A universal nucleic acid mass spectrometry detection kit and its application

Abstract

The present invention relates to the field of nucleic acid detection technology, and in particular to a universal nucleic acid mass spectrometry detection kit and its application. The resins used in the resin purification system of the universal detection kit include: a first resin, a second resin, and water; the first resin is a nucleic acid mass spectrometry amino purification resin, and the second resin is a strongly acidic cation exchange resin with a DVB content of 6-10%; the volume ratio of the first resin to the second resin is 1:(2-5). The present invention provides a universal kit for nucleic acid mass spectrometry detection. When applied to nucleic acid mass spectrometry detection, based on specific resin purification and other processes, it has excellent universality, can be applied to a variety of detection purposes, and can be used in conjunction with any model of nucleic acid mass spectrometry equipment. It has important value for the application of existing nucleic acid mass spectrometers and supporting technical systems.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular to a universal nucleic acid mass spectrometry detection kit and applications thereof. Background Art

[0002] Key research areas in public health include developing technologies for the detection and identification of pathogenic microorganisms, developing sensitive and rapid detection platforms, conducting research on epidemic monitoring, early warning, early detection, traceability, and on-site detection for public health emergencies, developing new technologies for multidrug resistance detection and evaluation, and developing rapid, high-throughput pathogen detection technologies. These technologies are crucial for the clinical diagnosis and treatment of infectious diseases, routine population testing and monitoring, and the prevention, control, and emergency response of infectious diseases. Multiplex PCR-mass spectrometry microsequencing (nucleic acid mass spectrometry) is a novel technology system that lies between PCR amplification combined with first-generation sequencing and whole-genome sequencing. It can simultaneously detect up to 50 mutations, addressing both the former's limitation of a small number of mutations detected in a single test and the latter's technical bottleneck of a long 48-hour detection time and associated costs.

[0003] Multiplex PCR-mass spectrometry microsequencing technology can perform single nucleotide polymorphism (SNP) analysis, gene mutation analysis, DNA methylation analysis, and DNA copy number variation analysis. It can also simultaneously detect multiple pathogens and multiple testing purposes, such as pathogen identification, typing and tracing, drug resistance, infection-related diseases, and vector-borne diseases. Compared to nucleic acid detection technologies such as probe-based fluorescent PCR amplification of SNP sites, PCR-based first-generation sequencing, and metagenomic sequencing, multiplex PCR-mass spectrometry microsequencing technology offers significant advantages: high throughput, low cost, ease of use, and high accuracy. It can also perform multi-pathogen and multi-purpose joint testing, making it more suitable for routine testing.

[0004] Currently, the mass spectrometry technology within multiplex PCR-mass spectrometry microsequencing has matured. However, unlike mass spectrometry-based microbial identification, which can be widely applied as long as a commercial database is available, multiplex PCR-mass spectrometry microsequencing requires the development of corresponding detection methods based on application needs, the determination of specific loci and primers, and the optimization of the detection system to form a detection system for a specific detection purpose. This has significantly limited the widespread application of multiplex PCR-mass spectrometry microsequencing technology.

[0005] During the method development and testing process, various institutions have been repeatedly exploring. There are no standardized universal analysis kits other than specific primers, resulting in unstable analysis, high costs, and time-consuming problems. In the application of multiplex PCR-mass spectrometry microsequencing technology, each set of detection kits is independent, and different primers correspond to different detection reagents, resulting in waste of resources and increased overall detection costs, which is not conducive to the promotion and application of technology. Summary of the Invention

[0006] To address the challenges of the prior art, the present invention provides a universal nucleic acid mass spectrometry detection kit and its applications. This kit provides a specific resin purification system, defines reagent types, dosages, and analytical conditions in the detection process, and expands the versatility of the detection kit. The resulting universal nucleic acid mass spectrometry detection kit can be applied to a variety of detection purposes and analyses.

[0007] In a first aspect, the present invention provides a universal nucleic acid mass spectrometry detection kit, wherein the resin used in the resin purification system of the universal detection kit comprises: a first resin, a second resin and water;

[0008] The first resin is a nucleic acid mass spectrometry amino purification resin, and the second resin is a strongly acidic cation exchange resin having a divinylbenzene (DVB) content of 6-10%;

[0009] The volume ratio of the first resin to the second resin is 1:(2-5), preferably 1:(2-4).

[0010] Furthermore, the amount of water used is 2.5 to 4 times the sum of the first resin and the second resin in terms of volume.

[0011] Furthermore, the nucleic acid mass spectrometry universal detection kit includes:

[0012] Multiplex PCR amplification system, SAP reaction system, MPE reaction system, resin purification system and target point system.

[0013] Furthermore, the multiplex PCR amplification system comprises: 40-50 v / v% PCR amplification premix, 1-8 v / v% primer mixture, 5-25 v / v% template DNA mixture, and the remainder is nuclease-free water; and / or,

[0014] The SAP reaction system comprises: 5-20 v / v% SAP and 15-30 v / v% SAP buffer, with the remainder being nuclease-free water; and / or,

[0015] The MPE reaction system comprises: 35-55 v / v% MPE buffer, 2-12 v / v% MPE enzyme, 35-45 v / v% MPE probe mixture, and the balance is nuclease-free water; and / or,

[0016] The target system includes 3-HPA and nucleic acid-free water.

[0017] Furthermore, the PCR amplification premix includes: deoxyribonucleoside triphosphates, DNA polymerase, and 10× DNA polymerase buffer. The MPE buffer includes: dideoxynucleoside triphosphates, 10× MPE enzyme buffer, and 100 mM magnesium chloride. Both can be purchased commercially.

[0018] In a second aspect, the present invention provides a nucleic acid mass spectrometry detection method, comprising:

[0019] The universal detection kit is used to detect the sample to be tested.

[0020] Furthermore, it includes: multiplex PCR amplification, SAP reaction, MPE reaction, resin purification and target spotting.

[0021] Further, the resin purification includes:

[0022] The resin and the product of the MPE reaction are mixed, mixed, centrifuged, and the supernatant is collected;

[0023] The mixing includes mixing at 75-150 rpm for 20-40 minutes, and the centrifuging includes centrifuging at 1000-2500 rpm for 1-3 minutes.

[0024] Furthermore, the multiplex PCR amplification includes: adding a primer pair and a template DNA to the multiplex PCR amplification system, and performing amplification according to an amplification program;

[0025] The SAP reaction comprises: mixing the products obtained by the multiplex PCR amplification with the SAP reaction system, and then centrifuging and performing the following reaction: reacting at 25-30° C. for 25-40 minutes and reacting at 60-70° C. for 3-10 minutes;

[0026] The MPE reaction comprises: adding an MPE probe to the MPE reaction system and performing a reaction according to an MPE reaction procedure;

[0027] The target dot method includes: dotting 3-HPA matrix solution on a sample target to form crystals, and after the crystals are dried, covering the mixed sample to be tested and 3-HPA on the dried matrix crystals and drying naturally.

[0028] Furthermore, the mixing ratio of the sample to be tested and 3-HPA is 1: (0.5~2)

[0029] When using the universal nucleic acid mass spectrometry detection kit provided by the present invention, technicians only need to provide specific primers and probes to complete efficient detection of the sample to be tested. In actual production processes, specific primers and probes can be further added to the universal nucleic acid mass spectrometry detection kit provided by the present invention to obtain a nucleic acid mass spectrometry detection kit for a specific detection purpose.

[0030] The present invention has the following beneficial effects:

[0031] The present invention provides a specific resin purification system, determines the reagent type, dosage and analysis conditions in the detection process, expands the versatility of the detection kit, and prepares a universal nucleic acid mass spectrometry detection kit that can be applied to various detection purposes and analyses.

[0032] The present invention, based on the developed universal nucleic acid mass spectrometry detection kit, tested four detection systems already constructed by the project team: a joint detection system for Mycobacterium tuberculosis drug resistance, a system for identifying Escherichia coli in marmots, a system for detecting mutant strains of the novel coronavirus, and a system for detecting resistance to pyrethroid insecticides in Aedes albopictus. The highest detection sensitivity can detect mutations as low as 20 copies, and 96 samples can be tested within 7 hours. The detection throughput far exceeds that of q-PCR, and the detection time is much shorter than the whole genome sequencing method for multi-site analysis. The detection cost is approximately 10 RMB per sample. The kit is also highly stable and can be used stably for one year at -20°C. The universal nucleic acid mass spectrometry detection kit provided by the present invention is compatible with all currently commercialized MALDI-TOF mass spectrometers domestically and internationally, and is therefore universally applicable. The detection kit provided by the present invention covers all detection reagents and detection conditions, except for method-specific primers, and is applicable to all multiplex PCR-mass spectrometry microsequencing technology detection systems. It provides an economical and efficient universal kit for the construction of methods based on this technology system, which can improve the development of target supporting kits for this technology and has important market application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0034] Figure 1 This is the result of detecting drug resistance of Mycobacterium tuberculosis using the kit provided in Example 1, provided in Example 2 of the present invention.

[0035] Figure 2 This is the mass spectrum result of detecting Escherichia coli in marmota using the kit provided in Example 1, provided in Example 2 of the present invention.

[0036] Figure 3 This is the result of using the novel coronavirus pan-mutant infection system provided in Example 3 of the present invention to verify the accuracy of the nucleic acid mass spectrometry universal detection kit provided by the present invention.

[0037] Figure 4 This is the result of using the Aedes albopictus insecticide resistance gene detection system provided in Example 3 of the present invention to verify the accuracy of the nucleic acid mass spectrometry universal detection kit provided by the present invention.

[0038] Figure 5 These are the results of detecting Mycobacterium tuberculosis DNA samples using different resins provided in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0039] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0040] Unless otherwise specified, the experimental methods involved in the following examples are all conventional methods in the art. For example, reference can be made to experimental manuals in the art, or the conditions recommended by the manufacturer's instructions.

[0041] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0042] Example 1

[0043] The present invention provides a universal nucleic acid mass spectrometry detection kit and a corresponding detection method, including the following:

[0044] 1. Multiplex PCR amplification

[0045] (1) Multiplex PCR amplification system includes:

[0046] Reagent 1, PCR amplification premix;

[0047] Reagent 6, nuclease-free water;

[0048] 20 μM primer mix system: The kit contains a primer mix (nuclease-free water), and the primers are diluted (according to the user's detection purpose) to 20 μM;

[0049] Template DNA: Template DNA of the sample to be tested.

[0050] The details are as follows:

[0051] Table 1 Multiplex PCR amplification system

[0052]

[0053] (2) Multiplex PCR amplification process

[0054] Gently vortex the mixed multiplex PCR amplification system in a vortexer and centrifuge at 1000 rpm for 1 minute. After mixing, perform the multiplex PCR reaction according to the reaction process that matches the primers used according to the user's detection purpose. The following is an example:

[0055] Table 2 Multiplex PCR reaction process

[0056]

[0057] 2. Shrimp alkaline phosphatase (SAP) reaction

[0058] (1) The SAP reaction system includes:

[0059] Reagent 2-1, shrimp alkaline phosphatase (SAP);

[0060] Reagent 2-2, shrimp alkaline phosphatase buffer (SAP buffer);

[0061] Reagent 6, nuclease-free water.

[0062] The details are as follows:

[0063] Table 3 SAP reaction system

[0064]

[0065] (2) SAP reaction process

[0066] Add the above SAP reaction system to the tube containing the PCR product obtained in the previous step, vortex gently to mix, and centrifuge at 1000 rpm for 1 minute. Perform the SAP reaction according to the following procedure.

[0067] Table 4 SAP reaction process

[0068]

[0069] 3. Mass Probe Extension (MPE) Reaction

[0070] (1) The MPE reaction system includes:

[0071] Reagent 3-1, MPE enzyme;

[0072] Reagent 3-2, MPE buffer;

[0073] 200μM MPE probe mixture system: The kit contains MPE probe mixture (nuclease-free water), and the MPE probe is diluted (according to the user's detection purpose) to 200μM.

[0074] The details are as follows:

[0075] Table 5 MPE reaction system

[0076]

[0077] (2) The MPE extension reaction process includes:

[0078] Add the above MPE reaction system to each tube of SAP reaction product (obtained from the SAP reaction in the previous step), gently vortex to mix, and centrifuge at 1000 rpm for 1 minute. Perform the MPE reaction using the MPE probe described above based on the user's detection purpose, as shown below:

[0079] Table 6 MPE reaction procedure

[0080]

[0081] 4. Resin purification

[0082] (1) Resin purification system

[0083] Reagent 4, ion exchange resin, specifically, Resin-1 and Resin-2 were swollen in a resin:nuclease-free water (volume ratio) of 1:3, and then the swollen Resin-1 was mixed with Resin-2 in a volume ratio of 1:3;

[0084] Reagent 6, nuclease-free water.

[0085] Resin 1 is a nucleic acid mass spectrometry amino purification resin (purchased from Jiangsu Wiki, product number NAM606), and resin 2 is a strongly acidic cation exchange resin with a DVB content of 8% (purchased from Jiangsu Jinshan, product number PCG984).

[0086] (2) Resin purification process

[0087] Pipette 5 μL of the mixed resin and add it to the MPE reaction product tube (the MPE reaction product obtained in the previous step); use a palm centrifuge to centrifuge briefly to prevent the resin from adhering to the tube wall; place the sample plate with the resin in a flip mixer and mix at 100 rpm for 30 minutes; after mixing, centrifuge the sample at 2000 rpm for 1 minute, and collect the supernatant for subsequent processes.

[0088] 5. Target Pointing

[0089] (1) Target system

[0090] Reagent 5, 3-hydroxypicolinic acid (3-HPA);

[0091] Reagent 6, nuclease-free water.

[0092] (2) Targeting process

[0093] Take 1 μL of matrix solution (3-HPA) and drop it on the target plate to wait for it to form crystals; mix the purified sample supernatant obtained in the previous step and 3-HPA in equal proportions, then spot it on the matrix crystallization point of the sample target), and then naturally dry it before proceeding with the subsequent mass spectrometry detection process.

[0094] For example, MALDI-TOF MS is used to collect data. Before sample collection, the mass axis of the mass spectrometer is calibrated using a standard. Data is collected in positive ion mode with a mass range of 4000-9500 Da. Users analyze the demonstration data based on their own testing objectives.

[0095] Example 2

[0096] The present invention further verifies the effectiveness of the nucleic acid mass spectrometry universal detection kit provided in Example 1, including the following process:

[0097] 1. The present invention utilizes the universal nucleic acid mass spectrometry detection kit provided in Example 1 to develop a joint detection system for Mycobacterium tuberculosis resistance to first- and second-line antibiotics. This joint detection system targets nine clinical first- and second-line anti-tuberculosis drugs, specifically: first-line anti-tuberculosis drugs (isoniazid, rifampicin, ethambutol, pyrazinamide, and streptomycin) and second-line anti-tuberculosis drugs (moxifloxacin, levofloxacin, norfloxacin, and amikacin). The system detects 28 loci and 50 mutations in 10 resistance-associated genes.

[0098] The present study tested nucleic acid samples from five Mycobacterium tuberculosis strains and 100 samples from throat swabs and bronchoalveolar lavage fluid. A MALDI-TOF mass spectrometer was used in linear mode, calibrated with standards developed by the project team. Drug resistance in all samples was determined using molecular biology methods.

[0099] The results showed that the drug resistance of all samples was correctly identified using the reagents and detection parameters in the universal kit of the present invention. Figure 1 As one of the results, the MTB-positive sample results in the figure show that a mutation (C-15T) was detected in the inhA promoter region, a site associated with isoniazid resistance. The T base was detected, indicating a mutation at this site and isoniazid resistance in this sample. The MTB-blank control in the figure uses water instead of template DNA.

[0100] 2. The present invention further used the universal nucleic acid mass spectrometry detection kit provided in Example 1 to test the identification effect of the Escherichia coli identification system for marmots. To address the phenomenon that Escherichia coli is easily confused with Escherichia coli and Shigella, five SNP sites in three housekeeping genes (gyrA, secA, era) were used to identify Escherichia coli.

[0101] This kit is a universal detection reagent, and the specific primers are synthesized by our company. A total of 50 nucleic acid samples were tested, 5 of which were Escherichia coli. According to the detection process of this invention, all 5 positive samples were correctly detected. Figure 2 As one of the results, the mass spectrum of Escherichia coli in the figure detected that the extended bases at the five test sites were a combination of T, T, T, A, and A, and it was identified as Escherichia coli; the blank control was achieved by using water instead of template DNA.

[0102] Example 3

[0103] The present invention further conducts universal testing of the reaction conditions (reaction system, reaction process) of the kit, including the following process:

[0104] The present invention further uses the following primer pairs to verify the versatility of the nucleic acid mass spectrometry universal detection kit provided in Example 1 of the present invention, specifically including:

[0105] Table 7. Infection system of novel coronavirus pan-mutant strains

[0106]

[0107] Table 8. Detection system of insecticide resistance genes of Aedes albopictus

[0108]

[0109] The reaction systems and reaction processes in Tables 1 to 6 were used respectively, and the kit operation procedures were followed to detect the samples to be tested (84 nasopharyngeal swab samples from population screening and 83 mosquito samples collected from mosquito monitoring) using the above primer pairs.

[0110] A detection and typing system was constructed for 13 novel coronavirus subtypes, and the following results were obtained: Figure 3 The results shown are: MPE-1, MPE-4, and MPE-5 represent the test sites 614, 484, and 452, respectively. When the extended bases are all G, it indicates that the new coronavirus is the Delta subtype. Among the 84 samples tested, 12 Delta mutants and 6 Omicron mutants were found, which is consistent with the whole genome sequencing results.

[0111] The detection system for Aedes albopictus resistance to pyrethroid insecticides identified the resistance of Aedes albopictus by knocking down three SNP sites of the kdr gene, and the results of the 83 samples tested were completely correct. Figure 4 As shown: When the extended base at position 1534 of MPE-3 is C, it indicates that this position is a homozygous mutation, indicating that the sample is resistant to pyrethroid insecticides.

[0112] According to the above results, it can be seen that the universal nucleic acid mass spectrometry detection kit provided by the present invention has excellent versatility.

[0113] Comparative Example 1

[0114] During the research, the present invention screened six domestic and foreign cation exchange resins (Table 9). After a Mycobacterium tuberculosis DNA sample was subjected to multiplex PCR amplification, SAP digestion, and MPE extension, each of the six resins was used for desalting and purification (the experimental conditions for resins 1-6 were the same as those in Example 1, except that the resin purification steps used the conditions recommended in the instructions). Using the same tube of 3-HPA matrix target, mass spectra were acquired by MALDI-TOF mass spectrometry. This system includes a total of 10 extension probes.

[0115] Table 9 Resin information used

[0116]

[0117] The results showed that the mass spectrum of the finished resin-1 showed multiple adduct peaks after the main peak, with low resolution and a total of 9 extended probe peaks detected; the mass spectrum of the finished resin-2 showed a clear main peak, but the number of peaks was small, with only 4 extended probe peaks detected, and low detection sensitivity; Resin-3, Resin-4, Resin-5, and Resin-6 all had no effective peaks. The mass spectrum of the sample treated with the optimized resin of the present invention (Example 1) had a clear main peak, high resolution, and a total of 10 extended probe peaks detected, with high sensitivity and good accuracy (Table 9, Figure 5 ).

[0118] At the same time, the detection experiment was carried out using the new coronavirus mutant strain, Aedes albopictus resistance primer pair and probe in Example 3, and the results were the same as above. This shows that the resin provided in Example 1 of the present invention has excellent stability and versatility.

[0119] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A universal nucleic acid mass spectrometry detection kit, characterized in that: The resin used in the resin purification system of the universal detection kit includes: a first resin, a second resin and water; The first resin is a nucleic acid mass spectrometry amino purification resin with the product number NAM606 from Suzhou Wiki, and the second resin is a strong acid cation exchange resin with the product number PCG984 from Jiangsu Jinshan; The volume ratio of the first resin to the second resin is 1:

3.

2. The universal nucleic acid mass spectrometry detection kit according to claim 1, characterized in that: In terms of volume, the amount of water used is 2.5 to 4 times the sum of the first resin and the second resin.

3. The universal detection kit according to claim 1, characterized in that include: Multiplex PCR amplification system, SAP reaction system, MPE reaction system, resin purification system and target point system.

4. The universal detection kit according to claim 3, characterized in that The multiplex PCR amplification system comprises: 40-50 v / v% PCR amplification premix, 1-8 v / v% primer mixture, 5-25 v / v% template DNA mixture, and the remainder is nuclease-free water; and / or, The SAP reaction system comprises: 5-20 v / v% SAP and 15-30 v / v% SAP buffer, with the remainder being nuclease-free water; and / or, The MPE reaction system comprises: 35-55 v / v% MPE buffer, 2-12 v / v% MPE enzyme, 35-45 v / v% MPE probe mixture, and the balance is nuclease-free water; and / or, The target system includes 3-HPA and nucleic acid-free water.

5. A nucleic acid mass spectrometry detection method for purposes other than disease diagnosis and treatment, characterized in that: include: The universal detection kit according to any one of claims 1 to 4 is used to detect the sample to be tested.

6. The nucleic acid mass spectrometry detection method according to claim 5, characterized in that: include: Multiplex PCR amplification, SAP reaction, MPE reaction, resin purification and target spotting.

7. The nucleic acid mass spectrometry detection method according to claim 6, characterized in that: The resin purification comprises: The resin and the product of the MPE reaction are mixed, mixed, centrifuged, and the supernatant is collected; The mixing includes mixing at 75-150 rpm for 20-40 minutes, and the centrifuging includes centrifuging at 1000-2500 rpm for 1-3 minutes.

8. The nucleic acid mass spectrometry detection method according to claim 6 or 7, characterized in that: The multiplex PCR amplification comprises: adding a primer pair and a template DNA to the multiplex PCR amplification system, and performing amplification according to an amplification program; The amplification procedure includes: 93~97℃ 10~15min; 92-96°C for 30-60 s, 52-60°C for 30-60 s, 70-74°C for 30-60 s, for 30-40 cycles; 70~74℃ 5~10min; and / or, The SAP reaction comprises: mixing the products obtained by the multiplex PCR amplification with the SAP reaction system, and then centrifuging and performing the following reaction: reacting at 25-30° C. for 25-40 minutes and at 60-70° C. for 3-10 minutes; and / or, The MPE reaction comprises: adding an MPE probe to the MPE reaction system and performing a reaction according to an MPE reaction procedure; and / or, The target dot method includes: dotting 3-HPA matrix solution on a sample target to form crystals, and after the crystals are dried, covering the mixed sample to be tested and 3-HPA on the dried matrix crystals and drying naturally.

9. Use of the universal detection kit according to any one of claims 1 to 4 in nucleic acid mass spectrometry detection for purposes other than disease diagnosis and treatment.

10. Use of the universal detection kit according to any one of claims 1 to 4 in preparing specific nucleic acid mass spectrometry detection kits for different detection purposes.

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