A microdroplet digital PCR detection kit for detecting multiple viruses of central nervous system infection and application

Abstract

The present application relates to a kind of micro-droplet digital PCR detection kit and application for detecting central nervous system infection multiple viruses, belong to the field of reagent kit technology.The specific primers and detection probes of eight viruses EV, HPeV, HSV1, HSV2, VZV, CMV, HHV6 and EBV are designed and synthesized, and the detection method is optimized.The minimum detection limit of the method for EV, HPeV, HSV1, HSV2 is 5, 10, 5 and 10 copies per reaction respectively, and the minimum quantitative limit is 10, 10, 50 and 10 copies per reaction respectively.The minimum detection limit of VZV, CMV, HHV6 and EBV is 5 copies per reaction, and the minimum quantitative limit is 50 copies per reaction.The two groups are linear in the range of 2-2000 copies per reaction;14 other common pathogens of CNSI are detected, and no cross reaction is found, and the specificity is good.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of reagent kits, and particularly relates to a microdroplet digital PCR detection reagent kit for detecting multiple viruses causing central nervous system infection and application. BACKGROUND

[0002] The pathogens of central nervous system infection (CNSI) mainly include bacteria, viruses, fungi, spirochetes and protozoa, etc. When the pathogens enter the central nervous system, they can cause meningitis, encephalitis and myelitis, etc. The clinical symptoms include fever, headache, projectile vomiting, disturbance of consciousness, convulsions, etc. Among them, viral CNSI has been one of the main causes of children's nervous system infection and death in the world. The viruses that can cause CNSI mainly include enterovirus (EV), human parechovirus (HPeV), herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), varicella-zoster virus (VZV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6) and Epstein-Barr virus (EBV). The clinical manifestations caused by different viruses have no big difference. It is very important to determine the virus type for further diagnosis and treatment.

[0003] Currently, enzyme-linked immunosorbent assay (ELISA) or immunofluorescence method is mainly used in clinical laboratories to detect specific antibodies produced against viruses. This method is simple to operate, low in cost, short in time, and can be used for large-scale screening of antibody production in the population, but has low sensitivity, cross-reaction, false positive, etc. A single IgG cannot represent the symptomatic infection. IgM can be positive for one year after some viral infections. Some patients do not produce IgM antibodies or have low antibody titers after viral infection, which is not easy to detect.

[0004] Virus culture is the traditional gold standard for the diagnosis of viral CNSI. Its method is to add the sample into a specific cell line and observe the cytopathic effect. Because the virus culture cycle is long and some viruses are difficult to culture, currently, clinical laboratories have rarely carried out virus culture.

[0005] Real-time quantitative polymerase chain reaction (qPCR) is the most widely used method at present, and only enterovirus and herpes simplex virus fluorescent PCR single detection reagents have been approved and applied in the clinical central nervous system infection in China. There are problems of insufficient minimum detection limit and low virus detection rate. The reagents capable of detecting other central nervous system infection viruses are limited to scientific research, and there are almost no commercialized approved reagent kits.

[0006] Based on the above deficiencies of the prior art, there is an urgent need to provide a method with high sensitivity, high specificity and capable of simultaneously detecting multiple central nervous system infection viruses. SUMMARY

[0007] In view of the deficiencies of the prior art, the purpose of the present application is to provide a microdroplet digital PCR detection kit for detecting multiple viruses of central nervous system infection, which is used for the detection of central nervous system infection (CNSI) viruses, can simultaneously realize the detection of multiple viruses, and has the advantages of high detection sensitivity and strong specificity.

[0008] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] The present application provides a microdroplet digital PCR detection kit for detecting multiple viruses of central nervous system infection, which comprises a primer mixture I and a probe mixture I.

[0010] The primer mixture I comprises HSV1 detection primer, HSV2 detection primer, EV detection primer and HPeV detection primer.

[0011] The HSV1 detection primer comprises a forward primer shown in SEQ ID No. 2 and a reverse primer shown in SEQ ID No. 3; the HSV2 detection primer comprises a forward primer shown in SEQ ID No. 5 and a reverse primer shown in SEQ ID No. 6; the EV detection primer comprises a forward primer shown in SEQ ID No. 14 and a reverse primer shown in SEQ ID No. 15; and the HPeV detection primer comprises a forward primer shown in SEQ ID No. 17 and a reverse primer shown in SEQ ID No. 18.

[0012] The probe mixture I comprises HSV1 detection probe HSV1-FAM, HSV2 detection probe HSV2-FAM and HSV2-VIC, EV detection probe EV-FAM, HPeV detection probe HPeV-FAM and HPeV-VIC.

[0013] Wherein, the HSV1-FAM has the sequence shown in SEQ ID No. 1, and is labeled with FAM gene at the 5' end; the HSV2-FAM and the HSV2-VIC have the sequence shown in SEQ ID No. 4, and are labeled with FAM and VIC genes at the 5' end respectively; the EV-FAM has the sequence shown in SEQ ID No. 13, and is labeled with FAM gene at the 5' end; the HPeV-FAM and the HPeV-VIC have the sequence shown in SEQ ID No. 16, and are labeled with FAM and VIC genes at the 5' end respectively.

[0014] In the above technical solution of the present application, the specific detection primer pairs and probes for HSV1, HSV2, EV and HPeV are designed, and the design of primer and probe sequences is directly related to the specificity and sensitivity of ddPCR detection. The above specific primer and probe sequences of the present application are used for viral detection of central nervous system infection, and there is no interference between primers and probes, no cross reaction between different viral detections, no positive reaction to other pathogens causing central nervous system infection, and the advantages of strong detection specificity and high sensitivity.

[0015] Further, the concentration of each primer in the primer mixture I is 300-500 nM; preferably, the concentration of each primer in the primer mixture is 400 nM.

[0016] Further, the concentration of each probe in the probe mixture I is 50-500 nM.

[0017] Further, the concentration of EV-FAM in the probe mixture I is 200 nM; the concentration of HSV2-FAM is 100 nM; the concentration of HSV2-VIC is 100 nM; the concentration of HPeV-FAM is 50 nM; the concentration of HPeV-VIC is 150 nM; and the concentration of HSV1-VIC is 400 nM.

[0018] Further, the kit provided in the present application comprises a primer mixture II and a probe mixture II.

[0019] The primer mixture II comprises CMV detection primers, EBV detection primers, VZV detection primers and HHV6 detection primers.

[0020] The CMV detection primer comprises a forward primer shown in SEQ ID No. 8 and a reverse primer shown in SEQ ID No. 9; the EBV detection primer comprises a forward primer shown in SEQ ID No. 11 and a reverse primer shown in SEQ ID No. 12; the VZV detection primer comprises a forward primer shown in SEQ ID No. 20 and a reverse primer shown in SEQ ID No. 21; and the HHV6 detection primer comprises a forward primer shown in SEQ ID No. 23 and a reverse primer shown in SEQ ID No. 24.

[0021] The probe mixture II comprises CMV detection probes CMV-FAM and CMV-VIC, EBV detection probes EBV-FAM, VZV detection probes VZV-FAM, HHV6 detection probes HHV6-FAM and HHV6-VIC.

[0022] The CMV-FAM and the CMV-VIC have the sequence shown in SEQ ID No. 7, and are labeled with FAM and VIC genes at 5' ends, respectively; the EBV-FAM has the sequence shown in SEQ ID No. 10, and is labeled with FAM gene at 5' end; the VZV-FAM has the sequence shown in SEQ ID No. 22, and is labeled with FAM gene at 5' end; and the HHV6-FAM and the HHV6-VIC have the sequence shown in SEQ ID No. 25, and are labeled with FAM and VIC genes at 5' ends, respectively.

[0023] In the method, HSV1, HSV2, EV and HPeV are taken as group one, and CMV, EBV, VZV and HHV6 are taken as group two. The above-mentioned primer mixture II and probe mixture II are specific detection primers and probes for CMV, EBV, VZV and HHV6. By preparing the above-mentioned primer mixture I, probe mixture I, primer mixture II and probe mixture II, the detection of eight common viruses causing central nervous system infection, i.e., EV, HPeV, HSV1, HSV2, VZV, CMV, HHV6 and EBV, can be realized. The method is applied to clinical cerebrospinal fluid samples, and provides a more accurate and sensitive detection method for early etiological screening of viral CNSI.

[0024] Further, in the probe mixture II, the VZV-FAM has a concentration of 400 nM; the CMV-FAM has a concentration of 150 nM; the CMV-VIC has a concentration of 50 nM; the HHV6-FAM has a concentration of 50 nM; the HHV6-VIC has a concentration of 150 nM; and the EBV-VIC has a concentration of 200 nM.

[0025] Further, the use method of the kit comprises the following steps:

[0026] 1) Extract nucleic acid from the sample to be tested (for DNA viruses, extract DNA for step 2), and for RNA viruses, reverse transcription is also needed before step 2);

[0027] 2) Prepare a quadruple ddPCR amplification system, including primer mixture and probe mixture;

[0028] 3) Prepare microdroplets: transfer the prepared PCR reaction solution to the sample hole of the microdroplet generation card, then add microdroplet generation oil to the oil hole, cover the card with a rubber pad, and then place the card in the QX200TM microdroplet generator to prepare reaction microdroplets;

[0029] 4) PCR amplification: transfer the microdroplets of each sample to the corresponding reaction hole of the 96-well PCR reaction plate, heat seal, and then perform amplification on the PCR instrument;

[0030] 5) Perform microdroplet detection and data analysis.

[0031] Further, the sample to be tested is cerebrospinal fluid.

[0032] Further, the amplification procedure of step 4) is as follows: 96℃ pre-denaturation for 10 min; 98℃ denaturation for 30 s, 55℃ annealing for 1 min, a total of 40 cycles; 55℃ for 2 min.

[0033] The application also provides the use of the kit in the preparation of a viral central nervous system infection detection product.

[0034] Compared with the prior art, the application has the following beneficial effects:

[0035] 1) Design and synthesize specific primers and detection probes for EV, HPeV, HSV1, HSV2, VZV, CMV, HHV6 and EBV, and optimize the detection method. The minimum detection limit of the method for EV, HPeV, HSV1, HSV2 is 5, 10, 5 and 10 copies per reaction (copies / reaction), respectively, and the minimum quantification limit is 10, 10, 50 and 10 copies per reaction, respectively. The minimum detection limit of VZV, CMV, HHV6 and EBV is 5 copies per reaction, and the minimum quantification limit is 50 copies per reaction. Two groups show linearity in the range of 2-2000 copies per reaction; detection of another 14 common pathogens of CNSI (Staphylococcus hemolyticus, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Acinetobacter baumannii, Streptococcus pyogenes, Escherichia coli, Staphylococcus hominis, Pseudomonas aeruginosa, Klebsiella pneumoniae, influenza virus, respiratory syncytial virus, rotavirus, adenovirus) found no cross-reaction, good specificity.

[0036] 2) The application provides a two-group quadruplex ddPCR method for detecting common viruses of CNSI, which provides a new means for pathogenic diagnosis of viral CNSI, so as to realize early accurate diagnosis and treatment of CNSI patients and improve the prognosis of patients. The absolute quantification, high sensitivity and high specificity of the method make it have good clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 It is a two-dimensional graph of the ddPCR detection results of the four plasmid mixtures in group one in Example 1;

[0038] Figure 2 It is a two-dimensional graph of the ddPCR detection results of the four plasmid mixtures in group two in Example 1;

[0039] Figure 3 It is a two-dimensional graph of the linear test results in group one in Example 2;

[0040] Figure 4 It is a two-dimensional graph of the linear test results in group two in Example 2;

[0041] Figure 5 It is a two-dimensional graph of the specificity test results in group one in Example 3;

[0042] Figure 6 It is a two-dimensional graph of the specificity test results in group two in Example 3;

[0043] Figure 7 It is a two-dimensional graph of the sensitivity test results in group one in Example 4 at a concentration of 50 copies / reaction;

[0044] Figure 8 It is a two-dimensional graph of the sensitivity test results in group one in Example 4 at a concentration of 10 copies / reaction;

[0045] Figure 9 It is a two-dimensional graph of the sensitivity test results in group one in Example 4 at a concentration of 5 copies / reaction;

[0046] Figure 10 It is a two-dimensional graph of the sensitivity test results in group two in Example 4 at a concentration of 50 copies / reaction;

[0047] Figure 11 It is a two-dimensional graph of the sensitivity test results in group two in Example 4 at a concentration of 10 copies / reaction;

[0048] Figure 12 It is a two-dimensional graph of the sensitivity test results in group two in Example 4 at a concentration of 5 copies / reaction;

[0049] Figure 13 It is a two-dimensional graph of the blank detection limit results in group one in Example 5;

[0050] Figure 14 Two-dimensional plot of blank detection limit results for Example 5, Set 2;

[0051] Figure 15 Two-dimensional plot of first test results for Example 6, Set 1 probe concentration;

[0052] Figure 16 Two-dimensional plot of second test results for Example 6, Set 1 probe concentration;

[0053] Figure 17 Two-dimensional plot of first test results for Example 6, Set 2 probe concentration;

[0054] Figure 18 Two-dimensional plot of second test results for Example 6, Set 2 probe concentration;

[0055] Figure 19 Two-dimensional plot of third test results for Example 6, Set 2 probe concentration;

[0056] Figure 20 Two-dimensional plot of clinical test sample results for Example 7. DETAILED DESCRIPTION

[0057] The technical solutions of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. The described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0058] The methods are all conventional methods unless otherwise specified, and the raw materials can be obtained from public commercial channels unless otherwise specified.

[0059] Example 1

[0060] 1) Design and synthesis of primers, probes and plasmids

[0061] According to the 5'UTR sequence of HPeV, the glycoprotein D sequence of HSV1, the envelope glycoprotein G sequence of HSV2, the UL55 sequence of CMV, the EBNA-1 sequence of EBV, the 5'UTR sequence of EV, the ORF28 sequence of VZV, the U57 sequence of HHV6, the primer and probe sequences for HPeV, HSV1, HSV2, CMV, EBV, EV, VZV, and HHV6 were designed respectively by using the primer design software Primer Express 3.0.1. The designed primer and probe sequences were aligned by the BLAST online alignment function to preliminarily verify their specificity. The alignment results should be that each pair of primer and probe is completely complementary or basically complementary to all sequences of the corresponding virus in the database, and there is no cross with other species sequences. All primers and probes were synthesized by Shanghai Generay Biotech Co., Ltd.

[0062] According to the conserved sequences of each virus downloaded during primer design, the sequence containing the target fragment was selected as the target gene and inserted into the PUC57 vector. The plasmids of the above-mentioned 8 viruses were synthesized, and all the plasmids were synthesized by Shanghai Generay Biotech Co., Ltd.

[0063] The sequence information of the above-mentioned designed primers and probes is shown in Table 1 below, and the sequence information of the plasmids is shown in Table 2:

[0064] Table 1 Sequence information of primers and probes

[0065]

[0066] Table 2 Sequence information of plasmids

[0067]

[0068] 2) ddPCR reaction system

[0069] The ddPCR reaction system of 20 μl was configured as shown in Table 3:

[0070] Table 3 Two groups of quadruple ddPCR amplification systems

[0071]

[0072] Among them, the concentration of the probe mixture is shown in Table 4 below:

[0073] Table 4 Concentration of probe mixture

[0074]

[0075] 3) Preparation of microdroplets

[0076] QX200 droplet digital PCR platform and ddPCR premix reagent kit of Bio-Rad Company were adopted. The microdroplet generation card (DG8 cartridge) was fixed in the generation card base, the prepared 20 μl PCR reaction solution was transferred to the microdroplet generation card sample hole, 70 μl droplet generation oil was added to the oil hole, the card was placed in the QX200 TM Microdroplet preparation reaction was prepared in the microdroplet generator. The microdroplet preparation of 8 samples can be completed at a time, and the time was about 2.5 min, about 20000 water-in-oil microdroplets were generated in each hole. When the number of microdroplets was <10000, it did not meet the principle of Poisson distribution, and should be regenerated.

[0077] 4) PCR amplification

[0078] The microdroplets of each sample were transferred to the corresponding reaction holes of the 96-hole PCR reaction plate, and the aluminum film was covered on the 96-hole plate. The PX1 TM After heat sealing in the PCR heat sealer (180℃, 5 sec), amplification was carried out on the Bio-Rad T100 PCR instrument. The amplification program is shown in Table 5 below:

[0079] Table 5 ddPCR amplification program

[0080]

[0081] 5) Microdroplet detection and data analysis

[0082] The 96-hole plate after PCR amplification was put into the QX200 TM Microdroplet analyzer, set the sample information, reagent information, etc. on the setting panel of QuantaSoft software, click the "run" button, the instrument automatically analyzes the fluorescence signal in each microdroplet of each sample, then the QuantaSoft completes the automatic processing of the data, and the copy number concentration (unit: copies / μl) of the target sequence in the PCR reaction system is obtained.

[0083] The above ddPCR method was used to detect the four plasmid mixtures of group one and group two respectively, and the results are shown in Figures 1-2 , Figure 1 The ddPCR detection results of the four plasmid mixtures of group one are shown in the two-dimensional graph, wherein Figure 1 Group A is the EV positive microdroplet group, group B is the HSV2 microdroplet group, group C is the HPeV microdroplet group, and group D is the HSV1 microdroplet group. Figure 2This is a two-dimensional graph showing the ddPCR detection results of a mixture of four plasmids in group 2; group A represents VZV positive droplets, group B represents CMV droplets, group C represents HHV6 droplets, and group D represents EBV droplets.

[0084] The type of virus can be determined by the location of droplet clusters.

[0085] Example 2: Linearity Test

[0086] Preparation of reference samples: Four plasmids were used in each group, with copy numbers of 2000, 200, 20, and 2 copies respectively. Each gradient was repeated three times. Linear regression curves were plotted, R0... 2 A value above 0.98 is considered to be within the linear range. The plasmid concentration used to prepare the reference sample should be based on singlet ddPCR quantification, and then serially diluted to the required concentration. The two-dimensional graph of the linearity test results is shown below. Figure 3 The two-dimensional graph of the linearity test results for Group II is shown below. Figure 4 The X-axis represents the theoretical concentration, and the Y-axis represents the measured concentration. There are four concentration gradients from 2 to 2000, with each gradient repeated three times. Unit: copy number / reaction.

[0087] from Figures 3-4 It can be seen that both groups exhibited linearity in the range of 2–2000 copies per reaction. The R² values ​​for linear regression between theoretical and detected concentrations were EV: 0.9991, HPeV: 0.9996, HSV1: 0.9995, HSV2: 1, CMV: 0.9997, VZV: 0.9997, HHV6: 1, and EBV: 0.9999.

[0088] Example 3: Specificity Test

[0089] Following the ddPCR detection method in Example 1, cross-reactivity verification was performed using the following 14 pathogens: Staphylococcus hemolyticus, Staphylococcus aureus (ATCC25923), Staphylococcus epidermidis, Streptococcus pneumoniae (ATCC49619), Acinetobacter baumannii, Streptococcus pyogenes, Escherichia coli (ATCC25922), Staphylococcus aureus, Pseudomonas aeruginosa (ATCC27853), Klebsiella pneumoniae, influenza virus, respiratory syncytial virus, rotavirus, and adenovirus. Results are shown below. Figures 5-6 As shown, Figure 5 A two-dimensional graph showing the results of the group 1 specificity test. Figure 6 This is a two-dimensional graph showing the results of the group 2 specificity test.

[0090] from Figures 5-6 As can be seen, the ddPCR detection method provided in this application is negative for all 14 pathogens mentioned above, with no cross-reaction, proving that the detection method in this application has good specificity.

[0091] Example 4: Sensitivity test

[0092] According to the ddPCR detection method in Example 1, the eight plasmid gradient dilution mixtures in the example were mixed as a positive template mixture to determine the limit of detection (LOD) and limit of quantification (LOQ) of the method. The reference was prepared, and the gradient of the four plasmid references in the same group was 50 copies, 10 copies, 5 copies, and 2 copies, respectively. In each group of 20 repeated tests, at least 19 tests were positive, and the lowest concentration was the sensitivity of the detection system. The lowest concentration gradient with a CV value of 20 quantification results of ≤25% was defined as the limit of quantification (LOQ). The plasmid concentration for the reference was quantified by single ddPCR, and then diluted to the required concentration.

[0093] The results of the sensitivity test are shown in Tables 6-7 and Figures 7-12 , wherein Figure 7 is a two-dimensional graph example of the concentration of 50 copies per reaction in group one sensitivity test results; Figure 8 is a two-dimensional graph example of the concentration of 10 copies per reaction in group one sensitivity test results; Figure 9 is a two-dimensional graph of the concentration of 5 copies per reaction in group one sensitivity test results; Figure 10 is a two-dimensional graph of the concentration of 50 copies per reaction in group two sensitivity test results; Figure 11 is a two-dimensional graph of the concentration of 10 copies per reaction in group two sensitivity test results; Figure 12 is a two-dimensional graph of the concentration of 5 copies per reaction in group two sensitivity test results; and Figures 7-12 the two-dimensional graphs above are all from one of the 20 repetitions.

[0094] Table 6 LOQ and LOD of group one virus

[0095]

[0096] Table 7 LOQ and LOD of group two virus

[0097]

[0098] From the results of Tables 6-7 and Figures 7-12 , it can be seen that the ddPCR method of the present application has good detection sensitivity.

[0099] Example 5: Blank detection limit determination

[0100] 10 cases of negative cerebrospinal fluid samples were confirmed to be free of 8 virus infections, ensuring that there was no virus mixing in the extraction process, reverse transcription process and environment. The 10 cases of cerebrospinal fluid samples were subjected to two groups of four ddPCR reactions, and according to the number of positive droplets in the detection results, the blank detection limit (limit of blank, LOB) of clinical interpretation was determined. The results are shown in Figures 13-14 , Figure 13 Figure 1 is a two-dimensional graph of the blank detection limit results of group one; Figure 14 Figure 2 is a two-dimensional graph of the blank detection limit results of group two; it can be seen from Figure 13 that the detection results of 10 cases of negative cerebrospinal fluid showed one positive droplet in some samples. It can be seen from Figure 14 that the detection results of 10 cases of negative cerebrospinal fluid were all negative.

[0101] Results: The blank detection limit (LOB) of EV, HPeV, HSV1 and HSV2 in group one was 1 positive droplet, and when the number of positive droplets was >1, it was judged to be positive, otherwise it was negative. The blank detection limit (LOB) of VZV, CMV, HHV6 and EBV in group two was 0, and the number of positive droplets ≥1 was positive.

[0102] Example 6: Optimization test

[0103] 1) Group one probe concentration optimization

[0104] First time group one probe concentration: 200 nM EV-FAM probe, 150 nM HSV2-FAM and 50 nM VIC probe (FAM:VIC=3:1), 50 nM HPeV-FAM probe and 150 nM VIC probe (FAM:VIC=1:3), 200 nM HSV1-VIC probe, Figure 15 Figure 3 shows a two-dimensional graph of the first time group one probe concentration test results.

[0105] Second time group one probe concentration: 200 nM EV-FAM probe, 100 nM HSV2-FAM and 100 nM VIC probe (FAM:VIC=1:1), 50 nM HPeV-FAM probe and 150 nM VIC probe (FAM:VIC=1:3), 400 nM HSV1-VIC probe, Figure 16 Figure 4 shows a two-dimensional graph of the second time group one probe concentration test results.

[0106] From the distribution of droplet groups shown in Figure 15 and Figure 16 , the second time probe concentration was finally selected as the group one probe concentration of the present application.

[0107] 2) Group two probe concentration optimization

[0108] Group 2 probe concentration first time: 200 nM of VZV-FAM probe, 150 nM of CMV-FAM and 50 nM of VIC probe (FAM:VIC = 3:1), 50 nM of HHV6-FAM probe and 150 nM of VIC probe (FAM:VIC = 1:3), 200 nM of EBV-VIC probe, Figure 17 The two-dimensional diagram of the test results of the group 2 probe concentration first time is shown.

[0109] Group 2 probe concentration second time: 200 nM of VZV-FAM probe, 150 nM of CMV-FAM and 50 nM of CMV-VIC probe (FAM:VIC = 3:1), 100 nM of HHV6-FAM probe and 100 nM of HHV6-VIC probe (FAM:VIC = 1:1), 200 nM of EBV-VIC probe, Figure 18 The two-dimensional diagram of the test results of the group 2 probe concentration second time is shown.

[0110] Group 2 probe concentration third time: 400 nM of VZV-FAM probe, 150 nM of CMV-FAM and 50 nM of VIC probe (FAM:VIC = 3:1), 50 nM of HHV6-FAM probe and 150 nM of VIC probe (FAM:VIC = 1:3), 200 nM of EBV-VIC probe, Figure 19 The two-dimensional diagram of the test results of the group 2 probe concentration third time is shown.

[0111] From Figures 17-19 The distribution of the microdroplet groups is shown, and the probe concentration of the third time is finally selected as the group 2 probe concentration of the application.

[0112] Example 7: Detection of clinical samples

[0113] According to the ddPCR detection method in Example 1, the cerebrospinal fluid of 100 patients suspected of viral CNSI was detected by two groups of quadruple ddPCR. At the same time, the positive sample well and the positive control well were selected, and the positive microdroplet group of the sample well was coincided with the control, so as to ensure the accuracy of the result interpretation. Figure 20 The two-dimensional diagram of the test results of VZV+HHV6 mixed infection (detection example) is shown.

[0114] The detection results show that the ddPCR detection results of the application are consistent with the results of other existing clinical detection methods, which indicates that the ddPCR detection method of the application can be well used for the detection of common viral pathogens of CNSI in clinical samples.

[0115] It should be noted that the above examples are only used to illustrate the technical solutions of the present application but not to limit the technical solutions of the present application; although the present application is explained in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the specific embodiments of the present application can be modified or some technical features can be replaced by equivalent ones, and all of them should be included in the technical solution range of the present application.

Claims

1. A droplet digital PCR detection kit for detecting multiple viruses infecting the central nervous system, characterized in that, Includes primer mixture I, probe mixture I, primer mixture II, and probe mixture II; The primer mixture I includes HSV1 detection primers, HSV2 detection primers, EV detection primers, and HPeV detection primers; The HSV1 detection primers include the forward primer shown in SEQ ID No. 2 and the reverse primer shown in SEQ ID No. 3; the HSV2 detection primers include the forward primer shown in SEQ ID No. 5 and the reverse primer shown in SEQ ID No. 6; the EV detection primers include the forward primer shown in SEQ ID No. 14 and the reverse primer shown in SEQ ID No. 15; and the HPeV detection primers include the forward primer shown in SEQ ID No. 17 and the reverse primer shown in SEQ ID No.

18. The probe mixture I includes HSV1 detection probe HSV1-FAM, HSV2 detection probes HSV2-FAM and HSV2-VIC, EV detection probe EV-FAM, and HPeV detection probes HPeV-FAM and HPeV-VIC; The HSV1-FAM sequence is shown in SEQ ID No. 1, with the 5' end marked with the FAM gene; the HSV2-FAM and HSV2-VIC sequences are shown in SEQ ID No. 4, with the 5' ends marked with the FAM and VIC genes, respectively; the EV-FAM sequence is shown in SEQ ID No. 13, with the 5' end marked with the FAM gene; and the HPeV-FAM and HPeV-VIC sequences are shown in SEQ ID No. 16, with the 5' ends marked with the FAM and VIC genes, respectively. The primer mixture II includes CMV detection primers, EBV detection primers, VZV detection primers, and HHV6 detection primers; The CMV detection primers include the forward primer shown in SEQ ID No. 8 and the reverse primer shown in SEQ ID No. 9; the EBV detection primers include the forward primer shown in SEQ ID No. 11 and the reverse primer shown in SEQ ID No. 12; the VZV detection primers include the forward primer shown in SEQ ID No. 20 and the reverse primer shown in SEQ ID No. 21; and the HHV6 detection primers include the forward primer shown in SEQ ID No. 23 and the reverse primer shown in SEQ ID No.

24. The probe mixture II includes CMV detection probes CMV-FAM and CMV-VIC, EBV detection probe EBV-FAM, VZV detection probe VZV-FAM, and HHV6 detection probes HHV6-FAM and HHV6-VIC. The CMV-FAM and CMV-VIC sequences are shown in SEQ ID No. 7, with FAM and VIC gene markers at their 5' ends, respectively; the EBV-FAM sequence is shown in SEQ ID No. 10, with FAM gene markers at its 5' end; the VZV-FAM sequence is shown in SEQ ID No. 22, with FAM gene markers at its 5' end; and the HHV6-FAM and HHV6-VIC sequences are shown in SEQ ID No. 25, with FAM and VIC gene markers at their 5' ends, respectively. EV-FAM concentration was 200 nM; HSV2-FAM concentration was 100 nM; HSV2-VIC concentration was 100 nM; HPeV-FAM concentration was 50 nM; HPeV-VIC concentration was 150 nM; HSV1-VIC concentration was 400 nM. The concentrations of VZV-FAM were 400 nM; CMV-FAM were 150 nM; CMV-VIC were 50 nM; HHV6-FAM were 50 nM; HHV6-VIC were 150 nM; and EBV-VIC were 200 nM.

2. The reagent kit according to claim 1, characterized in that, The concentration of each primer in primer mixture I is 300–500 nM.

3. The reagent kit according to claim 1, characterized in that, The concentration of each primer in primer mixture I is 400 nM.

4. The use of the kit according to any one of claims 1 to 3 in the preparation of a viral central nervous system infection detection product.

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