CRISPR multiple nucleic acid detection system and application

Abstract

The invention relates to the technical field of nucleic acid detection, in particular to a CRISPR multiple nucleic acid detection system and application, the CRISPR multiple nucleic acid detection system comprises a CRISPR / Cas two-step detection system and a conventional CRISPR two-step detection system, the reaction procedure comprises two steps, the first step is multiple amplification reaction; the reaction procedure in the second step comprises two stages, the first stage is detection by a conventional CRISPR two-step detection system, the design of PAM-free sgRNA or crRNA cannot cut a report probe due to low reaction temperature of the CRISPR / Cas two-step detection system, and a signal of the fluorescent probe in the first stage is generated by the conventional CRISPR two-step detection system; in the second stage, the temperature is rapidly increased to be within the reaction temperature range of the Cas protein 2, the CRISPR / Cas two-step method detection system plays a role, and meanwhile, the Cas protein 1 is inactivated due to high temperature, so that the conventional CRISPR two-step method system cannot cut the report probe, and a fluorescence signal in the stage is generated by the CRISPR / Cas two-step method detection system.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and specifically to a CRISPR multiplex nucleic acid detection system and its application. Background Art

[0002] The CRISPR system originated from the adaptive immune mechanism of bacteria and archaea. Through the synergistic effect of Cas proteins and guide RNAs, it specifically recognizes and cleaves target nucleic acid sequences. Based on its unique trans-cleavage activity, CRISPR technology has been widely applied in the field of nucleic acid detection. The core process of traditional CRISPR two-step detection includes two stages: nucleic acid amplification and CRISPR signal detection. First, target nucleic acids are exponentially amplified by nucleic acid amplification techniques such as PCR, RPA, and LAMP. Subsequently, the amplification products are added to the CRISPR reaction system to activate the trans-cleavage activity of Cas proteins (such as Cas12, Cas13), cleave the reporter probe, and achieve target detection through fluorescence signals or color development of test strips. By separating amplification from detection, this method takes into account both sensitivity and specificity and has become the mainstream design of early CRISPR diagnostic systems. Representative techniques include SHERLOCK (Cas13) and HOLMES (Cas12).

[0003] Although the CRISPR two-step detection system has made significant progress in nucleic acid detection, its limitations still restrict clinical applications and industrial development. Currently, most CRISPR two-step detection systems are designed for single targets, and the ability to detect multiple targets in a combined manner is limited. In addition, the recognition of target sequences by Cas proteins depends on specific PAM site sequences, which limits the design of sgRNA or crRNA. Therefore, optimizing and improving the multi-target detection and PAM-site-independent (PAM-free) performance of the CRISPR two-step detection system is of great significance. Summary of the Invention

[0004] The purpose of the present invention is to provide a CRISPR multiplex nucleic acid detection system and its application.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] A CRISPR multiplex nucleic acid detection system includes a CRISPR / Cas two-step detection system and a conventional CRISPR two-step detection system. The CRISPR / Cas two-step detection system includes an amplification system and a CRISPR reaction system. The amplification system contains a forward primer, a reverse primer, and nuclease-free water targeting the conserved region of the target gene. The CRISPR reaction system contains Cas protein 1, Cas protein 2, PAM-free sgRNA or PAM-free crRNA, a fluorescent reporter probe, and nuclease-free water. The Cas protein is selected from heat-resistant proteins or their mutants in the Cas12 and Cas13 families, and its optimal reaction temperature is 50-80°C. The highest reaction temperature of Cas protein 2 is higher than that of Cas protein 1.

[0007] Further, the optimal reaction temperature of the Cas protein is 55-75°C.

[0008] Furthermore, the optimal reaction temperature of the Cas protein is 60-70°C.

[0009] Among them, the amplification system includes an isothermal amplification system and a temperature-variable amplification system.

[0010] Among them, the conventional CRISPR two-step detection system includes a Cas protein, crRNA or sgRNA, a reporter probe, a reaction buffer, and an amplification product.

[0011] Among them, it includes two steps. The first step is a multiplex amplification reaction.

[0012] The second reaction procedure includes two stages: In the first stage, the conventional CRISPR two-step detection system is mainly used for detection. At this stage, due to the lower reaction temperature of the CRISPR / Cas two-step detection system, the design of PAM-free sgRNA or crRNA will not cleave the reporter probe, and the signal of the fluorescent probe in the first stage is generated by the conventional CRISPR two-step detection system. In the second stage, the temperature is rapidly increased to the reaction temperature range of Cas protein 2, and the CRISPR / Cas two-step detection system comes into play. At the same time, the high temperature inactivates Cas protein 1, so that the conventional CRISPR two-step system will not cleave the reporter probe, and the fluorescent signal in this stage is generated by the CRISPR / Cas two-step detection system.

[0013] Among them, the reaction temperature in the first stage is 37-65°C, and the time is 10-30 minutes. The reaction temperature in the second stage is 50-80°C, and the time is 5-30 minutes. The whole process is monitored by fluorescent signal.

[0014] The CRISPR multiplex nucleic acid detection system described in the present invention can be used to prepare a nucleic acid detection kit. Among them, the kit includes the above-mentioned nucleic acid detection system, positive control and negative control.

[0015] Compared with the prior art, the beneficial effects of the CRISPR multiplex nucleic acid detection system and its application of the present invention are as follows:

[0016] (1) Due to the trans-cleavage activity of the Cas protein, which can non-specifically cleave the reporter probe, the current CRISPR / Cas two-step system can only detect single targets and cannot detect multiple targets simultaneously. However, the CRISPR multiplex nucleic acid detection system of the present invention can detect multiple targets simultaneously using only the same type of Cas protein, solving this problem.

[0017] (2) The design of PAM-free sgRNA or crRNA greatly facilitates the selection of sgRNA or crRNA. At the same time, by raising the temperature, not only can the activity of PAM-free sgRNA or crRNA be regulated, but also the Cas protein with a lower reaction temperature can be inactivated, so that it will not interfere with the detection in the second stage. This design idea of staged detection broadens the design of the current CRISPR / Cas detection system.

[0018] (3) The present invention can achieve CRISPR / Cas multiplex detection using only a single reporter probe, which not only reduces the reagent complexity of the system, but also can reduce the reagent cost of the detection system. Description of the Drawings

[0019] Figure 1 It is the test result of the CRISPR two-stage variable-temperature two-step detection system for the conventional PAM sgRNA of monkeypox virus.

[0020] Figure 2 It is the test result of the CRISPR two-stage variable-temperature two-step detection system for PAM-free sgRNA of monkeypox virus.

[0021] Figure 3 It is the test result of the CRISPR two-stage variable-temperature two-step detection system for PAM-free sgRNA of Mycoplasma pneumoniae.

[0022] Figure 4 It is the test result of the CRISPR two-step multiplex detection system for monkeypox virus.

[0023] Figure 5 It is the test result of the CRISPR two-step multiplex detection system for Mycoplasma pneumoniae. Detailed Embodiments

[0024] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0025] Example 1 Temperature Regulation of Cas Enzyme Activity Based on PAM-free sgRNA Design

[0026] 1. Design of RPA Primers and PAM-free sgRNA for Monkeypox Virus

[0027] The conserved region of the G2R gene of the monkeypox virus was selected for the design of RPA amplification primers, and Cas12b PAM-free sgRNA was randomly designed at the middle position of the RPA amplification sequence. The specific sequences are shown in Table 1 below (shown as SEQ ID NO: 1-8).

[0028] Table 1 RPA Amplification Sequences, RPA Primers and PAM-free sgRNA of MPXV G2R Gene

[0029]

[0030]

[0031] Note: The underlined sequence of MPXV PAM-free sgRNA is the spacer sequence.

[0032] 2. Testing of the Two-stage Temperature-variable Two-step Detection System of Monkeypox Virus PAM-free sgRNA CRISPR

[0033] The ERA (Enzymatic Recombinase Amplification) technology, which is similar to the principle of RPA isothermal amplification technology, was used for the amplification of the reaction system.

[0034] The ERA reagent is from Suzhou Xianda Gene Technology Co., Ltd., with the product number KS301. AapCas12b (product number 32118 - 03), the reporter probe HOLMES ssDNA reporter (product number 31101), and 10×HOLMES Buffer for Cas12b (product number 32043) are from Anhui Tulugang Biotechnology Co., Ltd. The MPXV RPA amplification primers and the MPXV positive control plasmid (the pUC57 plasmid vector containing the RPA amplification sequence of the MPXV G2R gene) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The MPXV sgRNA was prepared by in vitro transcription using the Cas12b High Yield sgRNA Synthesis and Purification Kit (Anhui Tulugang Biotechnology Co., Ltd., product number: 31904).

[0035] (1) RPA reaction

[0036] ① Prepare the RPA reaction system: used to dissolve the RPA freeze - dried beads.

[0037] Table 2 Preparation of the MPXV RPA reaction system

[0038] Reagent Name Volume (μL) MPXV-G2R-F (10 μM) 1 MPXV-G2R-R (10 μM) 1 Nuclease-Free Water 17 Total 19 μL

[0039] ② Add 19 μL of the reaction solution to the RPA freeze - dried beads, gently flick by hand to make the freeze - dried beads fully uniform, and briefly centrifuge to collect the liquid at the bottom of the tube.

[0040] ③ Add 1 μL of the activator to the lid of each reaction tube, then add 5 μL of nuclease - free water as a negative control and 5 μL of the MPXV plasmid with a concentration of 2 copies / μL as a positive control to the reaction tube.

[0041] ④ Close the reaction tube lid, then perform a transient centrifugation in a hand - held centrifuge, and finally react the reaction tube at 37 °C for 20 min.

[0042] (2) MPXV CRISPR two - stage temperature - variable two - step method

[0043] ① MPXV conventional PAM sgRNA CRISPR two - stage temperature - variable two - step method:

[0044] Prepare the reaction system of the MPXV conventional PAM sgRNA CRISPR two-stage temperature-variable two-step method according to Table 3 below, and test it with dsDNA template (MPXV RPA amplification product) and ssDNA template (MPXV-ssDNA: 5’-CGTACATTGTGTATTAGTCTTGCTATCACATAATCTGGAA-3’, synthesized by Sangon Biotech (Shanghai) Co., Ltd., shown in SEQ ID NO: 9) respectively. Set the reaction program as the first stage: react at 37 °C for 10 min, and the second stage: react at 60 °C for 10 min, and collect FAM fluorescence signals once per minute.

[0045] Table 3 Preparation of the reaction system of the MPXV conventional PAM sgRNA CRISPR two-stage temperature-variable two-step method

[0046] Reagent Name Volume (μL) 10×HOLMES Buffer for Cas12b 1 AapCas12b (10 μM) 1 MPXV-sgRNA (10 μM) 1 HOLMES ssDNA reporter (10 μM, FAM) 1.25 Nuclease-Free Water 13.75 MPXV RPA Amplification Product (dsDNA) / MPXV-ssDNA (100 nM) 2 Total 20 μL

[0047] The test results of the MPXV conventional PAM sgRNA CRISPR two-stage temperature-variable two-step detection system are as Figure 1 shown. It can be seen from the test results that the conventional PAM sgRNA still has activity under lower temperature conditions (not the optimal reaction temperature of Cas12b), and both dsDNA and ssDNA templates can activate its trans-cleavage activity.

[0048] ② MPXV PAM-free sgRNA CRISPR two-stage temperature-variable two-step method

[0049] Prepare the reaction system of the MPXV PAM-free sgRNA CRISPR two-stage temperature-variable two-step method according to Table 4 below, and test it with dsDNA template (MPXV RPA amplification product) and ssDNA template (MPXV-ssDNA: 5’-CGTACATTGTGTATTAGTCTTGCTATCACATAATCTGGAA-3’, synthesized by Sangon Biotech (Shanghai) Co., Ltd.) respectively. Set the reaction program as the first stage: react at 37 °C for 10 min, and the second stage: react at 60 °C for 10 min, and collect FAM fluorescence signals once per minute.

[0050] Table 4 Preparation of the reaction system of the MPXV PAM-free sgRNA CRISPR two-stage temperature-variable two-step method

[0051] Reagent Name Volume (μL) 10×HOLMES Buffer for Cas12b 1 AapCas12b (10 μM) 1 MPXV-sgRNA1 / MPXV-sgRNA2 / MPXV-sgRNA3 / MPXV-sgRNA4 (10 μM) 1 HOLMES ssDNA reporter (10 μM, FAM) 1.25 Nuclease-Free Water 13.75 MPXV RPA Amplification Product / MPXV-ssDNA (100 nM) 2 Total 20 μL

[0052] The test results of the MPXV PAM-free sgRNA CRISPR two-stage temperature-variable two-step detection system are as Figure 2As shown. From the test results, it can be seen that the PAM-free sgRNA cannot activate its trans-cleavage activity with a dsDNA template under low temperature conditions (non-optimal reaction temperature of Cas12b), and only the ssDNA template can activate its trans-cleavage activity. When the temperature is increased, the activity of the PAM-free sgRNA can be activated. At this time, the dsDNA template can activate its trans-cleavage activity, so the temperature can be used to regulate the activity of the PAM-free sgRNA.

[0053] Example 2 Establishment of a two-stage temperature-variable two-step detection system for Mycoplasma pneumoniae (MP) PAM-free sgRNA CRISPR

[0054] 1. Design of Mycoplasma pneumoniae RPA primers and PAM-free sgRNA

[0055] The conserved region of the Mycoplasma pneumoniae P1 gene was selected for the design of RPA amplification primers, and Cas12b PAM-free sgRNA was randomly designed in the middle position of the RPA amplification sequence. The specific sequences are shown in Table 5 below (shown as SEQ ID NO: 10-15).

[0056] Table 5 RPA amplification sequence, RPA primers and PAM-free sgRNA of MP P1 gene

[0057]

[0058] Note: The underlined sequence of MP PAM-free sgRNA is the spacer sequence.

[0059] 2. Testing of the two-stage temperature-variable two-step detection system for Mycoplasma pneumoniae PAM-free sgRNA CRISPR

[0060] The MP RPA amplification primers and the MP positive control plasmid (pUC57 plasmid vector containing the RPA amplification sequence of the MP P1 gene) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The MP sgRNA was prepared by in vitro transcription using the Cas12b High Yield sgRNA Synthesis and Purification Kit (Anhui Tulugang Biotechnology Co., Ltd., product number: 31904). The sources of other reagents were the same as those in Example 1.

[0061] (1) RPA reaction

[0062] ① Preparation of the RPA reaction system: used to dissolve the RPA freeze-dried beads.

[0063] Table 6 Preparation of the MP RPA reaction system

[0064] Reagent Name Volume (μL) MP-P1-F (10 μM) 1 MP-P1-R (10 μM) 1 Nuclease-Free Water 17 Total 19 μL

[0065] ② Add 19 μL of the reaction solution to the RPA dry pellet, gently flick the tube by hand to fully and evenly disperse the dry pellet, and briefly centrifuge to collect the liquid at the bottom of the tube.

[0066] ③ Add 1 μL of the activator to the cap of each reaction tube, then add 5 μL of nuclease-free water to the reaction tube as a negative control and 5 μL of MP plasmid at a concentration of 2 copies / μL as a positive control.

[0067] ④ Close the reaction tube with its cap, then perform a brief centrifugation in a hand-held centrifuge, and finally incubate the reaction tube at 37 °C for 20 min.

[0068] (2) Two-step temperature-variable method for MP PAM-free sgRNA CRISPR

[0069] Prepare the reaction system for the two-step temperature-variable method of MP PAM-free sgRNA CRISPR according to Table 7 below. Set the reaction program as the first stage: react at 37 °C for 10 min, and the second stage: react at 60 °C for 10 min, with FAM fluorescence signals collected once per minute.

[0070] Table 7 Preparation of the reaction system for the two-step temperature-variable method of MP PAM-free sgRNA CRISPR

[0071] Reagent Name Volume (μL) 10×HOLMES Buffer for Cas12b 1 AapCas12b (10 μM) 1 MP-sgRNA1 / MP-sgRNA2 / MP-sgRNA3 (10 μM) 1 HOLMES ssDNA reporter (10 μM, FAM) 1.25 Nuclease-Free Water 13.75 MP RPA Amplification Product 2 Total 20 μL

[0072] The test results of the two-step temperature-variable detection system for Mycoplasma pneumoniae PAM-free sgRNA CRISPR are as Figure 3 shown. From the detection results, it can be seen that in the first stage, due to the design of PAM-free sgRNA and the reaction temperature being much lower than the optimal temperature of Cas12b, the activity of the CRISPR / Cas12b system is inhibited; after increasing the temperature in the second stage, the activity of Cas12b enzyme designed based on PAM-free sgRNA can be regulated, and the activity of the CRISPR / Cas12b system is restored, thus activating the trans-cleavage activity of CRISPR / Cas12b.

[0073] Example 3 Two-step CRISPR multiplex detection system for Monkeypox virus (Mpox virus, MPXV)

[0074] 1. Design of RPA primers for Monkeypox virus and internal reference gene (RNaseP, RP)

[0075] The conserved regions of the monkeypox virus G2R gene and the RNaseP internal reference gene were selected for the design of RPA amplification primers. The specific sequences are shown in Table 8 below (SEQ ID NO: 1-3, 16-18).

[0076] Table 8 Target Sequences and RPA Primers for Monkeypox Virus and RNaseP Internal Reference Gene

[0077]

[0078] 2. Design of Monkeypox Virus Cas12b PAM-free sgRNA and RNaseP Internal Reference Gene Cas12a crRNA

[0079] Cas12b PAM-free sgRNA was randomly designed at the middle position of the monkeypox virus RPA amplification sequence, and Cas12a crRNA was designed at the middle position of the RNaseP internal reference gene RPA amplification sequence. The specific sequences are shown in Table 9 below (SEQ ID NO: 19-20).

[0080] Table 9 Monkeypox Virus Cas12b PAM-free sgRNA and RNaseP Internal Reference Gene Cas12a crRNA

[0081]

[0082]

[0083] Note: The underlined sequences are spacer sequences.

[0084] 3. Testing of the Monkeypox Virus CRISPR Two-step Multiplex Detection System

[0085] The ERA (Enzymatic Recombinase Amplification) technology, which is similar to the principle of RPA isothermal amplification technology, was used for the amplification of the reaction system. The ERA reagent was from Suzhou Xianda Gene Technology Co., Ltd., with the product number KS301. AapCas12b (product number 32118-03), LbCas12a (product number 32108-03), the reporter probe HOLMES ssDNAreporter (product number 31101), and 10×HOLMES Buffer for Cas12b (product number 32043) were from Anhui Tulugang Biotechnology Co., Ltd. The MPXV and RNaseP internal reference RPA amplification primers, as well as the MPXV plasmid and RNaseP internal reference plasmid, were synthesized by Sangon Biotech (Shanghai) Co., Ltd. MPXV-PFsgRNA and RP-crRNA were respectively prepared by in vitro transcription using the Cas12b HighYield sgRNA Synthesis and Purification Kit (Anhui Tulugang Biotechnology Co., Ltd., product number: 31904) and the Cas12a High Yield crRNA Synthesis and Purification Kit (Anhui Tulugang Biotechnology Co., Ltd., product number: 31903).

[0086] (1) Multiplex RPA reaction

[0087] ① Preparation of the RPA reaction system: used to dissolve the RPA freeze-dried beads.

[0088] Table 10 MPXV and RP internal reference multiplex RPA reaction system

[0089] Reagent Name Volume (μL) MPXV-RPA-F (10 μM) 1 MPXV-RPA-R (10 μM) 1 RP-RPA-F (10 μM) 1 RP-RPA-R (10 μM) 1 Nuclease-Free Water 15 Total 19 μL

[0090] ② Add 19 μL of the reaction solution to the RPA freeze-dried beads, gently flick by hand to make the freeze-dried beads fully uniform, and briefly centrifuge to collect the liquid at the bottom of the tube.

[0091] ③ Add 1 μL of the activator to the cap of each reaction tube. Test according to the 4 possible situations of the monkeypox virus and the internal reference template: ① The template is 10 copies / μL MPXV plasmid and 10 copies / μL internal reference plasmid; ② The template is 10 copies / μL MPXV plasmid; ③ The template is 10 copies / μL internal reference plasmid; ④ The template is nuclease-free water.

[0092] ④ Close the reaction tube with the cap, then perform instantaneous centrifugation in a palm centrifuge, and finally react the reaction tube at 37 °C for 20 min.

[0093] (2) MPXV CRISPR Two-step Multiplex Detection System

[0094] Prepare the MPXV CRISPR two-step multiplex detection system according to Table 11 below. Set the reaction program as the first stage: react at 37°C for 5 min. At this stage, due to the low reaction temperature, the activity of AapCas12b is low and the MPXV PAM-free sgRNA is designed, so the activity of the MPXV Cas12b system is not activated. Only LbCas12a has the best activity, and RP-crRNA detects the internal reference RP in the first stage. The second stage: react at 70°C for 5 min. LbCas12a is inactivated at this temperature, and AapCas12b has activity at this temperature. Heating can regulate and activate the activity of MPXV PAM-free sgRNA, and MPXV is detected in the second stage. Collect FAM fluorescence signals once per minute.

[0095] Table 11 MPXV CRISPR Two-step Multiplex Detection System

[0096] Reagent Name Volume (μL) 10×HOLMES Buffer for Cas12b 2 LbCas12a (10 μM) 1 RP-crRNA (10 μM) 1 AapCas12b (10 μM) 1 MPXV-PFsgRNA (10 μM) 1 HOLMES ssDNA reporter (10 μM, FAM) 2 Nuclease-Free Water 10 RPA Multiplex Amplification Product 2 Total 20

[0097] From Figure 4 It can be seen that in the first stage, at 37°C, LbCas12a is used to detect the RP internal reference. At this stage, due to the low reaction temperature, the activity of AapCas12b is low and the MPXV PAM-free sgRNA is designed, so the activity of the MPXV Cas12b system is not activated. Only LbCas12a has the best activity, and RP-crRNA detects the internal reference RP in the first stage. In the second stage, at 70°C, LbCas12a is inactivated at this temperature, and AapCas12b still has activity at this temperature. Heating can regulate and activate the activity of MPXV PAM-free sgRNA, and MPXV is detected in the second stage.

[0098] Example 4 Mycoplasma pneumoniae (MP) CRISPR Two-step Multiplex Detection System

[0099] 1. Design of PCR Primers for Mycoplasma pneumoniae and Internal Reference Gene (RNaseP, RP)

[0100] Select the conserved regions of the Mycoplasma pneumoniae PIP gene (M. pneumoniae intracellular protease gene) and the RNaseP internal reference gene to design PCR amplification primers. The relevant sequences are shown in Table 12 below (SEQ ID NO: 21 - 26).

[0101] Table 12 Target Sequences of Mycoplasma pneumoniae and RNaseP Reference Gene and RPA Primers

[0102]

[0103] 2. Design of Cas12b PAM-free sgRNA of Mycoplasma pneumoniae and Cas12a crRNA of RNaseP Reference Gene

[0104] Randomly design Cas12b PAM-free sgRNA at the middle position of the Mycoplasma pneumoniae PCR amplification sequence, and design Cas12a crRNA at the middle position of the RNaseP reference gene PCR amplification sequence. The specific sequences are shown in Table 13 (SEQ ID NO: 27-28).

[0105] Table 13 Cas12b PAM-free sgRNA of Mycoplasma pneumoniae and Cas12a crRNA of RNaseP Reference Gene

[0106]

[0107]

[0108] Note: The underlined sequences are spacer sequences.

[0109] 3. Testing of the Two-step Multiplex Detection System for Mycoplasma pneumoniae CRISPR

[0110] The MP and RNaseP reference PCR amplification primers, as well as the MP plasmid and RNaseP reference plasmid, were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Taq HS DNA Polymerase (Nanjing Novoprotein Scientific Inc., P132-d2), and other reagents were the same as in Example 3.

[0111] (1) Multiplex PCR Reaction

[0112] ① Preparation of the MP Multiplex PCR Reaction System

[0113] Table 14 MP and RP Reference Multiplex PCR Reaction System

[0114] Reagent Name Volume (μL) <![CDATA[10×Taq HS Buffer (Mg 2+ plus)]]> 2.5 dNTP Mix (10 mM each) 0.5 Taq HS DNA polymerase (5 U / μl) 0.5 MP-F (10 μM) 1 MP-R (10 μM) 1 RP-F (10 μM) 1 RP-R (10 μM) 1 Nuclease-Free Water 12.5 Template 5 Total 25

[0115] ② Test according to the 4 possible situations of the Mycoplasma pneumoniae and reference templates: ① The template is 10 copies / μL MP plasmid and 10 copies / μL reference plasmid; ② The template is 10 copies / μL MP plasmid; ③ The template is 10 copies / μL reference plasmid; ④ The template is nuclease-free water.

[0116] ③PCR reaction procedure: 95°C for 30 s; 95°C for 30 s, 55°C for 30 s, 72°C for 10 s for a total of 35 cycles; 72°C for 5 min.

[0117] (2) MP CRISPR two-step multiplex detection system

[0118] Prepare the MP CRISPR two-step multiplex detection system according to Table 15 below. Set the reaction procedure as the first stage: react at 37°C for 5 min. At this stage, due to the relatively low reaction temperature, the activity of AapCas12b is low and the MP PAM-free sgRNA is designed, so the activity of the MP Cas12b system is not activated. Only LbCas12a has the best activity, and RP-crRNA detects the internal reference RP in the first stage. The second stage: react at 70°C for 5 min. LbCas12a is inactivated at this temperature, while AapCas12b is active at this temperature. Heating can regulate the activity of MP PAM-free sgRNA, and MP is detected in the second stage. Collect FAM fluorescence signals once per minute.

[0119] Table 15 MP CRISPR two-step multiplex detection system

[0120] Reagent Name Volume (μL) 10×HOLMES Buffer for Cas12b 2 LbCas12a (10 μM) 1 RP-crRNA (10 μM) 1 AapCas12b (10 μM) 1 MP-sgRNA (10 μM) 1 HOLMES ssDNA reporter (10 μM, FAM) 2 Nuclease-free water 10 PCR multiplex amplification product 2 Total 20

[0121] From Figure 5 It can be seen that in the first stage at 37°C, LbCas12a is used to detect the RP internal reference. At this stage, due to the relatively low reaction temperature, the activity of AapCas12b is low and the MP PAM-free sgRNA is designed, so the activity of the MP Cas12b system is not activated. Only LbCas12a has the best activity, and RP-crRNA detects the internal reference RP in the first stage. In the second stage at 70°C, LbCas12a is inactivated at this temperature, while AapCas12b is still active at this temperature. Heating can regulate and activate the activity of MP PAM-free sgRNA, and MP is detected in the second stage.

[0122] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A CRISPR multiplex nucleic acid detection system, characterized in that: CRISPR / Cas two-step detection system and conventional CRISPR two-step detection system. The CRISPR / Cas two-step detection system includes an amplification system and a CRISPR reaction system. The amplification system contains a forward primer, a reverse primer, and nuclease-free water targeting the conserved region of the target gene. The CRISPR reaction system contains Cas protein 1, Cas protein 2, PAM-free sgRNA or PAM-free crRNA, a fluorescent reporter probe, and nuclease-free water. The Cas protein 2 is selected from heat-resistant proteins or mutants thereof in the Cas12 and Cas13 families, and its optimal reaction temperature is 50 - 80 °C. The highest reaction temperature of the Cas protein 2 is higher than the highest reaction temperature of the Cas protein 1.

2. The CRISPR multiplex nucleic acid detection system according to claim 1, wherein: The optimal reaction temperature of the Cas protein 2 is 55 - 75 °C.

3. The CRISPR multiplex nucleic acid detection system according to claim 2, wherein: The optimal reaction temperature of the Cas protein 2 is 60 - 70 °C.

4. The CRISPR multiplex nucleic acid detection system according to claim 1, wherein: The amplification system includes an isothermal amplification system and a temperature-variable amplification system.

5. The CRISPR multiplex nucleic acid detection system according to claim 1, wherein: The conventional CRISPR two-step detection system includes a Cas protein, crRNA or sgRNA, a reporter probe, a reaction buffer, and an amplification product.

6. The CRISPR multiplex nucleic acid detection system according to claim 1, wherein: It includes two steps. The first step is a multiplex amplification reaction. The second reaction procedure includes two stages. In the first stage, the conventional CRISPR two-step detection system is mainly used for detection. At this stage, due to the relatively low reaction temperature of the CRISPR / Cas two-step detection system, the designed PAM-free sgRNA or crRNA will not cleave the reporter probe, and the signal of the fluorescent probe in the first stage is generated by the conventional CRISPR two-step detection system. In the second stage, the temperature is rapidly increased to the reaction temperature range of Cas protein 2, and the CRISPR / Cas two-step detection system comes into play. At the same time, the high temperature inactivates Cas protein 1, so that the conventional CRISPR two-step system will not cleave the reporter probe, and the fluorescent signal in this stage is generated by the CRISPR / Cas two-step detection system.

7. The CRISPR multiplex nucleic acid detection system according to claim 6, wherein: The reaction temperature in the first stage is 37 - 65 °C, and the time is 10 - 30 minutes. The reaction temperature in the second stage is 50 - 80 °C, and the time is 5 - 30 minutes. The whole process is monitored by fluorescent signal.

8. Use of the CRISPR multiplex nucleic acid detection system according to any one of claims 1 - 7 in a nucleic acid detection kit.

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