Primer probe composition and kit for organ transplantation susceptible virus nucleic acid detection

Abstract

The invention provides an organ transplantation susceptible virus nucleic acid detection primer probe composition and a kit, and belongs to the technical field of biological detection. The primer probe composition comprises an upstream primer, a downstream primer and a fluorescent probe sequence, wherein the upstream primer and the downstream primer are designed for human cytomegalovirus, JC virus, BK polyoma virus, EB virus, human herpes virus type 6, human herpes virus type 8, human parvovirus B19, torque teno virus, new coronavirus and hepatitis E virus respectively; an upstream primer, a downstream primer and a fluorescent probe sequence of an internal standard are designed aiming at a human housekeeping gene RNaseP. The kit can effectively distinguish 10 viruses, ensures high accuracy, specificity and sensitivity, can complete detection in a short time to obtain a result, and can be transported and stored at normal temperature.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a primer-probe composition and a kit for detecting nucleic acid of susceptible viruses for organ transplantation. Background Art

[0002] With the development of medical science, the number of organ transplants has increased year by year, and organ transplantation has been recognized as an effective treatment for end-stage diseases of organs such as the heart, pancreas, liver, lungs and kidneys. However, due to the lack of perfect preventive measures, rapid and effective diagnostic methods and reasonable treatment plans after solid organ transplantation, the morbidity and mortality of patients with postoperative infections are still high.

[0003] Postoperative infection is one of the important reasons for the ultimate failure of organ transplantation. With the emergence and combined use of immunosuppressive drugs, the early mortality rate of transplantation caused by infection has dropped significantly. However, due to the increase in the base of transplant patients, the postoperative threat caused by infection cannot be ignored. Among them, viral infections account for a large proportion, such as: human cytomegalovirus, JC virus, parvovirus, BK polyomavirus, Epstein-Barr virus, human parvovirus, human herpes virus type 6, human herpes virus type 8, new coronavirus, hepatitis E virus, etc.

[0004] Currently, the commonly used detection methods for pathogens include: culture method, serological test, molecular biology test method, etc. The culture method is the most reliable method for diagnosing viral infection, but this method is complex, cumbersome, long cycle, low sensitivity, high sample requirements, and prone to false negative results. Serological testing is based on the principle of antigen-antibody reaction, with a short detection cycle, but low specificity and sensitivity, prone to false negative and false positive results, and has certain limitations. Fluorescence PCR is a molecular biological method for early rapid diagnosis of pathogens that is currently widely used in clinical practice, but it can usually only detect one pathogen at a time. Gene chip technology can realize the simultaneous detection of multiple pathogens, but the existing chip technology is basically amplification followed by hybridization, which is relatively complicated and time-consuming. In addition, the process of opening the tube to take the PCR product will expose the high-concentration product to the air, which can easily cause cross-contamination between samples.

[0005] It can be seen that it is difficult to identify the type of infectious pathogen through clinical symptoms and routine laboratory tests.

[0006] Chinese invention patent application CN202310182324.0 discloses a specific primer, probe and kit for simultaneously detecting TTV, BKV and JCV after transplantation. Only cyclovirus, BK polyomavirus and JC polyomavirus can be detected simultaneously using a single-hole reaction. The reagent is a liquid reagent with low sensitivity of 5.85E+03 copies / mL.

[0007] Chinese invention patent application CN202210170155.4 discloses a primer and probe for rapid screening and quantitative detection of multiple viruses before and after organ transplantation; it only involves 6 pathogens, namely HCMV, EBV, HHV6, HHV8, BKV, and JCV, and a single well can detect up to 3 pathogens. The reagent is a liquid reagent with low sensitivity of 25 copies / mL.

[0008] Therefore, there is an urgent need in this field for a simple, rapid, and objective method for detecting pathogens to meet the detection needs of organ transplant patients, thereby achieving the identification of pathogens, early diagnosis, and reasonable guidance of clinical drug use. Summary of the invention

[0009] In view of the above technical defects, the present invention provides a primer probe composition and a kit for detecting nucleic acid of susceptible viruses in organ transplantation. The kit of the present invention is a kit for detecting human cytomegalovirus ( human cytomegalovirus , HCMV), JC virus, BK polyomavirus (BK p olyoma virus ), Epstein-Barr virus (EBV), human herpes virus type 6 ( human herpes virus 6, HHV-6), human herpes virus type 8 ( human herpes virus 8, HHV-8), human parvovirus B19 ( human bocavirus B19), Torque teno virus (TTV), Corona Virus Disease 2019 (COVID-19), and Hepatitis E Virus (HEV). The single-hole reaction is used to detect 10 viruses at the same time, which can effectively distinguish the 10 viruses, ensure high accuracy, specificity and sensitivity, complete the test results in a short time, and can be transported and stored at room temperature.

[0010] The technical solution adopted by the present invention is: According to the NCBI GeneBank database, human cytomegalovirus ( human cytomegalovirus , HCMV), JC virus, BK polyomavirus (BK p olyoma virus ), Epstein-Barr virus (EBV), human herpes virus type 6 ( human herpes virus 6, HHV-6), human herpes virus type 8 ( human herpes virus 8, HHV-8), human parvovirus B19 ( human bocavirusB19), Torque teno virus (TTV), Corona Virus Disease 2019 (COVID-19), and Hepatitis E Virus (HEV) gene sequences were compared and the following primer probes were designed: SEQ ID NO.1: an upstream primer designed for human cytomegalovirus, the sequence of which is 5'-GTTTTACAGGCGTGGTCAAC-3'; referred to as HCMV-F; SEQ ID NO.2: A downstream primer designed for human cytomegalovirus, the sequence of which is 5'-CTGAAAAGTTGTCTTTGATGAACC-3'; referred to as HCMV-R; SEQ ID NO.3: A fluorescent probe designed for human cytomegalovirus, the sequence of which is 5'-TGCCGTGTCTGCCCATCGCC-3'; referred to as HCMV-P; the 5' end of the fluorescent probe is labeled with a reporter group FAM, and the 3' end is labeled with a quencher group BHQ1; SEQ ID NO.4: An upstream primer designed for JC virus, the sequence of the upstream primer is 5'-TGTGATAGGTGCAGTGTTCC-3'; referred to as JC-F; SEQ ID NO.5: A downstream primer designed for JC virus, the sequence of which is 5'-ACTCTGGACATGGATCAAGC-3'; referred to as JC-R; SEQ ID NO.6: A fluorescent probe designed for JC virus, the sequence of which is 5'-CAATCACAATGCTCTTCCCAGGTCTCAG-3'; referred to as JC-P; the 5' end of the fluorescent probe is labeled with a reporter group FAM, and the 3' end is labeled with a quencher group BHQ1; SEQ ID NO.7: An upstream primer designed for BK polyomavirus, the sequence of the upstream primer is 5'-GGCATAACAAGTACCTCAGAG-3'; referred to as BK-F; SEQ ID NO.8: A downstream primer designed for BK polyomavirus, the sequence of which is 5'-CAGCAAACCCAGCAATAGC-3'; referred to as BK-R; SEQ ID NO.9: A fluorescent probe designed for BK polyomavirus, the sequence of which is 5'-CCAGGAGCACCAGCAATTACAGCATAT-3'; referred to as BK-P; the 5' end of the fluorescent probe is labeled with a reporter group FAM, and the 3' end is labeled with a quencher group BHQ1; SEQ ID NO.10: An upstream primer designed for Epstein-Barr virus, the sequence of which is 5'-TCACATGGAGGATTGAGGAC-3'; referred to as EBV-F; SEQ ID NO.11: A downstream primer designed for Epstein-Barr virus, the sequence of which is 5'-AGGCATACATATGAAGTCAAACC-3'; referred to as EBV-R; SEQ ID NO.12: A fluorescent probe designed for Epstein-Barr virus, the sequence of which is 5'-TCCATCACAGAGGCACACTAACCGTAA-3'; referred to as EBV-P; the 5' end of the fluorescent probe is labeled with a reporter group ROX, and the 3' end is labeled with a quencher group BHQ2; SEQ ID NO.13: An upstream primer designed for human herpesvirus type 6, the sequence of the upstream primer is 5'-CATAACCGCATAACTCTGTCC-3'; abbreviated as HHV-6-F; SEQ ID NO.14: A downstream primer designed for human herpesvirus type 6, the sequence of which is 5'-GCGAGATGGAGGAAGAAGG-3'; referred to as HHV-6-R; SEQ ID NO.15: A fluorescent probe designed for human herpesvirus type 6, the sequence of which is 5'-ACACTAATCCTCGCATCCGCACCT-3'; referred to as HHV-6-P; the 5' end of the fluorescent probe is labeled with a reporter group ROX, and the 3' end is labeled with a quencher group BHQ2; SEQ ID NO.16: An upstream primer designed for human herpesvirus type 8, the sequence of the upstream primer is 5'-TGATTTCAACGCCTTACACG-3'; abbreviated as HHV-8-F; SEQ ID NO.17: A downstream primer designed for human herpesvirus type 8, the sequence of which is 5'-GTCCCACACAACTAAAATTGC-3'; abbreviated as HHV-8-R; SEQ ID NO.18: A fluorescent probe designed for human herpesvirus type 8, the sequence of which is 5'-CCGTCTAATACATCCTTGCCAACATCCTG-3'; referred to as HHV-8-P; the 5' end of the fluorescent probe is labeled with a reporter group ROX, and the 3' end is labeled with a quencher group BHQ2; SEQ ID NO.19: An upstream primer designed for human parvovirus B19, the sequence of the upstream primer is 5'-TAGAGGGGTGCTTCAAGTTTC-3'; referred to as B19-F; SEQ ID NO.20: A downstream primer designed for human parvovirus B19, the sequence of which is 5'-CCAGTCAGAGGTGTCTAAATCC-3'; referred to as B19-R; SEQ ID NO.21: A fluorescent probe designed for human parvovirus B19, the sequence of which is 5'-CACCACCAGTTATCGTTAGCACAGTCC-3'; referred to as B19-P; the 5' end of the fluorescent probe is labeled with a reporter group CY5, and the 3' end is labeled with a quencher group BHQ3; SEQ ID NO.22: An upstream primer designed for the trichothivirus, the sequence of which is 5'-AAGTACGAGCCACCAGAAG-3'; referred to as TTV-F; SEQ ID NO.23: A downstream primer designed for the parvovirus, the sequence of which is 5'-CTCTTCTTCCTCTTCTGATTGG-3'; referred to as TTV-R; SEQ ID NO.24: A fluorescent probe designed for trichothivirus, the sequence of which is 5'-TGTCTCTTCTTCCTGGCTGCTCTCC-3'; referred to as TTV-P; the 5' end of the fluorescent probe is labeled with a reporter group CY5, and the 3' end is labeled with a quencher group BHQ3; SEQ ID NO.25: An upstream primer designed for the novel coronavirus, the sequence of which is 5'-CAGTGGCTAACTAACATCTTTGG-3'; referred to as COVID-19-F; SEQ ID NO.26: A downstream primer designed for the novel coronavirus, the sequence of which is 5'-TCTCTAAGAAACTCTACACCTTCC-3'; referred to as COVID-19-R; SEQ ID NO.27: A fluorescent probe designed for the novel coronavirus, the sequence of which is 5'-ACTTCTCTTCAAGCCAATCAAGGACGG-3'; referred to as COVID-19-P; the 5' end of the fluorescent probe is labeled with a reporter group CY5, and the 3' end is labeled with a quencher group BHQ3; SEQ ID NO.28: An upstream primer designed for hepatitis E virus, the sequence of the upstream primer is 5'-CATCCCTTCTGATCTTGCTG-3'; referred to as HEV-F; SEQ ID NO.29: A downstream primer designed for hepatitis E virus, the sequence of the downstream primer is 5'-GGTGGTCTTACAGGTGAGG-3'; referred to as HEV-R; SEQ ID NO.30: A fluorescent probe designed for hepatitis E virus, the sequence of which is 5'-AGCCGCCTTACTGCCACCGTT-3'; referred to as HEV-P; the 5' end of the fluorescent probe is labeled with a reporter group CY5, and the 3' end is labeled with a quencher group BHQ3; SEQ ID NO.31: An upstream primer designed for the human housekeeping gene RNaseP, the sequence of which is 5'-CATCCCTTCTGATCTTGCTG-3'; referred to as RNaseP-F; SEQ ID NO.32: A downstream primer designed for the human housekeeping gene RNaseP, the sequence of which is 5'-GGTGGTCTTACAGGTGAGG-3'; referred to as RNaseP-R; SEQ ID NO.33: A fluorescent probe designed for the human housekeeping gene RNaseP, the sequence of which is 5'-AGCCGCCTTACTGCCACCGTT-3'; referred to as RNaseP-P; the 5' end of the fluorescent probe is labeled with a reporter group VIC, and the 3' end is labeled with a quencher group BHQ1; The present invention uses the human housekeeping gene RNaseP as an internal standard and sample to simultaneously perform nucleic acid extraction, PCR amplification and other processes, and can monitor the entire detection process to ensure the validity of the results.

[0011] The probes of the present invention are modified with FAM, ROX, CY5 and VIC fluorescent groups respectively, meeting the requirements of multi-channel and high-throughput detection.

[0012] An organ transplant susceptible virus nucleic acid detection kit comprises: a freeze-dried reaction tube, a negative quality control, a positive quality control and a diluent.

[0013] The reagents in the freeze-drying reaction tube include the following components: One Step Lyo-Enzyme Mix 1.2μL, 5×One Step Mix 5μL, 50μM HCMV-F, 5μM HCMV-R, 5μM HCMV-P, 50μM JC-F, 5μM JC-R, 5μM JC-P, 50μM BK-F, 5μM BK-R, 5μM BK-P, 50μM EBV-F, 5μM EBV-R, 5μM EBV-P, 50μM HHV-6-F, 5μM HHV-6-R, 5μM HHV-6-P, 50μM HHV-8-F, 5μM HHV-8-R, 5μM HHV-8-P, 50μM B19-F, 5μM B19-R, 5μM B19-P, 50μM TTV-F, 5μM TTV-R, 5μM TTV-P, 50μM COVID-19-F, 5μM COVID-19-R, 5μM COVID-19-P, 50μM HEV-F, 5μM HEV-R, 5μM HEV-P, 5μM IC-F, 5μM IC-R, 5μM IC-P, m / v 10% lyoprotectant.

[0014] The freeze-drying protective agent comprises: 20% by mass of dextran, 2% by mass of bovine serum albumin (BSA), 2% by mass of polyvinylpyrrolidone (PVP), 1% by mass of sorbitol, 20% by mass of polyadenylic acid (Ploy A), 2% by mass of glycine, and 2% by mass of sodium citrate.

[0015] The positive quality control was 1×10 6 The lyophilized powder contains 10 copies / mL of artificially synthesized plasmids of 10 viruses and RNaseP artificially synthesized plasmids.

[0016] The negative quality control uses TE Buffer.

[0017] The components contained in the diluent are: formamide 1M, tris-HCl (Tris-HCl) 1M, EDTA-2Na 2M, sodium chloride (NaCl) 5M, Triton X-100 6M, and isopropanol 10M.

[0018] The beneficial effects of the present invention are as follows: the present invention utilizes a single-hole reaction to simultaneously detect 10 viruses, can effectively distinguish the 10 viruses, ensures high accuracy, specificity and sensitivity, can complete the detection and obtain results in a short time, and can be transported and stored at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1This is a picture of the test kit test results.

[0020] Figure 2 Synthesize plasmids for 10 viruses 2 copies / mL sensitivity test result graph and amplification graph.

[0021] Figure 3 Synthesize plasmids for 10 viruses 1 copies / mL sensitivity test result graph and amplification graph.

[0022] Figure 4 Synthesize plasmids for 10 viruses 0 copies / mL sensitivity test result graph and amplification graph.

[0023] Figure 5 The figure shows the kit-specific amplification results.

[0024] Figure 6 This is a graph showing the amplification results of the blood sample after dilution with diluent.

[0025] Figure 7 This is the amplification result of the blood sample without dilution with diluent. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the invention and are not intended to limit the scope of the invention. In addition, it should be understood that after reading the present invention, those skilled in the art can make various technical changes or modifications to the present invention through technical attempts, and these equivalent forms generally fall within the scope defined by the appended claims of the present application.

[0027] Example 1 A primer-probe composition for detecting nucleic acid of susceptible viruses in organ transplantation According to the NCBI GeneBank database, human cytomegalovirus ( human cytomegalovirus , HCMV), JC virus, BK polyomavirus (BK p olyoma virus ), Epstein-Barr virus (EBV), human herpes virus type 6 ( human herpes virus 6, HHV-6), human herpes virus type 8 ( human herpes virus 8, HHV-8), human parvovirus B19 ( human bocavirus B19), Torque teno virus (TTV), Corona Virus Disease 2019 (COVID-19), and Hepatitis E Virus (HEV) gene sequences were compared and the following primer probes were designed: SEQ ID NO.1: an upstream primer designed for human cytomegalovirus, the sequence of which is 5'-GTTTTACAGGCGTGGTCAAC-3'; referred to as HCMV-F; SEQ ID NO.2: A downstream primer designed for human cytomegalovirus, the sequence of which is 5'-CTGAAAAGTTGTCTTTGATGAACC-3'; referred to as HCMV-R; SEQ ID NO.3: A fluorescent probe designed for human cytomegalovirus, the sequence of which is 5'-TGCCGTGTCTGCCCATCGCC-3'; referred to as HCMV-P; the 5' end of the fluorescent probe is labeled with a reporter group FAM, and the 3' end is labeled with a quencher group BHQ1; SEQ ID NO.4: An upstream primer designed for JC virus, the sequence of the upstream primer is 5'-TGTGATAGGTGCAGTGTTCC-3'; referred to as JC-F; SEQ ID NO.5: A downstream primer designed for JC virus, the sequence of which is 5'-ACTCTGGACATGGATCAAGC-3'; referred to as JC-R; SEQ ID NO.6: A fluorescent probe designed for JC virus, the sequence of which is 5'-CAATCACAATGCTCTTCCCAGGTCTCAG-3'; referred to as JC-P; the 5' end of the fluorescent probe is labeled with a reporter group FAM, and the 3' end is labeled with a quencher group BHQ1; SEQ ID NO.7: An upstream primer designed for BK polyomavirus, the sequence of the upstream primer is 5'-GGCATAACAAGTACCTCAGAG-3'; referred to as BK-F; SEQ ID NO.8: A downstream primer designed for BK polyomavirus, the sequence of which is 5'-CAGCAAACCCAGCAATAGC-3'; referred to as BK-R; SEQ ID NO.9: A fluorescent probe designed for BK polyomavirus, the sequence of which is 5'-CCAGGAGCACCAGCAATTACAGCATAT-3'; referred to as BK-P; the 5' end of the fluorescent probe is labeled with a reporter group FAM, and the 3' end is labeled with a quencher group BHQ1; SEQ ID NO.10: An upstream primer designed for Epstein-Barr virus, the sequence of which is 5'-TCACATGGAGGATTGAGGAC-3'; referred to as EBV-F; SEQ ID NO.11: A downstream primer designed for Epstein-Barr virus, the sequence of which is 5'-AGGCATACATATGAAGTCAAACC-3'; referred to as EBV-R; SEQ ID NO.12: A fluorescent probe designed for Epstein-Barr virus, the sequence of which is 5'-TCCATCACAGAGGCACACTAACCGTAA-3'; referred to as EBV-P; the 5' end of the fluorescent probe is labeled with a reporter group ROX, and the 3' end is labeled with a quencher group BHQ2; SEQ ID NO.13: An upstream primer designed for human herpesvirus type 6, the sequence of the upstream primer is 5'-CATAACCGCATAACTCTGTCC-3'; abbreviated as HHV-6-F; SEQ ID NO.14: A downstream primer designed for human herpesvirus type 6, the sequence of which is 5'-GCGAGATGGAGGAAGAAGG-3'; referred to as HHV-6-R; SEQ ID NO.15: A fluorescent probe designed for human herpesvirus type 6, the sequence of which is 5'-ACACTAATCCTCGCATCCGCACCT-3'; referred to as HHV-6-P; the 5' end of the fluorescent probe is labeled with a reporter group ROX, and the 3' end is labeled with a quencher group BHQ2; SEQ ID NO.16: An upstream primer designed for human herpesvirus type 8, the sequence of the upstream primer is 5'-TGATTTCAACGCCTTACACG-3'; abbreviated as HHV-8-F; SEQ ID NO.17: A downstream primer designed for human herpesvirus type 8, the sequence of which is 5'-GTCCCACACAACTAAAATTGC-3'; abbreviated as HHV-8-R; SEQ ID NO.18: A fluorescent probe designed for human herpesvirus type 8, the sequence of which is 5'-CCGTCTAATACATCCTTGCCAACATCCTG-3'; referred to as HHV-8-P; the 5' end of the fluorescent probe is labeled with a reporter group ROX, and the 3' end is labeled with a quencher group BHQ2; SEQ ID NO.19: An upstream primer designed for human parvovirus B19, the sequence of the upstream primer is 5'-TAGAGGGGTGCTTCAAGTTTC-3'; referred to as B19-F; SEQ ID NO.20: A downstream primer designed for human parvovirus B19, the sequence of which is 5'-CCAGTCAGAGGTGTCTAAATCC-3'; referred to as B19-R; SEQ ID NO.21: A fluorescent probe designed for human parvovirus B19, the sequence of which is 5'-CACCACCAGTTATCGTTAGCACAGTCC-3'; referred to as B19-P; the 5' end of the fluorescent probe is labeled with a reporter group CY5, and the 3' end is labeled with a quencher group BHQ3; SEQ ID NO.22: An upstream primer designed for the trichothivirus, the sequence of which is 5'-AAGTACGAGCCACCAGAAG-3'; referred to as TTV-F; SEQ ID NO.23: A downstream primer designed for the parvovirus, the sequence of which is 5'-CTCTTCTTCCTCTTCTGATTGG-3'; referred to as TTV-R; SEQ ID NO.24: A fluorescent probe designed for trichothivirus, the sequence of which is 5'-TGTCTCTTCTTCCTGGCTGCTCTCC-3'; referred to as TTV-P; the 5' end of the fluorescent probe is labeled with a reporter group CY5, and the 3' end is labeled with a quencher group BHQ3; SEQ ID NO.25: An upstream primer designed for the novel coronavirus, the sequence of which is 5'-CAGTGGCTAACTAACATCTTTGG-3'; referred to as COVID-19-F; SEQ ID NO.26: A downstream primer designed for the novel coronavirus, the sequence of which is 5'-TCTCTAAGAAACTCTACACCTTCC-3'; referred to as COVID-19-R; SEQ ID NO.27: A fluorescent probe designed for the novel coronavirus, the sequence of which is 5'-ACTTCTCTTCAAGCCAATCAAGGACGG-3'; referred to as COVID-19-P; the 5' end of the fluorescent probe is labeled with a reporter group CY5, and the 3' end is labeled with a quencher group BHQ3; SEQ ID NO.28: An upstream primer designed for hepatitis E virus, the sequence of the upstream primer is 5'-CATCCCTTCTGATCTTGCTG-3'; referred to as HEV-F; SEQ ID NO.29: A downstream primer designed for hepatitis E virus, the sequence of the downstream primer is 5'-GGTGGTCTTACAGGTGAGG-3'; referred to as HEV-R; SEQ ID NO.30: A fluorescent probe designed for hepatitis E virus, the sequence of which is 5'-AGCCGCCTTACTGCCACCGTT-3'; referred to as HEV-P; the 5' end of the fluorescent probe is labeled with a reporter group CY5, and the 3' end is labeled with a quencher group BHQ3; SEQ ID NO.31: An upstream primer designed for the human housekeeping gene RNaseP, the sequence of which is 5'-CATCCCTTCTGATCTTGCTG-3'; referred to as RNaseP-F; SEQ ID NO.32: A downstream primer designed for the human housekeeping gene RNaseP, the sequence of which is 5'-GGTGGTCTTACAGGTGAGG-3'; referred to as RNaseP-R; SEQ ID NO.33: A fluorescent probe designed for the human housekeeping gene RNaseP, the sequence of which is 5'-AGCCGCCTTACTGCCACCGTT-3'; referred to as RNaseP-P; the 5' end of the fluorescent probe is labeled with a reporter group VIC, and the 3' end is labeled with a quencher group BHQ1; The above primers and probe plasmids were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0028] Example 2 A nucleic acid detection kit for organ transplant susceptible viruses This kit includes: lyophilized reaction tube, negative quality control, positive quality control and diluent.

[0029] 1. Prepare freeze-dried reaction tubes The reagent in the freeze-dried reaction tube is prepared using the primer-probe combination provided in Example 1, and the reagent includes the following components: One Step Lyo-Enzyme Mix 1.2μL, 5×One Step Mix 5μL, 50μM HCMV-F, 5μM HCMV-R, 5μM HCMV-P, 50μM JC-F, 5μM JC-R, 5μM JC-P, 50μM BK-F, 5μM BK-R, 5μM BK-P, 50μM EBV-F, 5μM EBV-R, 5μM EBV-P, 50μM HHV-6-F, 5μM HHV-6-R, 5μM HHV-6-P, 50μM HHV-8-F, 5μM HHV-8-R, 5μM HHV-8-P, 50μM B19-F, 5μM B19-R, 5μM B19-P, 50μM TTV-F, 5μM TTV-R, 5μM TTV-P, 50μM COVID-19-F, 5μM COVID-19-R, 5μM COVID-19-P, 50μM HEV-F, 5μM HEV-R, 5μM HEV-P, 5μM IC-F, 5μM IC-R, 5μM IC-P, 10% m / v lyoprotectant, wherein the lyoprotectant ingredients are: 20% dextran by mass, 2% bovine serum albumin (BSA) by mass, 2% polyvinylpyrrolidone (PVP) by mass, 1% sorbitol by mass, 20% polyadenylic acid (Ploy A) by mass, 2% glycine by mass, and 2% sodium citrate by mass. Weigh each reagent according to the corresponding mass percentage concentration and dissolve it with sterile enzyme-free water to obtain 2X lyoprotectant. The freeze-dried protective agent is mixed with DNA polymerase, PCR reaction buffer and the aforementioned primer-probe combination, and then freeze-dried.

[0030] The One Step Lyo-Enzyme Mix and 5×One Step Mix were purchased from Novozymes Biotech Co., Ltd., dextran, sorbitol, and sodium citrate were purchased from Sigma, bovine serum albumin was purchased from Shanghai Roche Pharmaceuticals Co., Ltd., polyvinyl pyrrolidone was purchased from Sinopharm Chemical Reagent Co., Ltd., polyadenylic acid was purchased from Suzhou Keer Life Science Co., Ltd., and glycine was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0031] The negative quality control used TE Buffer, and the positive quality control used 1×10 6 The lyophilized powder containing 10 kinds of virus synthetic plasmids and RNaseP synthetic plasmid at a concentration of 10 copies / mL was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0032] The diluent is used for diluting a blood direct amplification sample, and the components contained in the diluent are: formamide 1M, tris-hydroxymethylaminomethane hydrochloric acid (Tris-HCl) 1M, ethylenediaminetetraacetic acid disodium (EDTA-2Na) 2M, sodium chloride (NaCl) 5M, Triton X-100 6M, and isopropanol 10M.

[0033] Formamide was purchased from Thermo Fisher Scientific (China) Co., Ltd., tris(hydroxymethylaminomethane) hydrochloride was purchased from AMRESCO, and disodium ethylenediaminetetraacetic acid, sodium chloride, Triton X-100, and isopropanol were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0034] 2. Template preparation Take 10 μL of blood sample and dilute it with 100 μL of diluent; Add 25 μL of template to the freeze-dried reaction tube, where the amount of template is the amount of the sample to be tested.

[0035] 3. Build a reaction program The reaction procedure was as follows: 50 °C for 5 min, 95 °C for 30 s, 95 °C for 5 s, 60 °C for 15 s, 40 cycles, 95 °C, 1 min, 58 °C, 3 min, 58-80 °C, and a heating rate of 0.05 °C / s.

[0036] Figure 1 This is a picture of the test kit test results.

[0037] The results show: The target to be detected is HCMV, the detection channel is FAM, and the detection Tm value is: 63.4±0.5℃; the target to be detected is JC, the detection channel is FAM, and the detection Tm value is: 68.2±0.5℃; the target to be detected is BK, the detection channel is FAM, and the detection Tm value is: 69.8±0.5℃; The target to be detected is EBV, the detection channel is ROX, and the detection Tm value is: 64.5±0.5℃; the target to be detected is HHV-6, the detection channel is ROX, and the detection Tm value is: 66.5±0.5℃; the target to be detected is HHV-8, the detection channel is ROX, and the detection Tm value is: 69.5±0.5℃; The target to be detected is B19, the detection channel is CY5, and the detection Tm value is: 66.5±0.5℃; the target to be detected is TTV, the detection channel is CY5, and the detection Tm value is: 68.5±0.5℃; the target to be detected is COVID-19, the detection channel is CY5, and the detection Tm value is: 70.5±0.5℃; the target to be detected is HEV, the detection channel is CY5, and the detection Tm value is: 72.5±0.5℃.

[0038] When the target gene to be detected is RNaseP and the detection channel is VIC, the detected Tm value is: 70±0.5℃.

[0039] Example 3 Kit sensitivity determination The artificially synthesized plasmids for 10 viruses in Experimental Example 2 were diluted in a gradient to prepare standard products for the investigation of the sensitivity of the kit. 2 copies / mL, 10 1 copies / mL, 10 0 copies / mL, and the kit was used to detect them one by one to evaluate the sensitivity of the kit.

[0040] Test results such as Figure 2-4 ,Depend on Figure 2-4 It can be seen that the kit has a 1 When the detection rate of all plasmids was 100%, the detection limit of the primers was finally confirmed to be 1×10 1 copies / mL.

[0041] The reaction system is as follows: dilute to 10 2 copies / mL, 10 1 copies / mL, 10 0 Add 25 μL of each of the 10 artificially synthesized viral plasmids with a concentration of copies / mL into the freeze-dried PCR reaction tube of the kit, and perform PCR reaction at 50°C for 5 min, 95°C for 30 s, 95°C for 5 s, 60°C for 15 s, 40 cycles, 95°C for 1 min, 58°C for 3 min, 58-80°C, and a heating rate of 0.05°C / s.

[0042] Example 4 Kit specificity experiment A specific experimental group for detecting 10 viral primers was set up, and Mycobacterium tuberculosis, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Enterococcus faecium, Stenotrophomonas maltophilia, Staphylococcus aureus, human herpes virus type 1, influenza A virus, and influenza B virus were selected as interference strains in reaction tubes, and detection was performed according to the method of Example 2.

[0043] The results are as follows Figure 5As shown, from the results, it can be seen that the test results of the test kit for Mycobacterium tuberculosis, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Enterococcus faecium, Stenotrophomonas maltophilia, Staphylococcus aureus, human herpes virus type 1, influenza virus A, and influenza virus type B experimental group were negative, and the test results for human cytomegalovirus, JC virus, cyclovirus, BK polyomavirus, Epstein-Barr virus, human parvovirus, human herpes virus type 6, human herpes virus type 8, new coronavirus, and hepatitis E virus experimental group were positive, indicating that this kit has good specificity.

[0044] Example 5 Direct amplification experiment of blood samples in the kit The 10 kinds of virus synthetic plasmids were mixed with negative human whole blood samples at a ratio of 1:10 (10 μL: 100 μL). 10 μL of the mixed whole blood sample was added with 100 μL of diluent, and the remaining whole blood sample was not diluted. The diluted whole blood sample and the undiluted whole blood sample were tested according to the method of Example 2 for a control experiment.

[0045] The results are as follows Figure 6-7 As shown, it can be seen from the results that the blood sample diluted with the diluent can be amplified directly, while the undiluted blood sample is not amplified.

[0046] It should be noted that the above-described embodiments of the present invention do not limit the protection scope of the present invention. Any technical solution obtained by equivalent replacement or equivalent transformation should be included in the protection scope of the present invention.

Claims

1. A primer-probe composition for detecting nucleic acid of susceptible viruses in organ transplantation, characterized in that: The primer-probe composition comprises an upstream primer designed for human cytomegalovirus, a downstream primer designed for human cytomegalovirus, a fluorescent probe designed for human cytomegalovirus, an upstream primer designed for JC virus, a downstream primer designed for JC virus, a fluorescent probe designed for JC virus, an upstream primer designed for BK polyomavirus, a downstream primer designed for BK polyomavirus, a fluorescent probe designed for BK polyomavirus, an upstream primer designed for Epstein-Barr virus, a downstream primer designed for Epstein-Barr virus, a fluorescent probe designed for Epstein-Barr virus, an upstream primer designed for human herpesvirus type 6, a downstream primer designed for human herpesvirus type 6, a fluorescent probe designed for human herpesvirus type 6, an upstream primer designed for human herpesvirus type 8, a downstream primer designed for human herpesvirus type 8, A fluorescent probe designed for human herpesvirus type 8, an upstream primer designed for human parvovirus B19, a downstream primer designed for human parvovirus B19, a fluorescent probe designed for human parvovirus B19, an upstream primer designed for cyclovirus, a downstream primer designed for cyclovirus, a fluorescent probe designed for cyclovirus, an upstream primer designed for the new coronavirus, a downstream primer designed for the new coronavirus, a fluorescent probe designed for the new coronavirus, an upstream primer designed for hepatitis E virus, a downstream primer designed for hepatitis E virus, a fluorescent probe designed for hepatitis E virus, an upstream primer designed for an internal standard of the human housekeeping gene RNaseP, a downstream primer designed for an internal standard of the human housekeeping gene RNaseP, and a fluorescent probe designed for an internal standard of the human housekeeping gene RNaseP; The upstream primer designed for human cytomegalovirus: the sequence is shown in SEQ ID NO.1; The downstream primer designed for human cytomegalovirus: the sequence is shown in SEQ ID NO.2; The fluorescent probe designed for human cytomegalovirus: the sequence is shown in SEQ ID NO.3; The upstream primer designed for JC virus: the sequence is shown in SEQ ID NO.4; The downstream primer designed for JC virus: the sequence is shown in SEQ ID NO.5; The fluorescent probe designed for JC virus: the sequence is shown in SEQ ID NO.6; The upstream primer designed for BK polyomavirus: the sequence is shown in SEQ ID NO.7; The downstream primer designed for BK polyomavirus: the sequence is shown in SEQ ID NO.8; The fluorescent probe designed for BK polyomavirus: the sequence is shown in SEQ ID NO.9; The upstream primer designed for Epstein-Barr virus: the sequence is shown in SEQ ID NO.10; The downstream primer designed for EB virus: the sequence is shown in SEQ ID NO.11; The fluorescent probe designed for EB virus: the sequence is shown in SEQ ID NO.12; The upstream primer designed for human herpesvirus type 6: the sequence is shown in SEQ ID NO.13; The downstream primer designed for human herpesvirus type 6: the sequence is shown in SEQ ID NO.14; The fluorescent probe designed for human herpesvirus type 6: the sequence is shown in SEQ ID NO.15; The upstream primer designed for human herpesvirus type 8: the sequence is shown in SEQ ID NO.16; The downstream primer designed for human herpesvirus type 8: the sequence is shown in SEQ ID NO.17; The fluorescent probe designed for human herpesvirus type 8: the sequence is shown in SEQ ID NO.18; The upstream primer designed for human parvovirus B19: the sequence is shown in SEQ ID NO.19; The downstream primer designed for human parvovirus B19: the sequence is shown in SEQ ID NO.20; The fluorescent probe designed for human parvovirus B19: the sequence is shown in SEQ ID NO.21; The upstream primer designed for the microcircuivirus: the sequence is shown in SEQ ID NO.22; The downstream primer designed for the microcircuivirus: the sequence is shown in SEQ ID NO.23; The fluorescent probe designed for the microcircuivirus: the sequence is shown in SEQ ID NO.24; The upstream primer designed for the novel coronavirus: the sequence is shown in SEQ ID NO.25; The downstream primer designed for the new coronavirus: the sequence is shown in SEQ ID NO.26; The fluorescent probe designed for the new coronavirus: the sequence is shown in SEQ ID NO.27; The upstream primer designed for hepatitis E virus: the sequence is shown in SEQ ID NO.28; The downstream primer designed for hepatitis E virus: the sequence is shown in SEQ ID NO.29; The fluorescent probe designed for hepatitis E virus: the sequence is shown in SEQ ID NO.30; The upstream primer designed for the internal standard of the human housekeeping gene RNaseP: the sequence is shown in SEQ ID NO.31; The downstream primer designed for the internal standard of the human housekeeping gene RNaseP: the sequence is shown in SEQ ID NO.32; The fluorescent probe designed as an internal standard for the human housekeeping gene RNaseP: the sequence is shown in SEQ ID NO.

33.

2. A nucleic acid detection kit for susceptible viruses in organ transplantation, characterized in that: include: Lyophilized reaction tubes, negative controls, positive controls, and diluents.

3. The organ transplant susceptible virus nucleic acid detection kit according to claim 2, characterized in that: The reagents in the freeze-drying reaction tube include the following components: One Step Lyo-Enzyme Mix 1.2μL, 5×One Step Mix 5μL, 50μM HCMV-F, 5μM HCMV-R, 5μM HCMV-P, 50μM JC-F, 5μM JC-R, 5μM JC-P, 50μM BK-F, 5μM BK-R, 5μM BK-P, 50μM EBV-F, 5μM EBV-R, 5μM EBV-P, 50μM HHV-6-F, 5μM HHV-6-R, 5μM HHV-6-P, 50μM HHV-8-F, 5μM HHV-8-R, 5μM HHV-8-P, 50μM B19-F, 5μM B19-R, 5μM B19-P, 50μM TTV-F, 5μM TTV-R, 5μMTTV-P, 50μM COVID-19-F, 5μM COVID-19-R, 5μM COVID-19-P, 50μM HEV-F, 5μM HEV-R, 5μM HEV-P, 5μM IC-F, 5μM IC-R, 5μM IC-P, m / v 10% lyoprotectant.

4. The organ transplant susceptible virus nucleic acid detection kit according to claim 3, characterized in that: The freeze-drying protective agent comprises: 20% by mass of dextran, 2% by mass of bovine serum albumin (BSA), 2% by mass of polyvinylpyrrolidone (PVP), 1% by mass of sorbitol, 20% by mass of polyadenylic acid (PloyA), 2% by mass of glycine, and 2% by mass of sodium citrate.

5. The organ transplant susceptible virus nucleic acid detection kit according to claim 4, characterized in that: The positive quality control was 1×10 6 The lyophilized powder contains 10 copies / mL of artificially synthesized plasmids of 10 viruses and RNaseP artificially synthesized plasmids.

6. The organ transplant susceptible virus nucleic acid detection kit according to claim 5, characterized in that: The negative quality control uses TE Buffer.

7. The organ transplant susceptible virus nucleic acid detection kit according to claim 6, characterized in that: The components contained in the diluent are: formamide 1M, tris-HCl (Tris-HCl) 1M, EDTA-2Na 2M, sodium chloride (NaCl) 5M, Triton X-100 6M, and isopropanol 10M.

Images