Nucleic acid mass spectrum general detection kit and application thereof

Abstract

The invention relates to the technical field of nucleic acid detection, in particular to a nucleic acid mass spectrum general detection kit and application thereof. Resin used by a resin purification system in the general detection kit comprises first resin, second resin and water, the first resin is nucleic acid mass spectrum amino purification resin, and the second resin is strong acid cation exchange resin with the DVB content of 6-10%; the volume ratio of the first resin to the second resin is 1: (2-5). When the universal kit for nucleic acid mass spectrometry detection is applied to nucleic acid mass spectrometry detection, based on specific resin purification and other processes, the universal kit has excellent universality, can be suitable for various detection purposes, can be used in cooperation with nucleic acid mass spectrometry equipment of any model, and has wide application prospects. The method has an important value for the application of the existing nucleic acid mass spectrometer and a matched technical system.

Description

Technical Field

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

[0002] The development of pathogen detection and identification technology, sensitive and rapid detection technology platforms, epidemic monitoring, early warning, early detection, traceability and emergency on-site detection technology research for public health emergencies, and new technology development for multi-drug resistance detection and evaluation, as well as clinical rapid high-throughput pathogen detection technology, are key research directions in the current public health field, and are also in line with the needs of clinical diagnosis and treatment of infectious diseases, routine population detection, monitoring, and infectious disease prevention and control and emergency response to epidemics. Multiplex PCR-mass spectrometry microsequencing technology (nucleic acid mass spectrometry technology) is a new technology system between PCR amplification combined with first-generation sequencing technology and whole genome sequencing technology. It can detect up to 50 mutations at a time, which can solve the shortcoming of the former's small number of mutations detected in a single test, and the latter's technical bottleneck of 48 hours of long detection time and detection cost.

[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 perform pathogen identification, typing and tracing, drug resistance, infection-related diseases, vectors and other multiple pathogens, multiple detection purposes, and multiple tests in one test. Compared with nucleic acid detection technologies such as probe-based SNP site fluorescence PCR amplification technology, PCR amplification-based first-generation sequencing technology, and metagenomic sequencing technology, multiplex PCR-mass spectrometry microsequencing technology has outstanding advantages: high detection throughput, low detection cost, easy operation, high accuracy, and can realize multi-pathogen joint detection and multi-purpose joint detection, which is more suitable for routine detection.

[0004] At present, the mass spectrometry technology in multiplex PCR-mass spectrometry microsequencing technology has become mature, but unlike mass spectrometry microbial identification technology, which can be promoted and applied as long as there is a commercial database, multiplex PCR-mass spectrometry microsequencing technology must develop corresponding detection methods in combination with application requirements, determine specific sites and primers, and optimize the detection system to form a detection system for a certain detection purpose. This has largely limited the promotion and application of multiplex PCR-mass spectrometry microsequencing technology.

[0005] In the process of method development and testing, various institutions are repeatedly exploring. There are no standardized universal analysis kits other than specific primers, resulting in unstable analysis, high cost, and time-consuming problems. In the application process 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] In order to solve the problems existing in the prior art, the present invention provides a nucleic acid mass spectrometry universal detection kit and its application. A specific resin purification system is provided, the reagent type, dosage and analysis conditions in the detection process are determined, the versatility of the detection kit is expanded, and the prepared nucleic acid mass spectrometry universal 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; 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%; The volume ratio of the first resin to the second resin is 1:(2-5), preferably 1:(2-4).

[0008] Furthermore, in terms of volume multiple, the amount of water used is 2.5 to 4 times the sum of the first resin and the second resin.

[0009] Furthermore, the nucleic acid mass spectrometry universal detection kit comprises: Multiplex PCR amplification system, SAP reaction system, MPE reaction system, resin purification system and target spot system.

[0010] Further, 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 nucleic acid-free water; and / or, The SAP reaction system comprises: 5-20 v / v% SAP and 15-30 v / v% SAP buffer, and the balance is nucleic acid-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 remainder is nucleic acid-free water; and / or, The target point system includes: 3-HPA and nucleic acid-free water.

[0011] 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.

[0012] In a second aspect, the present invention provides a nucleic acid mass spectrometry detection method, comprising: The universal detection kit is used to detect the sample to be detected.

[0013] Furthermore, it includes: multiplex PCR amplification, SAP reaction, MPE reaction, resin purification and target spotting.

[0014] Further, the resin purification comprises: The resin and the product of the MPE reaction are mixed, and the supernatant is collected after mixing and centrifugation; The mixing includes: mixing at 75-150 rpm for 20-40 minutes, and the centrifugation includes: centrifuging at 1000-2500 rpm for 1-3 minutes.

[0015] 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; The SAP reaction comprises: mixing the products obtained by the multiplex PCR amplification with the SAP reaction system, and performing the following reaction after centrifugation: reacting at 25-30° C. for 25-40 minutes and reacting at 60-70° C. for 3-10 minutes; The MPE reaction comprises: adding an MPE probe to the MPE reaction system and performing a reaction according to an MPE reaction procedure; The target dot method comprises: 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 them naturally.

[0016] Furthermore, the mixing ratio of the sample to be tested and 3-HPA is 1: (0.5~2) When the nucleic acid mass spectrometry universal detection kit provided by the present invention is used, the technician only needs to provide specific primers and probes to complete the efficient detection of the sample to be tested. In the actual production process, specific primers and probes can be further added to the nucleic acid mass spectrometry universal detection kit provided by the present invention to obtain a nucleic acid mass spectrometry detection kit for a specific detection purpose.

[0017] The present invention has the following beneficial effects: 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 a variety of detection purposes and analyses.

[0018] The present invention is based on the developed nucleic acid mass spectrometry universal detection kit to detect four detection systems that the project team has constructed: Mycobacterium tuberculosis resistance joint detection system, Marmot Escherichia coli identification system, new coronavirus mutant strain detection system and Aedes albopictus resistance to pyrethroid insecticide detection system. The highest detection sensitivity can detect mutations with a minimum of 20 copies, and 96 samples can be detected within 7 hours. The detection throughput far exceeds q-PCR, and the detection time is much shorter than the whole genome sequencing method of multi-site analysis. The detection cost is about 10 yuan / sample; the kit has high stability and can be used stably for 1 year at -20°C. The nucleic acid mass spectrometry universal detection kit provided by the present invention is suitable for all currently commercialized domestic and foreign MALDI-TOF mass spectrometers and has universal applicability. The detection kit provided by the present invention covers all detection reagents and detection conditions except method-specific primers, and is suitable for all multiplex PCR-mass spectrometry microsequencing technology detection systems, providing an economical and efficient universal kit for the construction of methods based on the technical system, which can improve the target matching kit research and development of the technology, and has important market application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces 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 creative work.

[0020] Figure 1 This is the result of detecting the drug resistance of Mycobacterium tuberculosis using the kit provided in Example 1, provided in Example 2 of the present invention.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] Figure 5 This is the result of detecting Mycobacterium tuberculosis DNA samples using different resins provided in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0027] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0028] Example 1 The present invention provides a nucleic acid mass spectrometry universal detection kit and a corresponding detection method, including the following: 1. Multiplex PCR amplification (1) The multiplex PCR amplification system includes: Reagent 1, PCR amplification premix; Reagent 6, nucleic acid-free water; 20μM primer mixture system: The kit contains a primer mixture (nuclease-free water), and the primers are diluted (according to the user's detection purpose) to 20μM; Template DNA: Template DNA of the sample to be tested.

[0029] The details are as follows: Table 1 Multiplex PCR amplification system

[0030] (2) Multiplex PCR amplification process The mixed multiplex PCR amplification system was gently vortexed in a vortexer and centrifuged at 1000 rpm for 1 min. After mixing, the multiplex PCR reaction was performed according to the reaction process matching the primers used according to the user's detection purpose, as shown below: Table 2 Multiplex PCR reaction process

[0031] 2. Shrimp alkaline phosphatase (SAP) reaction (1) The SAP reaction system includes: Reagent 2-1, shrimp alkaline phosphatase (SAP); Reagent 2-2, shrimp alkaline phosphatase buffer (SAP buffer); Reagent 6, Nuclease-free Water.

[0032] The details are as follows: Table 3 SAP reaction system

[0033] (2) SAP reaction process Add the above SAP reaction system to the PCR reaction product tube obtained in the previous step, vortex gently to mix, centrifuge at 1000 rpm for 1 min, and perform SAP reaction according to the following procedure.

[0034] Table 4 SAP reaction process

[0035] 3. Mass Probe Extension (MPE) Reaction (1) The MPE reaction system includes: Reagent 3-1, MPE enzyme; Reagent 3-2, MPE buffer; 200μM MPE probe mixed system: The kit contains MPE probe mixed solution (nucleic acid-free water), and the MPE probe is diluted (according to the user's detection purpose) to 200μM.

[0036] The details are as follows: Table 5 MPE reaction system

[0037] (2) The MPE extension reaction process includes: Add the above MPE reaction system to each SAP reaction product tube (obtained from the SAP reaction in the previous step), vortex gently to mix, and centrifuge at 1000 rpm for 1 min. Perform MPE reaction according to the MPE probe used according to the user's detection purpose, as shown below: Table 6 MPE reaction procedure

[0038] 4. Resin purification (1) Resin purification system Reagent 4, ion exchange resin, specifically, resin-1 and resin-2 are swollen at a resin:nuclease-free water (volume ratio) of 1:3, and then the swollen resin-1 and -resin 2 are mixed at a volume ratio of 1:3; Reagent 6, Nuclease-free Water.

[0039] Resin 1 is a nucleic acid mass spectrometry amino purification resin (purchased from Jiangsu Wiki, product number NAM606), and resin 2 is a strong acid cation exchange resin with a DVB content of 8% (purchased from Jiangsu Jinshan, product number PCG984).

[0040] (2) Resin purification process Pipette 5 μL of 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 instantly 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 take the supernatant for subsequent processes.

[0041] 5. Target Point (1) Target system Reagent 5, 3-hydroxypicolinic acid (3-HPA); Reagent 6, Nuclease-free Water.

[0042] (2) Targeting process 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, and then spot it on the matrix crystal point of the sample target), dry it naturally, and then proceed with the subsequent mass spectrometry detection process.

[0043] For example, MALDI-TOF MS is used to collect data, and the mass axis of the mass spectrometer is calibrated using a standard before sample collection. Data collection is in positive ion mode with a mass range of 4000-9500Da. Users analyze the demonstration data according to their own testing purposes.

[0044] Example 2 The present invention further verifies the effect of the nucleic acid mass spectrometry universal detection kit provided in Example 1, including the following process: 1. The present invention uses the nucleic acid mass spectrometry universal detection kit provided in Example 1 to detect the joint detection system of Mycobacterium tuberculosis resistance to first-line and second-line antibiotics. The joint detection system targets nine clinical first-line and second-line anti-tuberculosis drugs, specifically: first-line anti-tuberculosis drugs (isoniazid, rifampicin, ethambutol, pyrazinamide, streptomycin), second-line anti-tuberculosis drugs (moxifloxacin, levofloxacin, norfloxacin, amikacin). The system detection sites include 28 sites and 50 mutant types of 10 resistance-related genes.

[0045] The present invention detected a total of 5 nucleic acid samples of Mycobacterium tuberculosis strains and 100 nucleic acid samples of throat swabs and alveolar lavage fluid. The MALDI-TOF mass spectrometer linear mode was used, and the calibration was performed using the standard products made by the project team. The drug resistance of all samples was determined by molecular biological methods.

[0046] 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, it can be seen from the MTB-positive sample results in the figure that a mutation (C-15T) was detected in the promoter region of the isoniazid resistance-related site inhA, and the T base was detected, indicating that there is a mutation at this site and the sample is resistant to isoniazid. The MTB-blank control in the figure uses water instead of template DNA.

[0047] 2. The present invention further uses the universal nucleic acid mass spectrometry detection kit provided in Example 1 to detect the identification effect of the Escherichia coli identification system for marmots. In view of the fact that Escherichia coli for marmots is easily confused with Escherichia coli and Shigella, Escherichia coli for marmots is identified through five SNP sites of three housekeeping genes (gyrA, secA, era).

[0048] This kit is used as a universal detection reagent, and the specific primers are synthesized by the company. A total of 50 nucleic acid samples were tested, 5 of which were Escherichia coli. According to the detection process of the present 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 of 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.

[0049] Example 3 The present invention further conducts universal testing of the reaction conditions (reaction system, reaction process) of the kit, including the following process: The present invention further uses the following primer pairs to verify the universality of the nucleic acid mass spectrometry universal detection kit provided in Example 1 of the present invention, specifically including: Table 7. Infection system of pan-mutant strains of novel coronavirus

[0050] Table 8. Detection system of insecticide resistance genes of Aedes albopictus

[0051] The reaction systems and reaction processes in Tables 1 to 6 were respectively used, and according to the operating procedures of the kit, the above primer pairs were used to detect the samples to be tested (84 nasopharyngeal swab samples from population screening and 83 mosquito samples collected for mosquito monitoring).

[0052] A detection and typing system was constructed for 13 subtypes of the novel coronavirus, 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, and when the extended bases are all G, it indicates that the new coronavirus is a Delta subtype. Among the 84 samples tested, 12 Delta mutants and 6 Omicron mutants were consistent with the results of whole genome sequencing.

[0053] The detection system for the resistance of Aedes albopictus to pyrethroid insecticides identified the resistance of Aedes albopictus through three SNP sites of the resistance knockdown 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.

[0054] It can be seen from the above results that the nucleic acid mass spectrometry universal detection kit provided by the present invention has excellent versatility.

[0055] Comparative Example 1 During the research, the present invention screened 6 kinds of cation exchange resins from home and abroad (Table 9), and after multiple PCR amplification-SAP digestion-MPE extension of Mycobacterium tuberculosis DNA samples, 6 kinds of resins were used for desalting and purification respectively (the experimental conditions of resins 1-6 were the same as those in Example 1, except that the resin purification steps adopted the conditions recommended in the instructions), the same tube of 3-HPA matrix target was used, and the mass spectrum was collected by MALDI-TOF mass spectrometry. This system includes a total of 10 extension probes.

[0056] Table 9 Resin information used

[0057] The results showed that the mass spectrum peak of the finished resin-1 showed that there were multiple adduct peaks behind the main peak, the resolution was low, and a total of 9 extended probe peaks were detected; the mass spectrum peak of the finished resin-2 showed a clear main peak, but the number of peaks was small, only 4 extended probe peaks were detected, and the detection sensitivity was low; resin-3, resin-4, resin-5, and resin-6 had no effective peaks. The mass spectrum of the sample treated with the optimized resin (Example 1) of the present invention had a clear main peak, high resolution, and a total of 10 extended probe peaks were detected, with high sensitivity and good accuracy (Table 9, Figure 5 ).

[0058] At the same time, the detection experiment was carried out using the mutant strain of the new coronavirus, the Aedes albopictus resistance primer pair and the probe in Example 3, and the results were the same as above. This can illustrate that the resin provided in Example 1 of the present invention has excellent stability and versatility.

[0059] 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 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, 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).

2. The universal nucleic acid mass spectrometry detection kit according to claim 1, characterized in that: In terms of volume multiple, 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 spot 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, and the balance is nucleic acid-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 remainder is nucleic acid-free water; and / or, The target point system includes: 3-HPA and nucleic acid-free water.

5. A nucleic acid mass spectrometry detection method, 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, and the supernatant is collected after mixing and centrifugation; The mixing includes: mixing at 75-150 rpm for 20-40 minutes, and the centrifugation 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℃ 30~60s, 52~60℃ 30~60s, 70~74℃ 30~60s, 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 performing the following reaction after centrifugation: 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 comprises: 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 them naturally.

9. Use of the universal detection kit according to any one of claims 1 to 4 in nucleic acid mass spectrometry detection.

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.

Images