Primer combination, probe set, detection product and application thereof

Abstract

The invention relates to the field of biological detection, in particular to a primer combination, a probe set, a detection product and application thereof. The invention provides a primer combination. The primer combination comprises one or more of a primer group 1 to a primer group 14. The invention provides a multiple detection kit for effectively improving the detection sensitivity and accuracy of common viruses infected by a central nervous system on the basis of a micro-droplet type digital PCR technology.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to primer combinations, probe sets, detection products and applications thereof. Background Art

[0002] Viral encephalitis (meningitis) is an infectious disease of the central nervous system, with an annual incidence of about 3.5 to 7.4 per 100,000 people, high mortality and disability rates, and an increasing trend in incidence. After the central nervous system is infected with a virus, the patient's clinical manifestations lack specificity, and some acutely infected patients are in critical condition and have a high mortality rate. It is difficult to diagnose and treat based on clinical manifestations alone. In addition, due to the low viral content in the cerebrospinal fluid (CSF) in the early stage of infection and the use of antimicrobial drugs, there is no obvious specific change in CSF laboratory testing, making early diagnosis difficult. Therefore, early, rapid and accurate diagnosis of viral encephalitis pathogens is particularly important for clinical diagnosis and treatment.

[0003] At present, it has been clearly reported that the pathogens that can cause viral encephalitis include more than 10 viruses and their subtypes, including herpes simplex virus type Ⅰ and type Ⅱ, cytomegalovirus, varicella-zoster virus, Epstein-Barr virus, enterovirus and mumps virus. Domestic research data show that the main incidence of viral encephalitis (meningitis) is children, and enterovirus, herpes simplex virus, mumps virus, etc. are the main pathogens; foreign reports show that in addition to enterovirus, herpes simplex virus and mumps virus, cytomegalovirus, Epstein-Barr virus and varicella-zoster virus are all important pathogens causing viral encephalitis. Among them, herpes simplex virus encephalitis (HSE) is currently a disease with a high mortality rate. HSV1 / 2 is considered to be the most important pathogen of viral encephalitis, which often occurs in the elderly and children. Viral encephalitis caused by Epstein-Barr virus and cytomegalovirus often occurs in patients with immune damage or organ transplantation. Viral encephalitis caused by varicella-zoster virus is also very common, with approximately 1 million new cases each year in the United States alone. In addition, in epidemiological studies of viral encephalitis, the pathogen in approximately 25% of cases has no clear diagnosis.

[0004] Accurate detection of infectious pathogens is of great significance for symptomatic treatment of central nervous system infectious diseases. Currently, molecular detection methods are mostly used, and commonly used molecular detection methods include ordinary PCR, nested PCR and fluorescent quantitative PCR. Real-time fluorescent quantitative PCR technology (real-time PCR, RT-PCR) has the characteristics of rapidity, high efficiency, quantification and anti-pollution. Although the application of fluorescent quantitative PCR has become increasingly widespread, it only detects one specific pathogen each time, making it difficult to perform broad-spectrum screening.

[0005] Digital PCR is a new technology that has emerged in recent years. By processing samples into micro-units, the absolute number of target molecules to be detected as low as a single copy can be determined. Digital PCR can directly count the number of target molecules without relying on any calibrants or external standards to achieve the purpose of absolute quantification. Therefore, digital PCR is particularly suitable for application areas that cannot be well distinguished by Ct values: copy number variation, mutation detection, gene relative expression research, second-generation test result verification miRNA expression analysis, single-cell gene expression analysis, etc. Multiplex PCR is a more sensitive and efficient PCR method that uses multiple primers to detect multiple pathogens at one time. This method can quickly diagnose pathogens, especially for infections of unknown causes, and can diagnose pathogens sensitively, specifically, and broadly.

[0006] At present, the reagents disclosed in the prior art for nucleic acid detection of encephalitis and meningitis mainly include:

[0007] Prior art 1 discloses a central nervous system infection pathogen detection kit, including primers and probes for detecting human enterovirus, Epstein-Barr virus, herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, mumps virus and human herpes virus type 6. The detection method is a melting curve method, and the amplification result is detected by the melting peak curve; this method can only perform qualitative detection, and the detection sensitivity of this method is low, and its application range in central nervous system infection scenarios is limited.

[0008] Prior art 2 discloses a primer-probe combination, a kit and a method for detecting multiple meningitis pathogens. The primer-probe combination includes primers and probes for detecting Neisseria meningitidis, group B Streptococcus, Listeria monocytogenes, herpes simplex virus type 1 / 2, enterovirus, human double echovirus, cytomegalovirus, varicella-zoster virus, human herpes virus type 6, and epidemic Japanese encephalitis virus, and the detection method is a PCR fluorescent probe method. This method can only achieve relative quantification, and does not cover viral encephalitis pathogens such as EBV virus, adenovirus, herpes virus type 7 and type 8, and cannot meet the clinical demand for accurate and quantitative detection of pathogens.

[0009] Prior art 3 discloses an EB virus detection kit based on droplet digital PCR, and prior art 4 discloses a droplet digital PCR kit for detecting human herpes virus type 6B in human body fluids. Both patents include: a reaction premix for droplet digital PCR; a target fragment detection mixture, including amplification primers and probes of the target fragment; an internal standard and an internal standard detection mixture; a positive control premix; a negative control premix, which can detect EBV, and the detection method is a digital PCR method. Since there are far more than one common pathogens for central nervous system infection, and most central nervous system samples are difficult to sample and the sample volume is small, it is not cost-effective to detect only one pathogen at a time.

[0010] Although there are many pathogen detection kits for encephalitis and meningitis in the prior art, there are no reports on multiple detection methods for common viral encephalitis and meningitis pathogenic viruses: HSV1, HSV2, VZV, EBV, CMV, HHV6A, HHV6B, HHV7, HHV8, ADV, EV-A71, COV-A16, JEV, and MuV on the digital PCR technology platform. Therefore, it is urgent for those skilled in the art to use this platform to develop a highly sensitive, highly specific, and highly accurate multiplex digital PCR quantitative detection method and kit, so that a single test can detect or exclude common viral encephalitis and meningitis pathogenic viruses and make full use of limited sample size. Summary of the invention

[0011] In view of this, the present invention provides a primer combination, a probe set, a detection product and its application. Based on the droplet digital PCR technology, the present invention provides a multiplex detection kit that effectively improves the sensitivity and accuracy of common viruses infecting the central nervous system.

[0012] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0013] The present invention provides a primer combination, comprising: (a1) or (a2) or (a3) ​​as follows:

[0014] (a1), consisting of primer set 1, primer set 2, primer set 3, primer set 4, primer set 5, primer set 6, primer set 7, primer set 8, primer set 9, primer set 10, primer set 11, primer set 12, primer set 13 and primer set 14;

[0015] (a2), consisting of any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of the primer set 1, the primer set 2, the primer set 3, the primer set 4, the primer set 5, the primer set 6, the primer set 7, the primer set 8, the primer set 9, the primer set 10, the primer set 11, the primer set 12, the primer set 13, and the primer set 14;

[0016] (a3), comprising the primer set 1, the primer set 2, the primer set 3, the primer set 4, the primer set 5, the primer set 6, the primer set 7, the primer set 8, the primer set 9, the primer set 10, the primer set 11, the primer set 12, the primer set 13 and the primer set 14;

[0017] Wherein: the primer set 1: the primers for amplifying herpes simplex virus type 1 have:

[0018] (1) The nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2;

[0019] (2) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (1);

[0020] (3) a sequence having at least 80% homology with the nucleotide sequence shown in (1);

[0021] (4) a complementary sequence of the sequence shown in (1), (2) or (3); and / or

[0022] The primer set 2: primers for amplifying herpes simplex virus type 2 have:

[0023] (5) The nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4;

[0024] (6) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (5);

[0025] (7) a sequence having at least 80% homology to the nucleotide sequence shown in (5);

[0026] (8), a complementary sequence of the sequence shown in (5), (6) or (7); and / or

[0027] The primer set 3: primers for amplifying varicella-zoster virus have:

[0028] (9) the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6;

[0029] (10) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (9);

[0030] (11) a sequence having at least 80% homology to the nucleotide sequence shown in (9);

[0031] (12), a complementary sequence of the sequence shown in (9), (10) or (11); and / or

[0032] The primer set 4: the primers used to amplify Epstein-Barr virus have:

[0033] (13) The nucleotide sequences shown in SEQ ID NO:7 and SEQ ID NO:8;

[0034] (14) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (13);

[0035] (15) a sequence having at least 80% homology to the nucleotide sequence shown in (13);

[0036] (16), a complementary sequence of the sequence shown in (13), (14) or (15); and / or

[0037] The primer set 5: primers for amplifying human cytomegalovirus have:

[0038] (17), the nucleotide sequences shown in SEQ ID NO:9 and SEQ ID NO:10;

[0039] (18) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (17);

[0040] (19) A sequence having at least 80% homology to the nucleotide sequence shown in (17);

[0041] (20), a complementary sequence of the sequence shown in (17), (18) or (19); and / or

[0042] The primer set 6: primers for amplifying herpes simplex virus type 6A have:

[0043] (21), the nucleotide sequences shown in SEQ ID NO:11 and SEQ ID NO:12;

[0044] (22) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (21);

[0045] (23) a sequence having at least 80% homology to the nucleotide sequence shown in (21);

[0046] (24), a complementary sequence of the sequence shown in (21), (22) or (23); and / or

[0047] The primer set 7: primers for amplifying herpes simplex virus type 6B have:

[0048] (25), the nucleotide sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14;

[0049] (26) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (25);

[0050] (27) a sequence having at least 80% homology to the nucleotide sequence shown in (25);

[0051] (28), a complementary sequence of the sequence shown in (25), (26) or (27); and / or

[0052] The primer set 8: primers for amplifying herpes simplex virus type 7 have:

[0053] (29), the nucleotide sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16;

[0054] (30) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (29);

[0055] (31), a sequence having at least 80% homology to the nucleotide sequence shown in (29);

[0056] (32), a complementary sequence of the sequence shown in (29), (30) or (31); and / or

[0057] The primer set 9: primers for amplifying herpes simplex virus type 8 have:

[0058] (33), the nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18;

[0059] (34) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (33);

[0060] (35) a sequence having at least 80% homology to the nucleotide sequence shown in (33);

[0061] (36), a complementary sequence of the sequence shown in (33), (34) or (35); and / or

[0062] The primer set 10: primers for amplifying human adenovirus have:

[0063] (37), the nucleotide sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20;

[0064] (38) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (37);

[0065] (39), a sequence having at least 80% homology to the nucleotide sequence shown in (37);

[0066] (40), a complementary sequence of the sequence shown in (37), (38) or (39); and / or

[0067] The primer set 11: primers for amplifying enterovirus A-71 type have:

[0068] (41), the nucleotide sequences shown in SEQ ID NO:21 and SEQ ID NO:22;

[0069] (42) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (41);

[0070] (43) a sequence having at least 80% homology to the nucleotide sequence shown in (41);

[0071] (44), a complementary sequence of the sequence shown in (41), (42) or (43); and / or

[0072] The primer set 12: primers for amplifying Coxsackievirus A-16 type have:

[0073] (45), the nucleotide sequences shown in SEQ ID NO:23 and SEQ ID NO:24;

[0074] (46) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (45);

[0075] (47), a sequence having at least 80% homology to the nucleotide sequence shown in (45);

[0076] (48), a complementary sequence of the sequence shown in (45), (46) or (47); and / or

[0077] The primer set 13: primers for amplifying mumps virus have:

[0078] (49), the nucleotide sequences shown in SEQ ID NO:25 and SEQ ID NO:26;

[0079] (50) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (49);

[0080] (51), a sequence having at least 80% homology to the nucleotide sequence shown in (49);

[0081] (52), a complementary sequence of the sequence shown in (49), (50) or (51); and / or

[0082] The primer set 14: primers for amplifying Japanese encephalitis virus have:

[0083] (53), the nucleotide sequences shown in SEQ ID NO:27 and SEQ ID NO:28;

[0084] (54) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (53);

[0085] (55), a sequence having at least 80% homology to the nucleotide sequence shown in (53);

[0086] (56) A complementary sequence of the sequence shown in (53), (54) or (55).

[0087] In some embodiments of the present invention, the above primer combination further includes: an internal standard primer set;

[0088] The internal standard primer group includes: internal standard primer group 1 and / or internal standard primer group 2;

[0089] The internal standard primer set 1 has:

[0090] (57), the nucleotide sequences shown in SEQ ID NO:29 and SEQ ID NO:30;

[0091] (58) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (57);

[0092] (59), a sequence having at least 80% homology to the nucleotide sequence shown in (57);

[0093] (60), a complementary sequence of the sequence shown in (57), (58) or (59); and / or

[0094] The internal standard primer set 2 has:

[0095] (61), the nucleotide sequences shown in SEQ ID NO:31 and SEQ ID NO:32;

[0096] (62) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (61);

[0097] (63) a sequence having at least 80% homology to the nucleotide sequence shown in (61);

[0098] (64) A complementary sequence of the sequence shown in (61), (62) or (63).

[0099] The present invention also provides a probe set, comprising: the following (b1) or (b2) or (b3):

[0100] (b1), consisting of probe 1, probe 2, probe 3, probe 4, probe 5, probe 6, probe 7, probe 8, probe 9, probe 10, probe 11, probe 12, probe 13 and probe 14;

[0101] (b2), consisting of any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of the probe 1, the probe 2, the probe 3, the probe 4, the probe 5, the probe 6, the probe 7, the probe 8, the probe 9, the probe 10, the probe 11, the probe 12, the probe 13, and the probe 14;

[0102] (b3), including the probe 1, the probe 2, the probe 3, the probe 4, the probe 5, the probe 6, the probe 7, the probe 8, the probe 9, the probe 10, the probe 11, the probe 12, the probe 13 and the probe 14;

[0103] Wherein: the probe 1: a probe for specific identification of herpes simplex virus type 1 has:

[0104] (65), the nucleotide sequence shown in SEQ ID NO:33;

[0105] (66) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (65);

[0106] (67), a sequence having at least 80% homology to the nucleotide sequence shown in (65);

[0107] (68), a complementary sequence of the sequence shown in (65), (66) or (67); and / or

[0108] The probe 2: a probe for specifically identifying herpes simplex virus type 2 has:

[0109] (69), the nucleotide sequence shown in SEQ ID NO:34;

[0110] (70) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (69);

[0111] (71), a sequence having at least 80% homology to the nucleotide sequence shown in (69);

[0112] (72), a complementary sequence of the sequence shown in (69), (70) or (71); and / or

[0113] The probe 3: a probe for specifically identifying varicella-zoster virus has:

[0114] (73), the nucleotide sequence shown in SEQ ID NO:35;

[0115] (74) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (73);

[0116] (75), a sequence having at least 80% homology to the nucleotide sequence shown in (73);

[0117] (76), a complementary sequence of the sequence shown in (73), (74) or (75); and / or

[0118] The probe 4: a probe for specifically identifying EB virus has:

[0119] (77), the nucleotide sequence shown in SEQ ID NO:36;

[0120] (78) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (77);

[0121] (79), a sequence having at least 80% homology to the nucleotide sequence shown in (77);

[0122] (80), a complementary sequence of the sequence shown in (77), (78) or (79); and / or

[0123] The probe 5: a probe for specifically identifying human cytomegalovirus has:

[0124] (81), the nucleotide sequence shown in SEQ ID NO:37;

[0125] (82) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (81);

[0126] (83), a sequence having at least 80% homology to the nucleotide sequence shown in (81);

[0127] (84), a complementary sequence of the sequence shown in (81), (82) or (83); and / or

[0128] The probe 6: a probe for specifically identifying herpes simplex virus type 6A has:

[0129] (85), the nucleotide sequence shown in SEQ ID NO:38;

[0130] (86) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (85);

[0131] (87), a sequence having at least 80% homology to the nucleotide sequence shown in (85);

[0132] (88), a complementary sequence of the sequence shown in (85), (86) or (87); and / or

[0133] The probe 7: a probe for specifically identifying herpes simplex virus type 6B has:

[0134] (89), the nucleotide sequence shown in SEQ ID NO:39;

[0135] (90) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence as shown in (89);

[0136] (91), a sequence having at least 80% homology to the nucleotide sequence shown in (89);

[0137] (92), a complementary sequence of the sequence shown in (89), (90) or (91); and / or

[0138] The probe 8: a probe for specifically identifying herpes simplex virus type 7 has:

[0139] (93), the nucleotide sequence shown in SEQ ID NO:40;

[0140] (94) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (93);

[0141] (95), a sequence having at least 80% homology to the nucleotide sequence shown in (93);

[0142] (96), a complementary sequence of the sequence shown in (93), (94) or (95); and / or

[0143] The probe 9: a probe for specifically identifying herpes simplex virus type 8 has:

[0144] (97), the nucleotide sequence shown in SEQ ID NO:41;

[0145] (98) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (97);

[0146] (99), a sequence having at least 80% homology to the nucleotide sequence shown in (97);

[0147] (100), a complementary sequence of the sequence shown in (97), (98) or (99); and / or

[0148] The probe 10: a probe for specifically identifying human adenovirus has:

[0149] (101), the nucleotide sequence shown in SEQ ID NO:42;

[0150] (102) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (101);

[0151] (103), a sequence having at least 80% homology to the nucleotide sequence shown in (101);

[0152] (104), a complementary sequence of the sequence shown in (101), (102) or (103); and / or

[0153] The probe 11: a probe for specifically identifying enterovirus A-71 type has:

[0154] (105), the nucleotide sequence shown in SEQ ID NO:43;

[0155] (106) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (105);

[0156] (107), a sequence having at least 80% homology to the nucleotide sequence shown in (105);

[0157] (108), a complementary sequence of the sequence shown in (105), (106) or (107); and / or

[0158] The probe 12: a probe for specifically identifying Coxsackievirus A-16 type has:

[0159] (109), the nucleotide sequence shown in SEQ ID NO:44;

[0160] (110) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence as shown in (109);

[0161] (111), a sequence having at least 80% homology to the nucleotide sequence shown in (109);

[0162] (112), a complementary sequence of the sequence shown in (109), (110) or (111); and / or

[0163] The probe 13: a probe for specifically identifying mumps virus has:

[0164] (113), the nucleotide sequence shown in SEQ ID NO:45;

[0165] (114) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (113);

[0166] (115), a sequence having at least 80% homology to the nucleotide sequence shown in (113);

[0167] (116), a complementary sequence of the sequence shown in (113), (114) or (115); and / or

[0168] The probe 14: a probe for specifically identifying Japanese encephalitis virus has:

[0169] (117), the nucleotide sequence shown in SEQ ID NO:46;

[0170] (118) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence as shown in (117);

[0171] (119), a sequence having at least 80% homology to the nucleotide sequence shown in (117);

[0172] (120), a complementary sequence of the sequence shown in (117), (118) or (119).

[0173] In some embodiments of the present invention, in the above-mentioned probe, the 5' end of the nucleotide sequence is connected to a fluorescent reporter gene, and the 3' end is connected to a quencher gene; the quencher group includes any one of SQ, MGB or NFQ.

[0174] In some embodiments of the present invention, the above-mentioned probe further comprises: an internal standard probe;

[0175] The internal standard probe comprises: internal standard probe 1 and / or internal standard probe 2;

[0176] The internal standard probe 1 has:

[0177] (121), the nucleotide sequence shown in SEQ ID NO:47;

[0178] (122) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (121);

[0179] (123), a sequence having at least 80% homology to the nucleotide sequence shown in (121);

[0180] (124), a complementary sequence of the sequence shown in (121), (122) or (123); and / or

[0181] The internal standard probe 2 has:

[0182] (125), the nucleotide sequence shown in SEQ ID NO:48;

[0183] (126) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (125);

[0184] (127), a sequence having at least 80% homology to the nucleotide sequence shown in (125);

[0185] (128), a complementary sequence of the sequence shown in (125), (126) or (127).

[0186] The present invention also provides a primer-probe set, comprising: the above primer combination and the above probe set.

[0187] The present invention also provides a detection reagent, comprising: the above primer combination, the above probe group and / or the above primer probe group and an acceptable auxiliary agent.

[0188] The present invention also provides the use of the above primer combination, the above probe group, the above primer probe group and / or the above detection reagent in the preparation of a product for detecting viral encephalitis.

[0189] In some embodiments of the present invention, in the above application, the detection adopts a digital PCR method.

[0190] In some embodiments of the present invention, in the above application, the detection comprises the following steps: mixing the sample to be tested with the detection reagent, centrifuging, obtaining microdroplets, amplifying, and obtaining the detection result.

[0191] In some embodiments of the present invention, in the above application, the amplification conditions include: the annealing time is 30 to 60 seconds, preferably 30 seconds.

[0192] The present invention also provides a detection product, comprising: the above primer combination, the above probe, the above primer probe group and / or the above detection reagent and acceptable auxiliary agents, excipients and / or devices.

[0193] In some embodiments of the present invention, in the above detection product, the concentration of the primers in the above primer combination is 1000 nM; the concentration of the above probe is 250 nM.

[0194] The beneficial effects of the present invention include:

[0195] (1) Compared with the prior art 2, which can only perform relative quantitative PCR fluorescent probe method, to achieve accurate quantification, it is necessary to add accurately quantified gradient dilution standard products to the same batch of test samples as a control to generate a calibration curve, thereby achieving the purpose of accurate quantification. However, this technology does not need to rely on any calibrants or external standards, and can directly count target molecules; while improving the quantitative accuracy, it can effectively reduce the number of reference product reactions required for each quantitative experiment, effectively reducing the batch detection cost.

[0196] (2) Compared with the digital PCR detection method of prior art 3 which can only detect a certain virus, this method can detect multiple common meningitis virus targets in one tube reaction, which can fully improve the utilization rate of precious samples and save sample resources and the number of reactions.

[0197] (3) Compared with the detection using the melting curve method in the prior art 1, the digital PCR method has higher detection accuracy for low-abundance pathogens and is more suitable for the application scenario of central nervous system infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0198] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0199] Figure 1 Shows the genetic polymorphism of the EVA71 genome region; where: the horizontal axis is the genome position; the vertical axis is the genetic polymorphism (unit: number of variations / base);

[0200] Figure 2 The figure shows the test results of the positive control premix 1; the left figure shows the distribution of the droplet fluorescence signal of the FAM channel (EBV) and the VIC channel (HSV1+HSV2); the right figure shows the distribution of the droplet fluorescence signal of the CY5 channel (VZV) and the CY5.5 channel (CMV); the red line divides the signal area into positive and negative areas, and the positive signal can be well separated from the negative signal;

[0201] Figure 3The figure shows the test results of the positive control premix 2; among them: the left figure shows the distribution of the droplet fluorescence signal of the FAM channel (HHV6A+HHV6B) and the VIC channel (ADV); the right figure shows the distribution of the droplet fluorescence signal of the CY5 channel (HHV7+HHV8) and the CY5.5 channel (RPP30); the red line divides the signal area into positive and negative areas, and the positive signal can be well separated from the negative signal;

[0202] Figure 4 The figure shows the test results of the positive control premix 3; among them: the left figure shows the distribution of the droplet fluorescence signal of the FAM channel (EV-A71+CV-A16) and the VIC channel (MuV); the right figure shows the distribution of the droplet fluorescence signal of the CY5 channel (JEV) and the CY5.5 channel (RNP); the red line divides the signal area into positive and negative areas, and the positive signal can be well separated from the negative signal;

[0203] Figure 5 Figure 2 shows the detection of enterovirus targets before probe optimization; among them: upper figure: EV71 target detection before probe optimization, lower figure: CVA16 target detection before probe optimization. Although the signals of the two individual target positive droplets and negative droplets can be distinguished, the negative signal fluorescence value is high, which is easy to cause false positive sites and needs further optimization.

[0204] Figure 6 The results show that the optimization of the probe system using the SQ probe can distinguish positive droplets from negative droplets more clearly; among them: the upper figure: the comparison of the new system of single probe EVA71 (1st and 2nd channels) with the original system (3rd and 4th channels); the middle figure: the comparison of the new system of single probe CVA16 (1st, 2nd and 3rd channels) with the original system (4th, 5th and 6th channels); the lower figure: the comparison of the new system of combined probe EVA71+CVA16 (1st-6th channels) with the original system (7th-12th channels);

[0205] Figure 7 The results show that in the ddPCR experiment of 16 clinical samples, the fluorescence signals of positive and negative droplets were clearly distinguished, indicating that the experiment has high specificity;

[0206] Figure 8 Shows the LOD test results of HSV1;

[0207] Fig. 9 Shows the LOD test results of HSV2;

[0208] Fig.10 Shows the LOD test results of VZV;

[0209] Fig.11 Shows the LOD test results of EBV;

[0210] Fig.12 Shows the LOD test results of CMV;

[0211] Fig.13 Shows the LOD test results of HHV6A;

[0212] Fig.14 Shows the LOD test results of HHV6B;

[0213] Fig.15 Shows the LOD test results of HHV7;

[0214] Fig.16 Shows the LOD test results of HHV8;

[0215] Fig.17 Shows the LOD test results of ADV;

[0216] Fig.18 Shows the LOD test results of EV-A71;

[0217] Fig.19 Shows the LOD test results of CV-A16;

[0218] Fig. 20 Shows the LOD test results of MuV;

[0219] Fig.21 Shows the LOD test results of JEV;

[0220] Fig. 22 Shows the EBV target test results before optimization;

[0221] Fig.23 Shows the results of HSV1+HSV2 target test before optimization;

[0222] Fig.24 The results of VZV target test before optimization are shown;

[0223] Fig.25 The results of CMV target test before optimization are shown;

[0224] Fig.26 The results of HHV-6A+HHV-6B target test before optimization are shown;

[0225] Fig. 27 The results of HHV-7+HHV-8 target test before optimization are shown;

[0226] Fig.28 The ADV target test results before optimization are shown;

[0227] Fig.29 The results of RPP30 target test before optimization are shown;

[0228] Fig.30The results of EV-A71+CV-A16 target test before optimization are shown;

[0229] Fig.31 The results of MuV target test before optimization are shown;

[0230] Fig.32 The results of JEV target test before optimization are shown;

[0231] Fig.33 Shows the RNP target test results before primer optimization. DETAILED DESCRIPTION

[0232] The invention discloses a primer combination, a probe group, a detection product and applications thereof.

[0233] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0234] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0235] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0236] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0237] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0238] In Examples 1 to 5 and Verification Examples 1 to 3 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0239] The present invention will be further described below in conjunction with embodiments:

[0240] Example 1

[0241] (1) Reference genomes of 14 viral encephalitis and meningitis pathogens and region selection and design steps for primers and probes

[0242] Considering the high degree of variation of virus strains, especially RNA viruses, it is necessary to find a suitable genomic region before designing primers and probes. The target genomic region must be both species-specific and conservative among the genomes of each strain of the species to ensure the sensitivity of PCR detection. The specific steps are as follows:

[0243] The NCBI virus database (https: / / www.ncbi.nlm.nih.gov / labs / virus) was used to search for the published genome sequences of the corresponding virus species (“Search by virus name or taxonomy”) according to the scientific name of the virus.

[0244] If there are too many sequences (more than 200 records), consider setting up conditional screening. The screening conditions include a) sequence length / completeness of nucleic acid sequence, try to select fragments close to the complete genome; b) region, give priority to records submitted by Chinese samples, and consider other countries in East Asia and Southeast Asia; c) host, select human.

[0245] Align the obtained genome sequences. Alignment can be done on the NCBI virus page using mega or other software. After the alignment is completed, the genetic polymorphism of the sequence fragments can be calculated using the ape and pegas packages of the R software. Take Enterovirus A71 as an example to write an R analysis script:

[0246] dna_file=" / file path / EVA71CHINA.aln.fasta"

[0247] No_of_seq=324#aligned sequences from NCBI virus

[0248] seq=read.dna(dna_file,format="fasta")

[0249] No_of_Sliding_window=7000 / 50

[0250] sq=1;end=100

[0251] for(iin c(1:No_of_Sliding_window)){step=i*50;print(nuc.div(seq[,(sq+step):(end+step)]))}

[0252] ##theta_pi output

[0253] ##theta_pi=the sum of the number of differences between pairs of sequences divided by the number of comparisons(ien(n-1) / 2,where n is the number of sequences).

[0254] an=1:No_of_seq

[0255] for(iin c(1:No_of_Sliding_window)){step=i*50;print(length(seg.sites(seq[,(sq+step):(end+step)])) / (100*sum(1 / an)))}

[0256] ##theta_w output

[0257] The script mainly performs regional polymorphism analysis on sequences and calculates genetic polymorphism within species.

[0258] The genetics calculation of the research population consisting of 324 submitted sequences showed that Enterovirus A71 strain may be subdivided into subtypes. The primer probe design was carried out in the region with relatively low theta_pi.

[0259] Table 1

[0260]

[0261] The same idea was used to collect candidate fragment regions for other viral species for the design of multiplex PCR primer probes.

[0262] (2) Steps for developing multiplex PCR primer and probe design scripts.

[0263] The input of the multiplex PCR primer design software is: a) the genome to be designed for primers and the human reference genome, and b) the candidate pathogen fragment regions collected in step 1. The output is multiple pairs of primers designed to be amplified in one reaction. The specific implementation ideas are as follows:

[0264] 1) Use Primer3 software to design as many available primers as possible for each target region as candidates;

[0265] 2) Filtering the primers designed in step 1) according to conditions such as the specificity of primer amplification and whether the primers contain simple repeat sequences;

[0266] 3) For primers that meet the filtering conditions in the previous step, select a pair of primers with the highest Primer3 score for each target region. The primers selected for different target regions must meet the principle of "minimizing primer interactions", that is, minimizing the 3' end of the primers and the overall complementarity of the primers. The specific requirements are generally that the 3' end base complementarity number between primers is not higher than 10, and the overall complementarity between primers is not higher than 75%. This principle can effectively avoid the formation of primer dimers.

[0267] 4) For primers that meet the filtering conditions in the previous step, the annealing temperature of the primers themselves needs to be as close as possible. The fragment in the middle of the primer can be used for probe design. The specific requirements are generally that the primer annealing temperature is 56-60℃, and the probe sequence annealing temperature is 68-72℃.

[0268] Table 2

[0269]

[0270]

[0271]

[0272]

[0273] Example 2

[0274] The primers and multiplex droplet digital PCR kit for viral encephalitis and meningitis pathogens of the present application include:

[0275] 1. Target fragment detection mixture 1: including primers and probe combinations for detecting target fragments, and primers and probe combinations for detecting internal standards; the target fragments in this embodiment are DNA or DNA fragments of Epstein-Barr virus (EBV), human cytomegalovirus (CMV), herpes simplex virus type 1 (HSV1), herpes simplex virus type 2 (HSV2), and varicella-zoster virus (VZV); the target fragments in this embodiment also include human internal control RPP30, which is used to monitor the extraction and detection process;

[0276] Target fragment detection mixture 1: each primer contained has a concentration of 1000nM; each probe contained has a concentration of 250nM;

[0277] 2. Target fragment detection mixture 2: including primers and probe combinations for detecting target fragments, and primers and probe combinations for detecting internal standards; in this embodiment, the target fragments are DNA or DNA fragments of herpes simplex virus 6A (HHV6A), herpes simplex virus 6B (HHV6B), human adenovirus (ADV), herpes simplex virus 7 (HHV7), and herpes simplex virus 8 (HHV8); in this embodiment, the target fragments also include human internal control RPP30, which is used to monitor the extraction and detection process;

[0278] Target fragment detection mixture 2: including each primer at a concentration of 1000 nM; the probe concentrations of herpes simplex virus 6A (HHV6A) and herpes simplex virus 6B (HHV6B) are 150 nM, and the probe concentrations of human adenovirus (ADV), herpes simplex virus 7 (HHV7), herpes simplex virus 8 (HHV8), and human internal control RPP30 are 250 nM respectively;

[0279] 3. Target fragment detection mixture 3: including primers and probe combinations for detecting target fragments, and primers and probe combinations for detecting internal standards; the target fragments in this embodiment are RNA or cDNA fragments of enterovirus A-71 (EV-A71), coxsackievirus A-16 (CV-A16), mumps virus (MuV), and Japanese encephalitis virus (JEV); the target fragments in this embodiment also include human internal control RNPs for monitoring the extraction and detection process; the primers are also used in the reverse transcription process.

[0280] Target fragment detection mixture 3: including each primer at a concentration of 1000 nM; the probe concentration of enterovirus A-71 (EV-A71) is 300 nM; the probe concentrations of Coxsackievirus A-16 (CV-A16), mumps virus (MuV), Japanese encephalitis virus (JEV), and human internal control RNP are 250 nM respectively;

[0281] 4. Positive control premix 1: including DNA fragments of Epstein-Barr virus (EBV), human cytomegalovirus (CMV), herpes simplex virus type 1 (HSV1), herpes simplex virus type 2 (HSV2), and varicella-zoster virus (VZV); see the specific results. Figure 2 ;

[0282] 5. Positive control premix 2: including DNA fragments of herpes simplex virus 6A (HHV6A), herpes simplex virus 6B (HHV6B), human adenovirus (ADV), herpes simplex virus 7 (HHV7), and herpes simplex virus 8 (HHV8); see the specific results. Figure 3 ;

[0283] 6. Positive control premix 3: Enterovirus A-71 (EV-A71), detection of cDNA fragments of Coxsackievirus A-16 (CV-A16), mumps virus (MuV), and Japanese encephalitis virus (JEV); see Figure 4 ;

[0284] 7. Negative control premix: includes extracted and diluted human gDNA.

[0285] Example 3

[0286] 1. Optimization of reaction conditions of multiplex PCR system (annealing time optimization: 1.60S; 2.30S)

[0287] Table 3

[0288]

[0289]

[0290] Table 4

[0291]

[0292] RNA target (MUV RNA) and DNA target (CMV X12) were selected as representatives to optimize the annealing time in the PCR program. The number of target positive droplets detected was similar when the annealing time was 30S compared with that of the annealing time of 60S, with a slightly higher number under the 30S condition; the number of false positive droplets under the two conditions in the NTC group was similar.

[0293] Therefore, an annealing time of 30S was selected as the preferred reaction condition.

[0294] 2. Multiplex PCR system: probe optimization

[0295] Before optimization: EV71 target detection, although the signals of two separate target positive droplets and negative droplets can be distinguished, the negative signal fluorescence value is high, which is easy to cause false positive sites, and the primer system needs to be further optimized. ( Figure 5 (shown)

[0296] Table 5

[0297]

[0298] We optimized the probe and replaced the probe quenching group from BHQ1 to SQ.

[0299] Enterovirus 71 (P:5'-FAM-TTCGCACAGCACAGCTGAGACCACT+SQ-3')

[0300] Coxsackievirus group A type 16 (P: 5'-FAM-AGCCATTGGGAATTTCTTCAGCCG TGC+SQ-3')

[0301] Table 6

[0302]

[0303] From Table 6 and Figure 6 It can be seen that the SQ system probe can distinguish positive droplets from negative droplets more clearly.

[0304] Example 4

[0305] The primers for viral encephalitis and meningitis pathogens of the present application and the method for using the multiple droplet digital PCR kit include:

[0306] (1) Samples should be extracted immediately after collection. If they cannot be extracted immediately, they should be stored on ice or at 4°C. Samples should not be stored at 4°C for more than 72 hours. They can be stored at -20°C for several months. For long-term storage, please store the samples at -70°C.

[0307] (2) Sample processing and nucleic acid extraction (sample processing area)

[0308] Use the nucleic acid extraction kit produced by Novazon to extract total nucleic acid from clinical samples according to the instructions of the nucleic acid extraction kit, and measure the DNA concentration after extraction.

[0309] (3) Reagent preparation (reagent preparation area)

[0310] a. Positive quality control and negative quality control are set for the detection reaction. The positive quality control and negative quality control are respectively realized by the positive control premix 1-3 and the negative control premix of Example 2; the reaction premix for droplet digital PCR developed and produced by Guangdong Yongnuo Medical Technology Co., Ltd. is used in conjunction with the reaction premix for droplet digital PCR. The reaction premix for droplet digital PCR should be compatible with the equipment for digital PCR and can be replaced by other reaction premix for digital PCR developed and produced by Guangdong Yongnuo Medical Technology Co., Ltd.

[0311] b. Preparation process

[0312] 1) Thaw all components and equilibrate them to room temperature. After each component is fully dissolved, shake and mix evenly, and centrifuge briefly;

[0313] Determine the number of reactions N, N = number of samples to be tested + number of quality controls 2 (including 1 positive quality control and 1 negative quality control) + 1, calculate the amount of each reagent added to the reaction mixture, and calculate the components and volumes as shown in the following table:

[0314] Table 7

[0315] Components volume Reaction Master Mix for Droplet Digital PCR 10×NμL Target fragment detection mixture 1 / 2 / 3 2×NμL Nuclease-free water 3×NμL

[0316] The above-mentioned reaction premix for droplet digital PCR is a product, and is used in conjunction with the MicroDrop-400B droplet digital PCR system used in this example.

[0317] 2) Prepare the reaction system in a 1.5 mL sterile centrifuge tube. Add all reagents, shake and mix, and centrifuge for a few seconds.

[0318] 3) Then dispense 15 μL / tube of the above mixed solution into 0.2 mL PCR reaction tubes.

[0319] c. Sample addition (sample preparation area)

[0320] Take 5 μL of the positive control premix 1 / 2 / 3, negative control premix, and clinical sample DNA of Example 2 and add them to 0.2 mL PCR reaction tubes to form each reaction system. Then, cover the tube tightly, shake and mix evenly, centrifuge briefly to throw all the liquid on the tube wall to the bottom of the tube (to avoid bubbles), and then generate microdroplets.

[0321] d. Microdroplet generation (sample preparation area)

[0322] 1) Place the microdroplet generation chip in the chip holder, add 55 μL of microdroplet generation oil to the oil phase well, and add 20 μL of PCR reaction water phase with sample added to the sample well. After the oil phase and water phase are added, cover the microdroplet generation chip sealing pad and place the microdroplet generation chip in the MicroDrop-100A microdroplet generator for microdroplet generation;

[0323] 2) After the microdroplets are generated, carefully transfer the generated microdroplets (about 75 μL) to a 96-well PCR reaction plate (it is recommended to use an eight-row gun to transfer the microdroplets);

[0324] 3) After the microdroplets are transferred to the 96-well PCR reaction plate, cover the 96-well plate with a pierceable heat-sealing film and place it on a preheated heat sealer for sealing. After sealing the film, PCR amplification should be performed within 30 minutes, or placed in a 4°C refrigerator for PCR amplification within 4 hours.

[0325] e.PCR amplification (amplification detection area)

[0326] The sealed 96-well PCR reaction plate was subjected to PCR amplification, and the amplification procedure was as follows:

[0327] Procedure for using the target fragment detection mixture:

[0328] Table 8

[0329]

[0330] (4) Microdroplet detection and result analysis

[0331] After PCR amplification, place the 96-well PCR reaction plate in the MicroDrop-400B droplet detector for detection; after the detection is completed, click "Analysis" to open and analyze the data. Click the one-dimensional graph (channel 1 channel 2) to display the single channel data of the selected well, and use the radio button to select the channel to be displayed; the threshold is set in the area where the negative droplets and positive droplets are separated. Click "2D Graph" to display the two-dimensional graph, and the four areas are shown in Figure 1 ; Click "Concentration" to display the number of DNA copies added to each well, in copies / μL.

[0332] (5) Determination of test results

[0333] a. Judgment of droplet generation effectiveness: The total number of droplets in each reaction tube is > 65,000. If the total number of droplets is < 65,000, the droplet generation of the reaction well is not ideal and droplet generation needs to be repeated.

[0334] b. Negative control validity judgment: the number of points falling in the "Ch1+" area is less than 3, the number of points falling in the "Ch2+" area is less than 3, the number of points falling in the "Ch3+" area is less than 3, and the number of points falling in the "Ch4+" area is ≥ 3;

[0335] c. Judgment of the effectiveness of the positive control: ≥3 points in the "Ch1+" area, ≥3 points in the "Ch2+" area, ≥3 points in the "Ch3+" area, and ≥3 points in the "Ch4+" area;

[0336] d. After meeting the above points a, b, and c, positive droplets are detected in the sample, and the sample concentration is calculated as follows:

[0337]

[0338] e. After meeting the above points a, b, and c, if no positive droplets are detected in the sample, it is judged to be below the detection limit.

[0339] Example 5

[0340] This example uses the kit described in this application and the method of use described in Example 3 to detect the cerebrospinal fluid or brain abscess drainage fluid of patients diagnosed with central nervous system infection. The specific detection method is:

[0341] (1) Sample extraction: The nucleic acid extraction kit produced by Novazon was used to extract total nucleic acid from 16 clinical samples according to the instructions of the nucleic acid extraction kit. 200 μL of sample was taken for extraction according to the instructions of the nucleic acid extraction kit. Nucleic acid was extracted according to the operating instructions of the kit. The elution volume was 50 μL. After extraction, the DNA concentration was measured.

[0342] (2) Preparation of reaction system:

[0343] Each sample was tested in 3 wells, and positive and negative control tests were performed in parallel in each well.

[0344] a. The system of PCR reaction solution for droplet digital PCR detection, the components and volumes are shown in the following table:

[0345] Table 9

[0346] Components volume Reaction Master Mix for Droplet Digital PCR 10×NμL Target fragment detection mixture 1 / 2 / 3 2×NμL Nuclease-free water 3×NμL

[0347] b. Prepare the reaction system in a 1.5mL sterile centrifuge tube. Add all the reagents and shake to mix. Centrifuge for a few seconds. Then dispense 15μL / tube of the above mixture into 0.2mL PCR reaction tubes.

[0348] c. Sample addition: Take 1 / 2 / 3 of the positive control premix, the negative control premix, and 12 μL of the clinical sample DNA in Example 2 and add them to 0.2 mL PCR reaction tubes to form each reaction system. Then, cover the tube tightly, shake and mix evenly, centrifuge briefly to throw all the liquid on the tube wall to the bottom of the tube (to avoid bubbles), and then prepare microdroplets.

[0349] 3) Microdroplet generation, PCR amplification, microdroplet detection and result analysis were all carried out according to the method described in Example 3.

[0350] The sample test results are as follows:

[0351] Table 10

[0352]

[0353]

[0354]

[0355]

[0356] The test results are as follows: Figure 7 shown.

[0357] Verification Example 1 Detection Limit Verification

[0358] 1. Sample source:

[0359] The nucleic acid extracted from the EBV inactivated culture medium and the CMV enterprise reference product provided by Guangdong Yongnuo Medical Co., Ltd., and the target sequence synthesized by the plasmid were used for the other 12 pathogenic microorganisms. The original sample value and extraction information are as follows:

[0360] Table 11

[0361] name Original value Extraction method Extraction volume Elution volume HSV1 E11 Self-developed column formulation 400 50 HSV2 E11 Self-developed magnetic bead method 400 50 V Z E11 Self-developed magnetic bead method 400 50 EBV 1.26×10^3~1.26×10^4IU / mL Self-developed column formulation 200 50 CMV 2×10^4cp / mL Self-developed magnetic bead method 300 80 HHV6A E11 Self-developed magnetic bead method 400 50 HHV6B E11 Self-developed magnetic bead method 400 50 HHV7 E11 Self-developed magnetic bead method 400 50 HHV8 E11 Self-developed magnetic bead method 400 50 ADV E11 Self-developed magnetic bead method 400 50 EV-A71 E11 Self-developed magnetic bead method 400 50 CV-A16 E11 Self-developed magnetic bead method 400 50 MuV E11 Self-developed magnetic bead method 400 50 JEV E11 Self-developed magnetic bead method 400 50

[0362] Note: 1IU≈7.5cp.

[0363] Each target template has been pre-diluted to a concentration below 100 cp / μL. As the stock solution for LOD sample configuration detection, each target detection kit provided by Guangdong Yongnuo Medical Co., Ltd. is used for quantitative detection. Sample concentration (cp / mL) = nucleic acid concentration (cp / μL) × elution volume / extraction volume × 1000. For plasmid samples, 400 μL is extracted and 50 μL is eluted. The quantitative detection results are as follows:

[0364] Table 12

[0365]

[0366]

[0367] 2. LOD sample configuration:

[0368] 1) Each target is diluted to an appropriate multiple, marked as target-LOD-dilution multiple. The theoretical concentration is shown in the following table:

[0369] Table 13 Theoretical concentration of each target in the first dilution

[0370] name Target Nucleic acid concentration (cp / μL) Converted sample concentration (cp / mL) HSV1-LOD-10 HSV1 1.31 163 HSV2-LOD-20 HSV2 1.13 141 VZV-LOD-10 V Z 1.46 183 EBV-LOD-5 EBV 11.80 2950 CMV-LOD-5 CMV 13.30 3547 HHV6A-LOD-40 HHV6A 0.83 103 HHV6B-LOD-20 HHV6B 1.05 131 HHV7-LOD-10 HHV7 1.00 125 HHV8-LOD-5 HHV8 1.82 228 ADV-LOD-30 ADV 1.00 125 EVA-LOD-10 EV-A71 1.30 163 CVA-LOD-10 CV-A16 1.40 175 MuV-LOD-10 MuV 1.20 150 JEV-LOD-10 JEV 1.40 175

[0371] Table 14 Theoretical concentration of each target in the second dilution

[0372]

[0373]

[0374] Table 15 Theoretical concentration of each target for the third dilution

[0375] name Target Nucleic acid concentration (cp / μL) Converted sample concentration (cp / mL) HSV1-LOD-40 HSV1 0.32 40 HSV2-LOD-40 HSV2 0.56 71 VZV-LOD-40 V Z 0.36 45 EBV-LOD-125 EBV 0.47 118 CMV-LOD-125 CMV 0.53 141 HHV6A-LOD-60 HHV6A 0.55 69 HHV6B-LOD-40 HHV6B 0.53 66 HHV7-LOD-20 HHV7 0.50 63 HHV8-LOD-15 HHV8 0.61 76 ADV-LOD-50 ADV 0.60 75 EVA-LOD-20 EV-A71 0.65 81 CVA-LOD-20 CV-A16 0.70 88 MuV-LOD-20 MuV 0.60 75 JEV-LOD-20 JEV 0.70 88

[0376] Table 16 Theoretical concentration of each target in the fourth dilution

[0377] name Target Nucleic acid concentration (cp / μL) Converted sample concentration (cp / mL) HSV1-LOD-50 HSV1 0.26 32 HSV2-LOD-50 HSV2 0.45 56 VZV-LOD-50 V Z 0.29 36 EBV-LOD-250 EBV 0.23 58 CMV-LOD-250 CMV 0.26 69 HHV6A-LOD-70 HHV6A 0.47 59 HHV6B-LOD-50 HHV6B 0.42 53 HHV7-LOD-25 HHV7 0.40 50 HHV8-LOD-20 HHV8 0.46 57 ADV-LOD-60 ADV 0.50 63 EVA-LOD-25 EV-A71 0.52 65 CVA-LOD-25 CV-A16 0.56 70 MuV-LOD-25 MuV 0.48 60 JEV-LOD-25 JEV 0.56 70

[0378] 3. LOD sample detection:

[0379] Use this system to detect the above target samples in parallel 3-5 times. The detection results are shown in the following table:

[0380] Table 17 HSV1 detection results

[0381]

[0382] The detection results are as follows Figure 8 shown.

[0383] Table 18 HSV2 detection results

[0384]

[0385]

[0386] The detection results are as follows Fig. 9 shown.

[0387] Table 19 VZV detection results

[0388]

[0389]

[0390] The detection results are as follows Fig.10 shown.

[0391] Table 20EBV detection results

[0392]

[0393] The detection results are as follows Fig.11 shown.

[0394] Table 21CMV detection results

[0395]

[0396] The detection results are as follows Fig.12 shown.

[0397] Table 22HHV6A detection results

[0398]

[0399]

[0400] The detection results are as follows Fig.13 shown.

[0401] Table 23HHV6B detection results

[0402]

[0403]

[0404] The detection results are as follows Fig.14 shown.

[0405] Table 24HHV7 detection results

[0406]

[0407] The detection results are as follows Fig.15 shown.

[0408] Table 25HHV8 detection results

[0409]

[0410]

[0411] The detection results are as follows Fig.16 shown.

[0412] Table 26 ADV detection results

[0413]

[0414] The detection results are as follows Fig.17 shown.

[0415] Table 27EV-A71 detection results

[0416]

[0417] The detection results are as follows Fig.18 shown.

[0418] Table 28 CV-A16 detection results

[0419]

[0420]

[0421] The detection results are as follows Fig.19 shown.

[0422] Table 29 MuV detection results

[0423]

[0424] The detection results are as follows Fig. 20 shown.

[0425] Table 30 JEV detection results

[0426]

[0427]

[0428] The detection results are as follows Fig.21 shown.

[0429] 4. Conclusion: Based on the above tests, the LOD values ​​of each target were obtained.

[0430] Table 31

[0431]

[0432]

[0433] Verification Example 2 Cross-reaction Verification

[0434] The most common pathogens of central nervous system infections are viruses, such as herpes virus types 1-8 and ADV. There are also some bacteria and fungi that are opportunistic pathogens, such as Cryptococcus neoformans, Candida albicans, Aspergillus, Talaromyces marneffei, Mycobacterium tuberculosis, Mycobacterium abscessus, Streptococcus agalactiae, Neisseria meningitidis, Staphylococcus aureus, Listeria monocytogenes, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Stenotrophomonas maltophilia, Enterococci, Serratia marcescens, Mycoplasma pneumoniae, Streptococcus pneumoniae, etc.

[0435] This system was used to detect clinical samples positive for pathogens such as Cryptococcus neoformans, Aspergillus fumigatus, Enterococcus faecalis, Penicillium marneffei, and Streptococcus pneumoniae, and cross-reactions were verified. The results showed that samples positive for non-target pathogens were all negative, as shown in the following table:

[0436] Table 32

[0437]

[0438] Verification Example 3 Repeatability Verification

[0439] The pre-packaged 12T CNS detection reagent was stored at -20℃ for 1.5 months and then sent to Guangdong Yongnuo Medical Technology Co., Ltd. (address: 8th Floor, Foshan Life Science Park, No. 82, Langbao West Road, Chancheng District, Foshan City) using dry ice + ice pack transportation mode. Different instruments and different personnel on the same model platform were used to test the same tube of positive control to test the stability of the reagent system.

[0440] Guangdong Yongnuo Medical Technology Co., Ltd. operates according to the SOP provided by the medical examination. The quantitative results of the positive control plasmid and the quantitative results of the medical examination institute are shown in the following table:

[0441] Table 33

[0442]

[0443] Based on the multiple parallel quantitative results of this laboratory, the fluctuation range of the quantitative value does not exceed 20%. The positive control results of the parallel detection of the Medical Inspection Institute are shown in the following table:

[0444] Table 34

[0445]

[0446]

[0447] Conclusion: After verification by the present invention and other prior arts, this system has good reproducibility.

[0448] Comparative Example

[0449] Before finalizing the final version of the viral encephalitis and meningitis pathogen detection kit of the present application, a large number of preliminary experiments and optimizations were performed on the primers and probes. The typical primer and probe sequences that have been designed are shown in the following table:

[0450] Table 35 Original version primer and probe combinations

[0451]

[0452]

[0453] The experimental results are shown in Tables 36 to 38 and Figure 22 to Figure 33 shown.

[0454] Table 36 Results before optimization of well 1

[0455] Location Test samples result C05 Clinical CMV positive samples CMV was detected, but the positive droplet signal was weak D05 Clinical HSV1 positive samples Normal detection E05 Clinical VZV-positive samples VZV was detected, but there was a non-specific amplification signal F05 Clinical EBV positive samples Normal detection G05 Clinical CMV positive samples CMV was detected, but the positive droplet signal was weak H05 NTC VZV has nonspecific amplification signal

[0456] Table 37 Results before optimization of hole 2

[0457]

[0458]

[0459] Table 38 Results before optimization of hole 3

[0460] Location Test samples result E02 EV-A17 plasmid Normal detection, but the bottom peak is too high F02 CV-A16 plasmid Normal detection, but the bottom peak is too high G02 MUV Plasmid There is a "raindrop" phenomenon H02 JEV Plasmid More non-specific signals A03 Normal whole blood gDNA normal B03 NTC normal

[0461] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer combination, characterized in that: include: As follows (a1) or (a2) or (a3): (a1), consisting of primer set 1, primer set 2, primer set 3, primer set 4, primer set 5, primer set 6, primer set 7, primer set 8, primer set 9, primer set 10, primer set 11, primer set 12, primer set 13 and primer set 14; (a2), consisting of any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of the primer set 1, the primer set 2, the primer set 3, the primer set 4, the primer set 5, the primer set 6, the primer set 7, the primer set 8, the primer set 9, the primer set 10, the primer set 11, the primer set 12, the primer set 13, and the primer set 14; (a3), comprising the primer set 1, the primer set 2, the primer set 3, the primer set 4, the primer set 5, the primer set 6, the primer set 7, the primer set 8, the primer set 9, the primer set 10, the primer set 11, the primer set 12, the primer set 13 and the primer set 14; Wherein: the primer set 1: the primers for amplifying herpes simplex virus type 1 have: (1) The nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2; (2) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (1); (3) a sequence having at least 80% homology with the nucleotide sequence shown in (1); (4) a complementary sequence of the sequence shown in (1), (2) or (3); and / or The primer set 2: primers for amplifying herpes simplex virus type 2 have: (5) The nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4; (6) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (5); (7) a sequence having at least 80% homology to the nucleotide sequence shown in (5); (8), a complementary sequence of the sequence shown in (5), (6) or (7); and / or The primer set 3: primers for amplifying varicella-zoster virus have: (9) the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6; (10) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (9); (11) a sequence having at least 80% homology to the nucleotide sequence shown in (9); (12), a complementary sequence of the sequence shown in (9), (10) or (11); and / or The primer set 4: the primers used to amplify Epstein-Barr virus have: (13) The nucleotide sequences shown in SEQ ID NO:7 and SEQ ID NO:8; (14) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (13); (15) a sequence having at least 80% homology to the nucleotide sequence shown in (13); (16), a complementary sequence of the sequence shown in (13), (14) or (15); and / or The primer set 5: primers for amplifying human cytomegalovirus have: (17), the nucleotide sequences shown in SEQ ID NO:9 and SEQ ID NO:10; (18) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (17); (19) A sequence having at least 80% homology to the nucleotide sequence shown in (17); (20), a complementary sequence of the sequence shown in (17), (18) or (19); and / or The primer set 6: primers for amplifying herpes simplex virus type 6A have: (21), the nucleotide sequences shown in SEQ ID NO:11 and SEQ ID NO:12; (22) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (21); (23) a sequence having at least 80% homology to the nucleotide sequence shown in (21); (24), a complementary sequence of the sequence shown in (21), (22) or (23); and / or The primer set 7: primers for amplifying herpes simplex virus type 6B have: (25), the nucleotide sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14; (26) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (25); (27) a sequence having at least 80% homology to the nucleotide sequence shown in (25); (28), a complementary sequence of the sequence shown in (25), (26) or (27); and / or The primer set 8: primers for amplifying herpes simplex virus type 7 have: (29), the nucleotide sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16; (30) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (29); (31), a sequence having at least 80% homology to the nucleotide sequence shown in (29); (32), a complementary sequence of the sequence shown in (29), (30) or (31); and / or The primer set 9: primers for amplifying herpes simplex virus type 8 have: (33), the nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18; (34) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (33); (35) a sequence having at least 80% homology to the nucleotide sequence shown in (33); (36), a complementary sequence of the sequence shown in (33), (34) or (35); and / or The primer set 10: primers for amplifying human adenovirus have: (37), the nucleotide sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20; (38) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (37); (39), a sequence having at least 80% homology to the nucleotide sequence shown in (37); (40), a complementary sequence of the sequence shown in (37), (38) or (39); and / or The primer set 11: primers for amplifying enterovirus A-71 type have: (41), the nucleotide sequences shown in SEQ ID NO:21 and SEQ ID NO:22; (42) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (41); (43) a sequence having at least 80% homology to the nucleotide sequence shown in (41); (44), a complementary sequence of the sequence shown in (41), (42) or (43); and / or The primer set 12: primers for amplifying Coxsackievirus A-16 type have: (45), the nucleotide sequences shown in SEQ ID NO:23 and SEQ ID NO:24; (46) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (45); (47), a sequence having at least 80% homology to the nucleotide sequence shown in (45); (48), a complementary sequence of the sequence shown in (45), (46) or (47); and / or The primer set 13: primers for amplifying mumps virus have: (49), the nucleotide sequences shown in SEQ ID NO:25 and SEQ ID NO:26; (50) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (49); (51), a sequence having at least 80% homology to the nucleotide sequence shown in (49); (52), a complementary sequence of the sequence shown in (49), (50) or (51); and / or The primer set 14: primers for amplifying Japanese encephalitis virus have: (53), the nucleotide sequences shown in SEQ ID NO:27 and SEQ ID NO:28; (54) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (53); (55), a sequence having at least 80% homology to the nucleotide sequence shown in (53); (56) A complementary sequence of the sequence shown in (53), (54) or (55).

2. The primer combination according to claim 1, characterized in that Also includes: Internal standard primer set; The internal standard primer group includes: internal standard primer group 1 and / or internal standard primer group 2; The internal standard primer set 1 has: (57), the nucleotide sequences shown in SEQ ID NO:29 and SEQ ID NO:30; (58) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (57); (59), a sequence having at least 80% homology to the nucleotide sequence shown in (57); (60), a complementary sequence of the sequence shown in (57), (58) or (59); and / or The internal standard primer set 2 has: (61), the nucleotide sequences shown in SEQ ID NO:31 and SEQ ID NO:32; (62) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (61); (63) a sequence having at least 80% homology to the nucleotide sequence shown in (61); (64) A complementary sequence of the sequence shown in (61), (62) or (63).

3. A probe set, characterized in that: include: As follows (b1) or (b2) or (b3): (b1), consisting of probe 1, probe 2, probe 3, probe 4, probe 5, probe 6, probe 7, probe 8, probe 9, probe 10, probe 11, probe 12, probe 13 and probe 14; (b2), consisting of any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of the probe 1, the probe 2, the probe 3, the probe 4, the probe 5, the probe 6, the probe 7, the probe 8, the probe 9, the probe 10, the probe 11, the probe 12, the probe 13, and the probe 14; (b3), including the probe 1, the probe 2, the probe 3, the probe 4, the probe 5, the probe 6, the probe 7, the probe 8, the probe 9, the probe 10, the probe 11, the probe 12, the probe 13 and the probe 14; Wherein: the probe 1: a probe for specific identification of herpes simplex virus type 1 has: (65), the nucleotide sequence shown in SEQ ID NO:33; (66) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (65); (67), a sequence having at least 80% homology to the nucleotide sequence shown in (65); (68), a complementary sequence of the sequence shown in (65), (66) or (67); and / or The probe 2: a probe for specifically identifying herpes simplex virus type 2 has: (69), the nucleotide sequence shown in SEQ ID NO:34; (70) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (69); (71), a sequence having at least 80% homology to the nucleotide sequence shown in (69); (72), a complementary sequence of the sequence shown in (69), (70) or (71); and / or The probe 3: a probe for specifically identifying varicella-zoster virus has: (73), the nucleotide sequence shown in SEQ ID NO:35; (74) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (73); (75), a sequence having at least 80% homology to the nucleotide sequence shown in (73); (76), a complementary sequence of the sequence shown in (73), (74) or (75); and / or The probe 4: a probe for specifically identifying EB virus has: (77), the nucleotide sequence shown in SEQ ID NO:36; (78) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (77); (79), a sequence having at least 80% homology to the nucleotide sequence shown in (77); (80), a complementary sequence of the sequence shown in (77), (78) or (79); and / or The probe 5: a probe for specifically identifying human cytomegalovirus has: (81), the nucleotide sequence shown in SEQ ID NO:37; (82) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (81); (83), a sequence having at least 80% homology to the nucleotide sequence shown in (81); (84), a complementary sequence of the sequence shown in (81), (82) or (83); and / or The probe 6: a probe for specifically identifying herpes simplex virus type 6A has: (85), the nucleotide sequence shown in SEQ ID NO:38; (86) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (85); (87), a sequence having at least 80% homology to the nucleotide sequence shown in (85); (88), a complementary sequence of the sequence shown in (85), (86) or (87); and / or The probe 7: a probe for specifically identifying herpes simplex virus type 6B has: (89), the nucleotide sequence shown in SEQ ID NO:39; (90) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence as shown in (89); (91), a sequence having at least 80% homology to the nucleotide sequence shown in (89); (92), a complementary sequence of the sequence shown in (89), (90) or (91); and / or The probe 8: a probe for specifically identifying herpes simplex virus type 7 has: (93), the nucleotide sequence shown in SEQ ID NO:40; (94) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (93); (95), a sequence having at least 80% homology to the nucleotide sequence shown in (93); (96), a complementary sequence of the sequence shown in (93), (94) or (95); and / or The probe 9: a probe for specifically identifying herpes simplex virus type 8 has: (97), the nucleotide sequence shown in SEQ ID NO:41; (98) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (97); (99), a sequence having at least 80% homology to the nucleotide sequence shown in (97); (100), a complementary sequence of the sequence shown in (97), (98) or (99); and / or The probe 10: a probe for specifically identifying human adenovirus has: (101), the nucleotide sequence shown in SEQ ID NO:42; (102) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (101); (103), a sequence having at least 80% homology to the nucleotide sequence shown in (101); (104), a complementary sequence of the sequence shown in (101), (102) or (103); and / or The probe 11: a probe for specifically identifying enterovirus A-71 type has: (105), the nucleotide sequence shown in SEQ ID NO:43; (106) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (105); (107), a sequence having at least 80% homology to the nucleotide sequence shown in (105); (108), a complementary sequence of the sequence shown in (105), (106) or (107); and / or The probe 12: a probe for specifically identifying Coxsackievirus A-16 type has: (109), the nucleotide sequence shown in SEQ ID NO:44; (110) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence as shown in (109); (111), a sequence having at least 80% homology to the nucleotide sequence shown in (109); (112), a complementary sequence of the sequence shown in (109), (110) or (111); and / or The probe 13: a probe for specifically identifying mumps virus has: (113), the nucleotide sequence shown in SEQ ID NO:45; (114) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (113); (115), a sequence having at least 80% homology to the nucleotide sequence shown in (113); (116), a complementary sequence of the sequence shown in (113), (114) or (115); and / or The probe 14: a probe for specifically identifying Japanese encephalitis virus has: (117), the nucleotide sequence shown in SEQ ID NO:46; (118) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence as shown in (117); (119), a sequence having at least 80% homology to the nucleotide sequence shown in (117); (120), a complementary sequence of the sequence shown in (117), (118) or (119).

4. The probe according to claim 3, characterized in that The 5' end of the nucleotide sequence is connected to a fluorescent reporter gene, and the 3' end is connected to a quencher gene; the quencher group includes any one of SQ, MGB or NFQ.

5. A primer probe set, characterized in that: include: The primer combination as claimed in claim 1 or 2 and the probe set as claimed in claim 3 or 4.

6. A detection reagent, characterized in that: include: The primer combination according to claim 1 or 2, the probe set according to claim 3 or 4 and / or the primer-probe set according to claim 5, and an acceptable auxiliary agent.

7. Use of the primer combination according to claim 1 or 2, the probe set according to claim 3 or 4, the primer-probe set according to claim 5 and / or the detection reagent according to claim 6 in the preparation of a product for detecting viral encephalitis.

8. The use according to claim 7, characterized in that The detection adopts the digital PCR method.

9. The use according to claim 7 or 8, characterized in that The detection comprises the following steps: mixing the sample to be detected with the detection reagent, centrifuging, obtaining microdroplets, amplifying, and obtaining the detection result.

10. The detection product is characterized in that, include: The primer combination according to claim 1 or 2, the probe according to claim 3 or 4, the primer-probe set according to claim 5 and / or the detection reagent according to claim 6, and acceptable auxiliary agents, excipients and / or devices.

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