a gBlock. According to a particularly preferred embodiment, the myco-DPC is a gBlock comprising SEQ ID NO: 70 and its reverse complementary sequence or consisting of SEQ ID NO: 70 and its reverse complementary sequence.

[0139] In embodiments used for detecting mycoplasma, an additional probe 4 may be used to detect myco-DPC. Probe 4 preferably binds to or anneals with myco-DPC in a region different from the region where probe 1 (if present) and probe 2 (if present) bind or anneal. Probe 4 preferably has one or more details described above in the context of probes 1 and 2 regarding its length, Tm, and labeling, provided that the labeling of probe 4 is different from the labeling chosen for probes 1, 2, and 3, thereby distinguishing probe 4 from other probes. Probe 4 may also contain one or more of LNA and / or MGB modifications. According to one embodiment, probe 4 has a length between 20 and 42 nucleotides, preferably between 21 and 30 nucleotides, and most preferably between 22 and 24 nucleotides. According to a preferred embodiment, probe 4 has a Tm between about 55°C and 70°C, preferably between about 58°C and 69°C, more preferably between about 59°C and 68°C, and most preferably about 60°C. Probe 4 preferably comprises the nucleic acid sequence of SEQ ID NO: 71 or its reverse complementary sequence, or is composed of the nucleic acid sequence of SEQ ID NO: 71 or its reverse complementary sequence. More preferably, probe 4 is composed of the nucleic acid sequence of SEQ ID NO: 71 or its reverse complementary sequence. According to a preferred embodiment, probe 4 comprises the fluorescent dye / quencher pair HEX / IABkFQ. According to a particularly preferred embodiment, probe 4 is composed of the nucleic acid sequence of SEQ ID NO: 71 and comprises the fluorescent dye / quencher pair HEX / IABkFQ. According to one embodiment, the reaction mixture further comprises myco-DPC and probe 4, both of which may be present in the premix of the reaction mixture or added before PCR begins. Alternatively, if myco-DPC is used in a separate control reaction, the separate control reaction uses the same parameters as the PCR used for mycoplasma detection, but contains myco-DPC instead of the sample, and probe 4.

[0140] A particularly preferred combination of primers and probes used in the method for detecting mycoplasma of the present invention comprises primer P1 consisting of the nucleic acid sequence of SEQ ID NO: 9, primer P2 consisting of the nucleic acid sequence of SEQ ID NO: 50, a first probe (probe 1) consisting of the nucleic acid sequence of SEQ ID NO: 60, and a second probe (probe 2) consisting of the nucleic acid sequence of SEQ ID NO: 67. In this particularly preferred combination of primers and probes for detecting mycoplasma in a sample, probe 1 preferably comprises the fluorescent dye / quencher pair FAM / IABkFQ, and probe 2 preferably comprises the fluorescent dye / quencher pair FAM / IABkFQ.

[0141] In embodiments used for detecting MMV, the differential positive control nucleic acid sequence or mmv-DPC comprises at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 109, at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 110, and an intercalation nucleic acid sequence of at least 18 nucleotides in length between the two nucleic acid sequences. The intercalation nucleic acid sequence preferably allows annealing or binding of probe 5 (if present) and preferably probe 3 (if present). Therefore, the intercalation sequence of mmv-DPC preferably has a length between 18 and about 200 nucleotides, such as between about 25 and 190, about 30 and 180, about 35 and 170, about 40 and 160, about 50 and 150, about 60 and 140, about 70 and 130, about 80 and 120, or about 90 and 110 nucleotides. According to a preferred embodiment, the intercalation nucleic acid sequence of mmv-DPC has a length of at least 29 nucleotides. According to a particularly preferred embodiment, the intercalated nucleic acid sequence of mmv-DPC is no different from SEQ ID NO: 111, enabling probe 5 to anneal with the intercalated nucleic acid sequence.

[0142] In embodiments used for detecting MMV, the differential positive control nucleic acid sequence or mmv-DPC preferably comprises or consists of: a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 113, or the reverse complementary sequence of a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 113. According to one embodiment, the mmv-DPC comprises or consists of: a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 114, or the reverse complementary sequence of a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 114. The mmv-DPC is preferably in the form of a double-stranded DNA molecule, more preferably in the form of a gBlock. According to a particularly preferred embodiment, the mmv-DPC is a gBlock comprising or consisting of SEQ ID NO: 114 and its reverse complementary sequence.

[0143] In embodiments used for detecting MMV, probe 5 is preferably bonded or annealed with the mmV-DPC in a region different from the region bonded or annealed with probe 3. Probe 5 preferably has one or more details described above, such as with respect to its length, Tm, and markings, provided that the markings on probe 5 are different from those selected for probe 3 and / or probe 4, thereby distinguishing probe 5 from other probes.

[0144] Those skilled in the art can readily select the concentrations of the various components in the reaction mixture. According to a particularly preferred embodiment, preferably in combination with the particularly preferred combination of primers and probes described above, the PCR reaction mixture contains primers, such as P1 and P2 or P3 and P4, at a concentration of about 400 nM; and probes 1 and 2 or probe 5 at a concentration of about 200 nM. In embodiments including an internal positive control (IPC), the PCR reaction mixture preferably further contains a control, such as probe 3 and optionally probe 4, at a concentration of about 200 nM. The IPC is preferably present in the PCR reaction mixture at an amount of about 200 copies. It should be noted that the concentrations shown refer to the corresponding concentrations in the final PCR reaction mixture used in the actual reaction. Those skilled in the art will readily understand that premixes or pre-mixtures of the reaction mixture or portions thereof can be prepared, wherein the concentrations deviate from the final concentrations described above. Such premixes or pre-mixtures are typically diluted with H2O and / or buffer solutions to obtain the final reaction mixture.

[0145] According to a preferred embodiment, the reaction mixture further comprises an enzyme for degrading unwanted DNA, such as residual and / or contaminating DNA. The preferred enzyme for degradation is UNGase. Therefore, according to a particularly preferred embodiment, in addition to the particularly preferred combination of primers, probes, and IPCs described above, the reaction mixture also comprises UNGase, preferably at a final concentration of 1 U / 100 μL of the reaction mixture.

[0146] The optimal combinations of primers, probes and other components used in polymerase chain reactions are listed in Table 3 below.

[0147] Table 3.1: Preferred concentrations in the reaction mixture for mycoplasma detection

[0148]

[0149] Table 3.2: Preferred concentrations in the reaction mixture for MMV detection

[0150]

[0151] The primer concentration for mycoplasma detection or MMV detection can be 400 nm for both primers, or alternatively 800 nm for both primers.

[0152] According to a particularly preferred embodiment of the present invention relating to mycoplasma detection, the concentrations of primers and probes are shown in Table 3.1 above, wherein primer 1 is Fw Myco7 and primer 2 is Rev Myco1. Probe 1 is Myco5 bis, probe 2 is Acho1, probe 3 (if present) is SAtPSY, probe 4 (if present) is StraA, and IPC gBlock (if present) contains SEQ ID NO: 68. Therefore, according to a particularly preferred embodiment, the present invention includes the combinations shown in Table 4.1 below.

[0153] Table 4.1: Preferred components and their preferred concentrations for mycoplasma detection

[0154]

[0155] According to a particularly preferred embodiment of the invention relating to MMV detection, the concentrations of primers and probes are as shown in Table 3.2 above, wherein primer 3 is Fw MMV, primer 4 is Rev MMV, probe 5 is probe MMV, probe 3 (if present) is SAtPSY, probe 4 (if present) is StraA, and IPC gBlock (if present) contains SEQ ID NO: 68. Therefore, according to a particularly preferred embodiment, the invention comprises the combinations shown in Table 4.2 below.

[0156] Table 4.2: Preferred components and their preferred concentrations for MMV detection

[0157]

[0158] According to a particularly preferred embodiment for detecting MMV, the reaction mixture comprises: primer 3 having SEQ ID NO: 104, primer 4 having SEQ ID NO: 105, probe 5 having SEQ ID NO: 106 and containing a fluorescent dye / quencher for FAM / IABkFQ, and mmv-IPC having SEQ ID NO: 68. The reaction mixture preferably further comprises probe 4 having SEQ ID NO: 71 and containing a fluorescent dye / quencher for HEX / IABkFQ. The reaction mixture optionally further comprises mmv-DPC having SEQ ID NO: 113 and preferably probe 3 having SEQ ID NO: 69 and containing a fluorescent dye / quencher for Cy5 / IABkRQ.

[0159] It should be understood that IPCs and DPCs can be present in the same reaction mixture as the PCR reaction mixture containing primers 1 and 2 or 3 and 4. Alternatively, IPCs and DPCs can be present in combination in separate reaction mixtures or separated in separate reaction mixtures.

[0160] The present invention further provides primer pairs comprising primer P1 annealed to the reverse complementary sequence of the nucleic acid sequence of SEQ ID NO: 1 and primer P2 annealed to the nucleic acid sequence of SEQ ID NO: 2. These primers can be used to detect mycoplasma in samples, and particularly in corresponding PCR assays for detecting mycoplasma as described herein. According to one embodiment, primers P1 and P2 have one or more of the properties described above for P1 and P2. P1 preferably comprises or consists of the nucleic acid sequence of any one of SEQ ID NO: 3 to 49. More preferably, primer P1 comprises or consists of the nucleic acid sequence according to SEQ ID NO: 9. Most preferably, P1 consists of the nucleic acid sequence according to SEQ ID NO: 9. Primer P2 preferably comprises or consists of the nucleic acid sequence of any one of SEQ ID NO: 50 to 52. More preferably, primer P2 comprises or consists of the nucleic acid sequence according to SEQ ID NO: 50. Most preferably, P2 consists of the nucleic acid sequence according to SEQ ID NO: 50.

[0161] The particularly preferred primer pair according to the invention comprises primer P1 consisting of SEQ ID NO: 9 and primer P2 consisting of SEQ ID NO: 50.

[0162] The present invention also provides primer pairs comprising primer P3 annealed to the reverse complementary sequence of the nucleic acid sequence of SEQ ID NO: 109 and primer P4 annealed to the nucleic acid sequence of SEQ ID NO: 110. These primers can be used to detect MMV in samples, and particularly in the corresponding PCR assays for detecting (and particularly MMV) as described herein. According to one embodiment, primers P3 and P4 have one or more of the properties described above for P3 and P4. P3 preferably comprises or is composed of the nucleic acid sequence of SEQ ID NO: 104. More preferably, P3 is composed of the nucleic acid sequence according to SEQ ID NO: 104. Primer P4 preferably comprises or is composed of the nucleic acid sequence of SEQ ID NO: 105. More preferably, primer P4 is composed of the nucleic acid sequence according to SEQ ID NO: 105.

[0163] The present invention further provides a kit for detecting mycoplasma in a sample. The kit comprises the primer pair of the present invention as described above, and at least a first nucleic acid probe (probe 1) annealed or bound to the nucleic acid sequence of SEQ ID NO: 53 or its reverse complementary sequence. According to one embodiment, the kit further comprises a second nucleic acid probe (probe 2) annealed or bound to SEQ ID NO: 53 or its reverse complementary sequence. Probe 1 and / or probe 2 preferably have one or more of the characteristics of probe 1 and probe 2 as described above. Probe 1 preferably comprises or is composed of the nucleic acid sequence of SEQ ID NO: 54 to 65. More preferably, probe 1 comprises or is composed of the nucleic acid sequence according to SEQ ID NO: 62, and most preferably, probe 1 is composed of the nucleic acid sequence according to SEQ ID NO: 62. Probe 1 may comprise one or more locked nucleic acids (LNAs). As a supplement to or alternative to LNAs, probe 1 may comprise one or more minor groove binding (MGB) moieties. According to a particularly preferred embodiment, probe 1 consists of the nucleic acid sequence according to SEQ ID NO: 62 and contains LNAs, preferably five LNAs, as shown in SEQ ID NO: 60 (underlined nucleotides represent LNA modifications). Probe 2 (if present) preferably contains or consists of the nucleic acid sequence of SEQ ID NO: 66. More preferably, probe 2 consists of the nucleic acid sequence according to SEQ ID NO: 66. Probe 2 may contain one or more LNAs. As a supplement to or alternative to LNAs, probe 2 may contain one or more small groove binding (MGB) moieties. According to a particularly preferred embodiment, probe 2 consists of the nucleic acid sequence according to SEQ ID NO: 66 and contains LNAs, preferably three LNAs, as shown in SEQ ID NO: 67 (underlined nucleotides represent LNA modifications).

[0164] According to a particularly preferred embodiment, the kit comprises primers P1, P2, probe 1, and probe 2 as described herein, more preferably primer P1 consisting of the nucleic acid sequence of SEQ ID NO: 9, primer P2 consisting of the nucleic acid sequence of SEQ ID NO: 50, probe 1 consisting of the nucleic acid sequence of SEQ ID NO: 60, and probe 2 consisting of the nucleic acid sequence of SEQ ID NO: 67. In this particularly preferred combination of primers and probes in the kit, probe 1 preferably comprises the fluorescent dye / quencher pair FAM / IABkFQ, and probe 2 preferably comprises the fluorescent dye / quencher pair FAM / IABkFQ.

[0165] The kit may further comprise one or more of the following: a PCR reaction mixture as described above, an internal positive control nucleic acid sequence (IPC) as described above, a differential positive control nucleic acid sequence (DPC) as described above, tools for extracting and / or purifying DNA from a sample as described above, and instructions for PCR using primers. If the IPC and / or DPC are part of the kit of the present invention, the kit preferably further comprises probe 3 and probe 4 as described herein, for alignment or binding to the IPC and DPC, respectively.

[0166] Using the present invention, namely the method of the present invention, the primer set of the present invention, or the kit of the present invention, mycoplasma in samples can be detected. The detectable mycoplasma are preferably one or more of the following mycoplasma species: *Mycoplasma argininae*, *Mycoplasma buccalis*, *Mycoplasma hominis*, *Mycoplasma oralis*, *Mycoplasma salivaria*, *Mycoplasma fermentans*, *Mycoplasma hyoides*, *Mycoplasma synovitis*, *Mycoplasma pneumoniae*, *Acholesterolus reesei*, *Mycoplasma gallisepticum*, and *Citrus spirochetes*.

[0167] The present invention further provides a kit for detecting MMV in a sample. The kit comprises the primer pair of the present invention as described above, and a nucleic acid probe (probe 5) annealed to the nucleic acid sequence of SEQ ID NO: 111 or its reverse complementary sequence.

[0168] Probe 5 preferably has one or more of the properties of probe 5 as described above. Probe 5 preferably comprises or is composed of the nucleic acid sequence of SEQ ID NO: 106. Probe 5 may comprise one or more locked nucleic acids (LNAs). As a supplement to or alternative to the LNA, probe 5 may comprise one or more minor groove binding (MGB) moieties. According to a particularly preferred embodiment, probe 5 is composed of the nucleic acid sequence of SEQ ID NO: 106 and comprises an LNA. As a supplement to or alternative to the LNA, probe 5 may comprise one or more minor groove binding (MGB) moieties.

[0169] According to a particularly preferred embodiment, the kit comprises primer P3, primer P4, and probe 5 as described herein, more preferably primer P3 consisting of the nucleic acid sequence of SEQ ID NO: 104, primer P4 consisting of the nucleic acid sequence of SEQ ID NO: 105, and probe 5 consisting of the nucleic acid sequence of SEQ ID NO: 106. In this particularly preferred combination of primers and probes in the kit, probe 5 preferably comprises a fluorescent dye / quencher pair FAM / IABkFQ.

[0170] According to one embodiment, the kit further comprises one or more of the following: a PCR reaction mixture as described above, an internal positive control nucleic acid sequence as described above, a differential positive control nucleic acid sequence as described above, tools for extracting and / or purifying DNA from a sample as described above, and instructions for performing PCR with primers. If the IPC and / or DPC (such as mmv-IPC and / or mmv-DPC as described above) is part of the kit of the present invention, the kit preferably further comprises probe 5, probe 3, and / or probe 4 as described herein.

[0171] According to one embodiment, in the method according to the invention, in the primer pair according to the invention, or in the kit according to the invention, the virus is selected from the group consisting of: Anelloviridae, Inoviridae, Parvoviridae, preferably Parvovirus, Erythovirus, Dependovirus, Amdovirus, and Bocavirus, more preferably Parvovirus, and most preferably Mouse parvovirus.

[0172] This document also discloses combinations of primer sets or kits for detecting mycoplasma and primer sets or kits for detecting viruses. In other words, the present invention also provides methods, primers, and kits for detecting mycoplasma and viruses in samples. According to one embodiment, the detection of mycoplasma and viruses is performed simultaneously, preferably in the same reaction vessel (such as a PCR tube). According to an alternative embodiment, the detection of mycoplasma and viruses is performed subsequently, preferably in different reaction vessels (such as PCR tubes). Example

[0173] Materials and methods

[0174] All sequences mentioned in this article were retrieved from the SILVA and NCBI websites (www.arb-silva.de and www.ncbi.nlm.nih.gov). Sequence analysis was performed using Vector NTI, Snap Gene, and Geneious software. Primer thermodynamic calculations (Tm, primer dimer formation, and hairpin Tm) were performed using PrimerExpress. ® 3.0 (Life Technologies), Geneious (Geneious Prime 2023.2; Dortmund Martix), and IDT (Integrated DNA Technologies, Inc.) online software. Settings are as follows:

[0175] • PrimerExpress: Standard Settings

[0176] •Geneious: Monovalent ion 50 mM; Divalent ion 3 mM; Primer 200 nM; dNTP 0.8 mM

[0177] •IDT: Parameter set, qPCR; Monovalent ion 50 mM; Divalent ion 3 mM; Primer 200 nM; dNTP 0.8 mM.

[0178] Similar parameters are used for probe thermodynamic calculations.

[0179] Example 1: Identification of regions used for primer design

[0180] The organisms used to identify primer design regions are listed in Table 5 below. These include mycoplasma species and other pathogens whose presence should be investigated in various cell substrates taken from the production workflow (master cell bank, working cell bank, unprocessed harvest fluid, end-of-production cells), as indicated in various guidelines.

[0181] Table 5: Organisms used to identify primer design regions

[0182]

[0183] To identify the genomic regions to be targeted by PCR and ensure specific mycoplasma detection while excluding signals from other organisms, 16S and 23S ribosomal RNA sequences from the organisms listed in Table 5 were retrieved from the SILVA and NCBI websites (www.arb-silva.de and www.ncbi.nlm.nih.gov) and compared. Sequences from 23S rRNA were excluded because they were too unique and therefore unsuitable for identifying virtually all mycoplasma species with only a limited number of primers and probes. On the other hand, 16S RNA showed some specificity but retained sufficient homology across mycoplasma species. Therefore, it was decided to focus on 16S rRNA sequences to identify regions common to all mycoplasma species, regions that should be recognizable by the primer and probe set, and further, significantly different from all other species. Using such sequence segments ensured specific mycoplasma detection without cross-reactivity with DNA from other species.

[0184] The first analysis focused on the mycoplasma 16S rRNA sequence, aiming to identify a 100 bp fragment with 80% homology across species. Primers and probes of 20 and 23 nucleotides respectively could be designed for PCR detection of mycoplasma. Since *Escherichia coli* is the closest organism to the *Mycoplasma* genus, domains with high homology to *E. coli* 16S rRNA were ignored to ensure no cross-reactivity with *E. coli* and other organisms listed in Table 5. As shown in Figure 2, three sequence segments meeting the above criteria were identified.

[0185] In the second analysis, sequences were analyzed within the three identified regions. Surprisingly, although all three regions showed homology among mycoplasma species, Figure 1 Primers and probes could not be designed in the first two regions highlighted in the figure (i.e., the region between 500 and 600 bp, and the region around 800 bp). Instead, primers and probes were designed in a 1100 bp sequence segment on the 16S rRNA in the third region, which necessitated widening the region to accommodate all three types of forward primers, probes, and reverse primers. Details of this region are shown in Figure 2.

[0186] Example 2: Primer Design

[0187] Several primers were designed for the identified region, and PCR fitness was determined. Regarding forward primers, strictly identical sequences across species could not be identified, and primers capable of accommodating mismatches with the target DNA had to be selected. As shown in Table 6 below, several primers were selected, depending on the mycoplasma target sequence, exhibiting one to three mismatches. A selection filter based on eliminating primers with unfavorable primer dimer formation and primers with heat-resistant hairpin structures (Tm > 50°C) resulted in the selection of a limited number of primers for subsequent experiments.

[0188] Table 6: Characteristics of forward primers

[0189]

[0190] A similar approach led to the identification of reverse primers with the sequences and characteristics shown in Table 7 below. Primer RevMyco1 It was selected because it exhibits the best Tm standard.

[0191] Table 7: Characteristics of reverse primers

[0192]

[0193] The forward and reverse primers that met all criteria are presented in Table 8 below. For clarity, it is assumed that there is no mismatch with the target when calculating Tm.

[0194] Table 8: Forward and reverse primers used for experimental evaluation.

[0195]

[0196] Example 3: Primer evaluation for various organisms using dye-incorporated PCR

[0197] Before designing, selecting, and optimizing probes for more specific TaqMan PCR, primers are first tested in SYBRgreen-based PCR to select the best set.

[0198] DNA samples from different organisms were obtained as described in Table 9 below. For some DNA samples, lyophilized genomic DNA was obtained at the concentration indicated by the manufacturer (10 ng per tube). Alternatively, lyophilized qPCR standards provided in 10⁸ copies / tube were used. For E. coli, Homo sapiens, and CHO samples, lyophilized genomic DNA was purchased and packaged in 7 × 10⁸ ng / tube containers. 10 7×10 9 and 1.6×10 6 Resuspend 1 copy of genome per µL. In all cases, dilute the DNA solution to achieve 3 × 10⁻⁶. 3 The concentration of one gene copy / µL was used for primer characterization.

[0199] Table 9: Biological and DNA Sources Used for Primer Evaluation

[0200]

[0201] All forward primers and the unique reverse primer RevMyco1 as shown in Table 8 Together, they were used in the PCR reaction. For each reaction, 20,000 copies of genomic DNA were used. The obtained PCR Cq values ​​are presented in Table 10 below.

[0202] Table 10: Cq values ​​of primer combinations used in qPCR experiments on reference DNA samples of specified organisms.

[0203]

[0204] Forward primers 27, 30, 39, and 46 produced the highest Cq values ​​across all studied species. Therefore, the optimal set of forward primers 7, 11, 14, and 17 was selected. qPCR using forward primer 7 (Fwd7) showed Cq values ​​significantly lower than 20 in all organisms except Mycoplasma hominis. Analysis of the PCR reactions by PAGE and melting curves further confirmed the presence of a single amplicon of the expected size and Tm in each reaction. Figure 3 ).

[0205] Example 4: Hydrolysis Probe Design

[0206] The target sequence for the probe was selected in the region shown in Figure 2. The task was to cover homologous sequences in mycoplasma species that significantly mismatch with other organisms. Due to the nature of DNA sequences, it was not possible to design probes of 23 nucleotides with a Tm of approximately 70°C. Therefore, attempts were made to design longer probes of up to 40 nucleotides, but such probes always covered non-homologous sequences in the 5' region. To overcome these limitations, two strategies were followed: 1) generating long probes with internal quenchers (ZEN probes), and 2) using short probes (approximately 23 nucleotides) and increasing the Tm by using chemical modifications such as locked nucleic acid (LNA) or minor groove binding (MGB) moieties to enhance probe / target interactions. Common long probes, probes with heat-resistant hairpin structures (Tm > approximately 45°C), and probes that were unfavorable for primer dimer formation were excluded. The designed probes are listed in Table 11 below.

[0207] Table 11: Hydrolysis probes designed for experimental evaluation

[0208]

[0209] Example 5: Probe evaluation of various organisms using PCR based on hydrolysis probes

[0210] With the unique Fwd7 / Rev1 The primer set (Table 10) was tested against the probes selected from Table 11 to determine the optimal primer / probe combination. As shown in Tables 12 and 13 below, all mycoplasma species except *Acholestearia repens* were detected by both LNA and MGB probes, although the *Smyco5* MGB probe failed to detect *Mycoplasma gallisepticum* DNA. No background signal or only a limited background signal (Cq > 37) was generated on DNA samples from other species.

[0211] Table 12: Evaluation of LNA and MGB probes on mycoplasma DNA samples

[0212]

[0213] Table 13: Evaluation of LNA and MGB probes on DNA samples from other organisms

[0214]

[0215] The SAcho1LNA probe, designed to be more specific to *Achoptophanaplasma reesei*, was also evaluated in similar assays. As shown in Tables 14 and 15 below, this probe successfully detected amplification of *Achoptophanaplasma reesei* DNA without detecting any other DNA samples and without background detection on DNA from other species. Furthermore, when used in a single mixture, both the Smyco5bis LNA and SAcho1LNA probes detected DNA from all mycoplasma species without background detection on DNA from other species.

[0216] Table 14: Evaluation of LNA probes on mycoplasma DNA samples

[0217]

[0218] Table 15: Evaluation of LNA probes on DNA samples from other organisms

[0219]

[0220] The preferred combinations of primers and probes for mycoplasma detection are shown in Table 16 below.

[0221] Table 16: Preferred primer / probe combinations

[0222]

[0223] Example 6: Control probe

[0224] Synthesis with Figure 4 The two gBlocks (double-stranded DNA fragments) of the structure described in Table 17 were used. An IPC (internal positive control) containing a sequence that allows for the annealing of mycoplasma PCR primers and unique probes (TraA) was added to all samples. This construct generated a positive signal in the StraA channel of all samples and served as a positive PCR control.

[0225] In a separate reaction, a DPC (discriminatory positive control) construct is used instead of the sample DNA, containing a sequence that allows annealing of the mycoplasma PCR primers, the mycoplasma-specific probe, and another unique probe (AtPSY). This construct generates a positive signal in the mycoplasma probe channel, confirming the presence of the mycoplasma-specific probe in the PCR mixture. Furthermore, fluorescence in the AtPSY probe channel should be detectable only in that sample. A positive result in the AtPSY probe channel indicates that the sample was contaminated with this construct.

[0226] All control constructs were purchased from IDT as double-stranded gBlocks.

[0227] Table 17: IPC and DPC sequences

[0228]

[0229] Example 7: Probe modification and PCR reaction parameters

[0230] All primers and probes were purchased from IDT. Probes were linked to fluorescent dyes and quenchers, as shown in Table 18.

[0231] Table 18: Probe sequences and chemical modifications

[0232]

[0233] The underlined nucleotide is an LNA modification.

[0234] Prepare the PCR premix and perform the PCR reaction as described in Table 19. Add 5 µL of unknown DNA sample or DPC. The code UNGase was purchased from ArcticZymes.

[0235] Table 19: PCR reaction mixture

[0236]

[0237] The loop conditions used are described in Table 20 below.

[0238] Table 20: Cycling conditions for PCR

[0239]

[0240] Example 8: Determining the limits of detection and quantitation

[0241] As described above, probes and primers were developed using purified genomic DNA (15,000 to 20,000 genomic copies per reaction) at known concentrations for use in PCR reactions. However, guidelines require assays capable of detecting a defined number of colony-forming units (CFU) / mL. Therefore, lyophilized mycoplasma samples with known CFU content were purchased from suppliers, as shown in Table 21 below, and tested together with various amounts of reference DNA (see Table 6) to link CFU to genomic copies and determine the limits of quantitation (LOQ) and detection (LOD) of the method of the present invention, expressed in CFU / mL.

[0242] Table 21: Mycoplasma colony reference standards for confirming LOD

[0243]

[0244] In the first step, the LOQ for this method was determined for each reference DNA sample listed in Table 9. In short, reference DNA of known concentrations was diluted, and PCR was performed as described herein to associate Cq with DNA copies. The results obtained for each mycoplasma DNA sample are shown in Table 22 below. All samples were positive in the IPC channel at the expected Cq value (data not shown). The LOQ for all mycoplasmas was set to the penultimate reference DNA dilution to which the qPCR software could calculate the Cq value. Therefore, all LOQs were set at 100 mycoplasma genome copies per reaction, except for Mycoplasma synovitis, where the LOQ was set at 1,000 mycoplasma genome copies per reaction.

[0245] Table 22: Calibration curves for mycoplasma reference DNA

[0246]

[0247] In the second step, the method was applied to the colony reference standards listed in Table 21, and all Cq values ​​were determined. The equivalence between CFUs and genomic copies was then determined using equations calculated from the calibration curves (Table 22). The number of CFUs corresponding to the LOQ determined by this method on the reference DNA standards was then calculated as the LOQ in CFU / mL, considering a maximum sample volume of 5 µL. As shown in Table 23 below, the LOQ and LOD are <100 and <10 CFU / mL, respectively. LOD < 10 CFU / mL meets the requirements described in Chapter 2.6.7 of the European Pharmacopoeia.

[0248] Table 23: Method LOQ and LOD determined on colony reference standards

[0249]

[0250] Example 9: Identification of genomic regions to be targeted by PCR

[0251] The organisms studied are listed in Table 24 below. These include four MMV serotypes (p, i, m, and c) and parvoviruses from closely related species, the presence of which should be investigated in various cell substrates taken from the production workflow: upstream and downstream processing (for virus clearance), master cell bank, working cell bank, unprocessed harvest fluid, and end-of-production cells for virus safety, as shown in various guidelines.

[0252] Table 24 - Sequences of the organisms involved in this study.

[0253]

[0254] To identify the optimal genomic regions to be targeted by PCR and to ensure specific MMV detection, the complete genomes of parvoviruses listed in Table 24 were retrieved from the NCBI website (www.ncbi.nlm.nih.gov) and compared.

[0255] The first round of analysis focused on the MMV genome, aiming to identify a 300 bp fragment with 80% homology across the genome, for which primers and hydrolysis probes of 20 and 23 nucleotides respectively could be designed. Figure 5 As shown, only one fragment meeting the above criteria was identified. A highly conserved region of approximately 315 bp (NC_001510.1) was identified at positions 2002-2316. The coding sequences for the proteins NS1, NS2, VP1, VP2, VP3, and VP4 are located at this strategic position, precisely before the “VP intron,” where various primers can be designed for future RT-qPCR.

[0256] In the second filtering method, sequences were analyzed within this 315 bp domain, where primers and probes could be designed for all serotypes, with Tm values ​​of 60°C and 70°C, respectively. Sequences generating primer dimers and hairpin structures >50°C and amplicon >100 bp were excluded. Once selected, the region of interest was again examined against the parvovirus sequences listed in Table 24. Details of this region are presented in... Figure 6 middle.

[0257] Example 10: Primer Design

[0258] The design and testing primer sets and probes are listed in Table 25 below.

[0259] Table 25: List of primers and probes and their properties

[0260]

[0261] The hairpin Tm was higher than the usual choice for probe design (>50°C), but it was decided to keep the sequence close to the target end of the ns1 gene. Table 26 shows the probe sequence and chemical modifications (if introduced).

[0262] Table 26: Probe sequences and chemical modifications

[0263]

[0264] The sequences used for both MMV and mycoplasma assays are shown in Table 27 below.

[0265] Table 27: List of common sequences used for MMV and mycoplasma assays

[0266]

[0267] Figure 7 The different positions of primers and probes on the control construct are shown.

[0268] Example 11: Qualification of MMV Detection Method

[0269] To demonstrate the specificity of the method, various gDNA extracts were used as matrices for parallel amplification of MMV vDNA (see Table 28 below). A specific triple assay was developed to allow detection of these gDNAs in samples where the MMV method specificity was being tested. MMV and gDNA quantifications were performed simultaneously and on the same plate using two PCR mixtures (mixture 1 targeting MMV and mixture 2 simultaneously targeting human, E. coli, and CHO gDNA). For comparison, 1,500 to 2,000 copies of each target were added to the matrix, taking into account the genomic type of each species (dsDNA, ssDNA, haploid, or diploid). Amplification results are shown in... Figure 8 and 9 middle. Figure 8 The amplification curves in the MMV mixture 1 were as expected: it amplified neither human gDNA nor E. coli or CHO gDNA. It only amplified the desired target at specific channels. For IPCs (Cy5 channels), not all amplifications reached the same plateau. This is due to probe and primer consumption attributable to the heterogeneity of the method design. This is not a problem because all Cqs are similar and, as expected, within SD < 0.5. Similarly, Figure 9 The amplification curves were as expected: gDNA mixture 2 amplified neither MMV, DPC, nor IPC. It amplified only gDNA, and amplification was observed in specific channels. In summary, this method is specific for MMV.

[0270] Table 28: Materials used as DNA matrices for method evaluation

[0271]

[0272] Subsequently, the amplification products were analyzed by 15% PAGE to assess the quality of the amplification. The results are shown below. Figure 10The visualization of the amplification products clearly shows the results for both the MMV mixture and the gDNA mixture. The number and size of the observed bands are consistent with the expected results. For PCR mixture 1, the IPC band (approximately 150 bp) was present in all wells (01 to 07) but not in the negative control well 08 (an MMV mixture containing neither DNA nor IPC). Well 02 contained a viral band of approximately 70 bp in addition to the IPC band, and well 03 contained a distinguishing band of approximately 90 bp. Wells 05, 06, and 07, which were gDNA, did not produce any other significant bands. Primer dimer bands were observed in all wells (01 to 08). For PCR mixture 2, several primer dimer bands were observed because no primer optimization was performed. The assay was designed to check for the presence of gDNA in the assay. No significant bands were observed in wells 09 to 12, which were reagents involved in the MMV assay. Different sizes of the expected bands were observed at approximately 59 bp in well 13 (human), approximately 62 bp in well 14 (E. coli), and approximately 81 bp in well 15 (CHO). 15% PAGE analysis confirmed that the qPCR amplification products were specific for MMV.

[0273] The detection limit of the qPCR method is defined as one copy per target DNA reaction. To determine the detection limit of this method, 10-fold serial dilutions of DPC and IPC were performed, down to approximately 0.08 copies / reaction. The last seven spots (referred to as G4 to G10) were analyzed in six copies. For each dilution, at least three replicates must be performed to account for the signal. The results are summarized in Tables 29 and 30 below.

[0274] Table 29: Average Cq values ​​of DPC series diluents

[0275]

[0276] Table 30: Average Cq values ​​of IPC series diluents

[0277]

[0278] In summary, the detection limit for both samples is fixed at one copy per reaction.

[0279] To establish the limit of quantitation, data from sample preparation as described in Example 11 above were used. Calibration curves were plotted for points (G4 to G8) where SD < 0.5. Intercepts for all targets were within the same range (mean 39.43), with an SD of 0.08 (< 0.5). While maintaining the last point G8 with SD > 0.5 for the Cy5 target, all parameters met the acceptance criteria for all targets.

[0280] The slope is -3.32 (±0.3) and r² > 0.98. (For example...) Figure 11 As shown, all curves are perfectly superimposed, with a slight increase in SD at lower concentrations. As expected, this observation is primarily observed for the Cy5 reporter gene. Table 31 below shows the variability at the lower point when there are eight copies. In summary, the limit of quantitation is estimated at ten copies per reaction.

[0281] Table 31: Variance at lower points: 8 copies

[0282]

[0283] The robustness of this method was evaluated by performing a 10-fold dilution of the virus sample. Then approximately 4 × 10⁻⁶ ppm was added. 5 DNA was extracted from cells or TE (10; 0.1) at pH 8.0. The extract was quantified by droplet digital PCR to determine TCID. 50 The / mL value is correlated with the copy number / µL unit. Each extracted DNA sample is then cycled in triplicate. For compliance, no significant difference in Cq values ​​of MMV extracted with cells or TE (10; 0.1) pH 8.0 should be observed. Additionally, ΔCq should be <1. Results are summarized in Tables 32 to 35 below.

[0284] Table 32: Extraction Series Dilutions

[0285]

[0286] At each dilution point, extractions of MMV with either added cells or TE returned similar Cq values. ΔCq < 0.5 was observed at 5 of the 6 dilution points, and ΔCq < 1 for all. This means that extraction of viral genetic material from either the buffer (TE) or the composite medium (cell suspension) had no significant impact on MMV detection and quantification.

[0287] Table 33: Dilution Curve Parameters

[0288]

[0289] Table 33 shows that, although this is an extraction, these parameters fully meet the specifications of the calibration curves described above. These observations lead to the conclusion that this method is robust.

[0290] The intercept value will depend on the unit of the variable x used for the curve, in this case, the infectious "TCID". 50 / mL". This unit is not typically used for qPCR, which is why it is preferred to reformat Tables 32 and 33 with the genomic equivalent "copy" unit determined by absolute quantification via ddPCR, as shown in Tables 34 and 35 below.

[0291] Table 34: Reformatted Extraction Series Dilutions

[0292]

[0293] Table 35: Reformatted dilution curve parameters (“copy” means a copy of each reaction)

[0294]

[0295] The reformatted table does not show changes to Cq, SD, slope, r², and E. As expected, only the intercept values ​​were modified in the calculations, and they are now very close to those shown in Table 33.

[0296] The calibration curves for two different data units are shown in Figure 11 In the meantime, a 10 Cq drift was observed after data reformatting, which translates to an absolute difference of approximately 3 Log10.

[0297] Considering MMV CoA and quantification, a relationship between infectious TCID50 / mL and genome copies / µL was established, which was specific to the analyzed viral samples. MMV virus stock solutions were prepared at 1.6 × 10⁻⁶. 6 TCID 50 Titrated at 2.24 × 10⁹ / mL by ddPCR. 11 Copy / mL of the same solution. This means one TCID 50 / mL equals 139,805 copies / mL (approximately 140 copies / µL).

Claims

1. A method for detecting microorganisms in a sample, the method comprising: (i) Performing PCR on a sample suspected of containing microbial DNA using a PCR reaction mixture, the PCR reaction mixture comprising: (1) A first primer (P1) annealed to the reverse complementary sequence of the nucleic acid sequence of SEQ ID NO: 1 and a second primer (P2) annealed to the nucleic acid sequence of SEQ ID NO: 2; and / or 2) The third primer (P3) annealed to the reverse complementary sequence of the nucleic acid sequence of SEQ ID NO: 109 and the fourth primer (P4) annealed to the nucleic acid sequence of SEQ ID NO: 110; and (ii) Detect PCR products The presence of PCR products indicates that the sample contains microorganisms, while the absence of PCR products indicates that the sample does not contain microorganisms.

2. A method for detecting mycoplasma in a sample, the method comprising: (i) PCR was performed on a sample suspected of containing mycoplasma DNA using a PCR reaction mixture comprising a first primer (P1) annealed to the reverse complementary sequence of the nucleic acid sequence of SEQ ID NO: 1 and a second primer (P2) annealed to the nucleic acid sequence of SEQ ID NO: 2; and (ii) Detect PCR products The presence of PCR products indicates that the sample contains mycoplasma, while the absence of PCR products indicates that the sample does not contain mycoplasma.

3. The method according to claim 2, wherein: (i) P1 has a length between 18 and 24 nucleotides, preferably between 19 and 21 nucleotides, and most preferably 19 nucleotides, and / or P2 has a length between 17 and 22 nucleotides, preferably between 18 and 20 nucleotides, and most preferably 20 nucleotides; and / or (ii) P1 has a Tm between 60°C and 70°C, preferably between 63°C and 69°C, more preferably between 66°C and 68°C, and / or P2 has a Tm between 60°C and 67°C, preferably between 62°C and 66°C, and most preferably between 64°C and 65°C; and / or (iii) P1 contains or is composed of the nucleic acid sequence of SEQ ID NO: 3 to 49 and / or P2 contains or is composed of the nucleic acid sequence of SEQ ID NO: 50 to 52.

4. The method according to claim 2 or 3, wherein the reaction mixture further comprises at least a first nucleic acid probe (probe 1) annealed to the nucleic acid sequence of SEQ ID NO: 53 or its reverse complementary sequence.

5. The method according to claim 4, wherein: (i) The reaction mixture further comprises at least a second nucleic acid probe (probe 2) annealed with SEQ ID NO: 53; and / or (ii) Probe 1 and / or probe 2 each have a length between 20 and 42 nucleotides, preferably between 21 and 30 nucleotides, and most preferably between 22 and 24 nucleotides; and / or (iii) Probe 1 and / or probe 2 each have a Tm between 55°C and 70°C, preferably between 58°C and 69°C, more preferably between 59°C and 68°C; and / or (iv) Probe 1 contains or is composed of the nucleic acid sequence of SEQ ID NO: 54 to 65 and / or probe 2 contains or is composed of the nucleic acid sequence of SEQ ID NO: 66; and / or (v) Probe 1 and / or probe 2 each contain one or more locked nucleic acid (LNA) and / or minor groove binding (MGB) moieties; and / or (vi) Probe 1 and / or probe 2 each contain a detectable marker, preferably wherein probe 1 and probe 2 contain the same detectable marker.

6. The method according to any one of claims 1 to 5, wherein: (i) The PCR is a qPCR, dPCR, ddPCR, or cdPCR; and / or (ii) The PCR uses an annealing temperature between 55°C and 65°C, preferably between 58°C and 60°C; and / or (iii) The PCR includes an activation step and / or a degradation step; and / or (iv) Annealing and elongation are carried out at the same temperature; and / or (v) The PCR consists of 40 to 50 denaturation and annealing / elongation cycles, preferably 45 cycles.

7. The method according to any one of claims 1 to 6, wherein the reaction mixture further comprises an internal positive control nucleic acid sequence comprising at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 1 and at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 2, and an intercalation nucleic acid sequence of at least 18 nucleotides in length between the two nucleic acid sequences, wherein the intercalation nucleic acid sequence is preferably different from SEQ ID NO: 53 in that probe 1 and probe 2 are not annealed to the intercalation nucleic acid sequence, wherein the reaction mixture preferably further comprises at least a third nucleic acid probe (probe 3) annealed to the intercalation nucleic acid sequence or its reverse complementary sequence.

8. The method according to claim 7, wherein i) Probe 3 has a length between 20 and 42 nucleotides, preferably between 21 and 30 nucleotides, and most preferably between 22 and 24 nucleotides; and / or (ii) The probe 3 has a Tm between 55°C and 70°C, preferably between 58°C and 69°C, more preferably between 59°C and 68°C; and / or (iii) Probe 3 contains the nucleic acid sequence of SEQ ID NO: 69 or its reverse complementary sequence, or is composed of the nucleic acid sequence of SEQ ID NO: 69 or its reverse complementary sequence; and / or (iv) Probe 3 contains one or more locked nucleic acid (LNA) and / or minor groove binding (MGB) moieties; and / or (v) Probe 3 contains a detectable marker that is different from the markers on probe 1 and probe 2.

9. The method according to claim 7 or 8, wherein the internal positive control nucleic acid sequence comprises or consists of the following: a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 68 or its reverse complementary sequence.

10. The method according to any one of claims 1 to 9, wherein the reaction mixture further comprises a differential positive control nucleic acid sequence comprising at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 1 and at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 2, and an intercalation nucleic acid sequence of at least 18 nucleotides in length between the two nucleic acid sequences, wherein the intercalation nucleic acid sequence is preferably identical to SEQ ID NO: 53, such that probe 1 and probe 2 can anneal with the intercalation nucleic acid sequence, wherein the reaction mixture preferably further comprises at least a fourth nucleic acid probe (probe 4) annealing with the intercalation nucleic acid sequence or its reverse complementary sequence at a sequence different from the sequence annealed with probe 1 and / or probe 2.

11. The method of claim 10, wherein i) Probe 4 has a length between 20 and 42 nucleotides, preferably between 21 and 30 nucleotides, and most preferably between 22 and 24 nucleotides; and / or (ii) The probe 4 has a Tm between 55°C and 70°C, preferably between 58°C and 69°C, more preferably between 59°C and 68°C; and / or (iii) Probe 4 contains the nucleic acid sequence of SEQ ID NO: 71 or its reverse complementary sequence, or is composed of the nucleic acid sequence of SEQ ID NO: 71 or its reverse complementary sequence; and / or (iv) Probe 4 contains one or more locked nucleic acid (LNA) and / or minor groove binding (MGB) moieties; and / or (v) Probe 4 contains a detectable marker that is different from the markers on probes 1, 2 and 3.

12. The method according to claim 10 or 11, wherein the differential positive control nucleic acid sequence comprises or consists of the following: A nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 70 or its reverse complementary sequence.

13. The method according to any one of claims 1 to 12, wherein the sample is a biological sample, preferably selected from the group consisting of: cell banks, culture media, cell cultures, cell culture supernatants, pharmaceutical products, vaccine preparations, blood, saliva, and sputum.

14. A primer pair for detecting mycoplasma in a sample, the primer pair comprising: primer P1 annealed to the reverse complementary sequence of the nucleic acid sequence of SEQ ID NO: 1 and primer P2 annealed to the nucleic acid sequence of SEQ ID NO:

2.

15. A kit for detecting mycoplasma in a sample, wherein the kit comprises the primer pair according to claim 9 and a first nucleic acid probe (probe 1) annealed to the nucleic acid sequence of SEQ ID NO: 53 or its reverse complementary sequence, and optionally a second nucleic acid probe (probe 2) annealed to SEQ ID NO: 53 or its reverse complementary sequence, preferably wherein the kit further comprises one or more of the following: (i) PCR reaction mixture; (ii) An internal positive control nucleic acid sequence comprising at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 1 and at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 2, and an intercalation nucleic acid sequence of at least 18 nucleotides in length between the two nucleic acid sequences, wherein the intercalation nucleic acid sequence is preferably different from SEQ ID NO: 53 in that neither probe 1 nor probe 2 is annealed to the intercalation nucleic acid sequence, wherein the kit preferably further comprises at least a third nucleic acid probe (probe 3) annealed to the intercalation nucleic acid sequence or its reverse complementary sequence. (iii) A differential positive control nucleic acid sequence comprising at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 1 and at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 2, and an intercalation nucleic acid sequence of at least 60 nucleotides in length between the two nucleic acid sequences, wherein the intercalation nucleic acid sequence is preferably identical to SEQ ID NO: 53, such that probe 1 and probe 2 can anneal to the intercalation nucleic acid sequence, wherein the kit preferably further comprises at least a fourth nucleic acid probe (probe 4) which anneals to the intercalation nucleic acid sequence or its reverse complementary sequence at a position different from the annealing of probe 1 and / or probe 2; (iv) Tools used for extracting and / or purifying DNA from the sample; and (v) Instructions for using primers for PCR.

16. The method according to any one of claims 1 to 13, the primer pair according to claim 14, or the kit according to claim 15, wherein the mycoplasma is one or more mycoplasma species selected from the group consisting of: Mycoplasma argininae, Mycoplasma buccalis, Mycoplasma hominis, Mycoplasma oralis, Mycoplasma salivariae, Mycoplasma fermentans, Mycoplasma hyoidosaurus, Mycoplasma synovitis, Mycoplasma pneumoniae, Acholestylasma reesei, Mycoplasma gallisepticum, and Mycoplasma citrinum.

17. A method for detecting a virus in a sample, the method comprising: (i) PCR was performed on a sample suspected of containing viral DNA using a PCR reaction mixture comprising a third primer (P3) annealed to the reverse complementary sequence of the nucleic acid sequence of SEQ ID NO: 109 and a fourth primer (P4) annealed to the nucleic acid sequence of SEQ ID NO: 110; and (ii) Detect PCR products The presence of PCR products indicates that the sample contains the virus, while the absence of PCR products indicates that the sample does not contain the virus.

18. The method of claim 17, wherein: (i) P3 has a length between 20 and 25 nucleotides, preferably between 21 and 24 nucleotides, and most preferably 23 nucleotides, and / or P4 has a length between 15 and 21 nucleotides, preferably between 16 and 20 nucleotides, and most preferably 23 nucleotides; and / or (ii) P3 has a Tm between 60°C and 70°C, preferably between 63°C and 69°C, more preferably between 66°C and 68°C, and / or P4 has a Tm between 60°C and 67°C, preferably between 62°C and 66°C, and most preferably between 64°C and 65°C; and / or (iii) P3 contains or is composed of the nucleic acid sequence of SEQ ID NO: 104 and / or P4 contains or is composed of the nucleic acid sequence of SEQ ID NO:

105.

19. The method according to claim 17 or 18, wherein the reaction mixture further comprises at least one nucleic acid probe (probe 5) annealed to the nucleic acid sequence of SEQ ID NO:111 or its reverse complementary sequence.

20. The method of claim 19, wherein: (i) The probe 5 has a length between 15 and 30 nucleotides, preferably between 18 and 22 nucleotides, and most preferably between 19 and 21 nucleotides; and / or (ii) The probe 5 has a Tm between 59°C and 71°C, preferably between 60°C and 70°C, more preferably between 63°C and 67°C; and / or (iii) Probe 5 contains or is composed of the nucleic acid sequence of SEQ ID NO: 106; and / or (iv) Probe 5 contains one or more locked nucleic acid (LNA) and / or minor groove binding (MGB) moieties; and / or (v) Probe 5 contains a detectable marker.

21. The method according to any one of claims 17 to 20, wherein: (i) The PCR is a qPCR, dPCR, ddPCR, or cdPCR; and / or (ii) The PCR uses an annealing temperature between 55°C and 65°C, preferably between 58°C and 60°C; and / or (iii) The PCR includes an activation step and / or a degradation step; and / or (iv) Annealing and elongation are carried out at the same temperature; and / or (v) The PCR consists of 40 to 50 denaturation and annealing / elongation cycles, preferably 45 cycles.

22. The method according to any one of claims 17 to 21, wherein the reaction mixture further comprises an internal positive control nucleic acid sequence comprising at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 68 or 112 or 115.

23. The method of claim 22, wherein the internal positive control nucleic acid sequence comprises or consists of the following: A nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 68 or 112 or 115 or its reverse complementary sequence.

24. The method according to any one of claims 17 to 23, wherein the reaction mixture further comprises a differential positive control nucleic acid sequence comprising at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 109 and at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 110, and an intercalation nucleic acid sequence of at least 18 nucleotides in length between the two nucleic acid sequences, wherein the reaction mixture preferably further comprises at least a third nucleic acid probe (probe 3) and / or a fourth nucleic acid probe (probe 4), the third nucleic acid probe and / or the fourth nucleic acid probe being annealed to the intercalation nucleic acid sequence or its reverse complementary sequence at a sequence different from the sequence annealed to probe 5.

25. The method of claim 24, wherein a) Probe 3: (ai) has a length between 20 and 42 nucleotides, preferably between 21 and 30 nucleotides, and most preferably between 22 and 24 nucleotides; and / or (aii) has a Tm between 55°C and 70°C, preferably between 58°C and 69°C, more preferably between 59°C and 68°C; and / or (aiii) Containing the nucleic acid sequence of SEQ ID NO: 69 or its reverse complementary sequence, or consisting of the nucleic acid sequence of SEQ ID NO: 69 or its reverse complementary sequence; and / or (aiv) contains one or more locked nucleic acid (LNA) and / or minor groove binding (MGB) moieties; and / or (av) contains detectable markers that are different from those of probes 4 and 5; And / or b) Probe 4: (bi) has a length between 20 and 42 nucleotides, preferably between 21 and 30 nucleotides, and most preferably between 22 and 24 nucleotides; and / or (bii) has a Tm between 55°C and 70°C, preferably between 58°C and 69°C, more preferably between 59°C and 68°C; and / or (biii) Containing the nucleic acid sequence of SEQ ID NO: 71 or its reverse complementary sequence, or consisting of the nucleic acid sequence of SEQ ID NO: 71 or its reverse complementary sequence; and / or (biv) Contains one or more locked nucleic acid (LNA) and / or minor groove binding (MGB) moieties; and / or (bv) contains detectable markers that are different from those of probes 3 and 5.

26. The method according to claim 24 or 25, wherein the differential positive control nucleic acid sequence comprises or consists of the following: A nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 113 or its reverse complementary sequence.

27. The method according to any one of claims 17 to 26, wherein the sample is a biological sample, preferably selected from the group consisting of: cell banks, culture media, cell cultures, cell culture supernatants, pharmaceutical products, vaccine preparations, blood, saliva, and sputum.

28. A primer pair for detecting MMV in a sample, the primer pair comprising: primer P3 annealed to the reverse complementary sequence of the nucleic acid sequence of SEQ ID NO: 109 and primer P4 annealed to the nucleic acid sequence of SEQ ID NO:

110.

29. A kit for detecting MMV in a sample, wherein the kit comprises the primer pair according to claim 28 and a nucleic acid probe (probe 5) annealed to the nucleic acid sequence of SEQ ID NO: 111 or its reverse complementary sequence, preferably wherein the kit further comprises one or more of the following: (i) PCR reaction mixture; (ii) An internal positive control nucleic acid sequence comprising at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 109 and at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 110, and an intercalated nucleic acid sequence of at least 18 nucleotides in length between the two nucleic acid sequences; (iii) A differential positive control nucleic acid sequence comprising at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 109 and at least 18 consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 110, and an intercalation nucleic acid sequence of at least 29 nucleotides in length between the two nucleic acid sequences, wherein the intercalation nucleic acid sequence is preferably identical to SEQ ID NO: 111, such that probe 5 can anneal to the intercalation nucleic acid sequence; (iv) Tools used for extracting and / or purifying DNA from the sample; and (v) Instructions for using primers for PCR.

30. The method according to any one of claims 17 to 27, the primer pair according to claim 28, or the kit according to claim 29, wherein the virus is selected from the group consisting of: circoviridae, filoviridae, parvoviridae, preferably parvovirus, erythrovirus, dependent virus, Aleutian virus, and bocavirus, more preferably parvovirus, and most preferably mouse parvovirus.

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