Kit for detecting multiple nucleic acids based on CRISPR / Cas12a combined with universal crRNA

Abstract

The invention discloses a kit for detecting multiple nucleic acids based on CRISPR / Cas12a in combination with universal crRNA, and belongs to the field of nucleic acid detection.The kit comprises crRNA, Cas12a, a buffer solution 3.1, a buffer solution 2, an RNA enzyme inhibitor, BstDNA polymerase and dNTP mixed.The Cas12a trans-cleavage activity is utilized, crRNA of a single type is applied to nucleic acid detection of different types of viruses for the first time, multiple viral nucleic acids can be detected by using the same crRNA, and the crRNA of a single type can be applied to detection of different types of viruses. Comprising but not limited to multiple subtypes of SARS-CoV-2 and HPV viruses, tumor markers miRNA and the like, the design thought and method of CRISPR / Cas nucleic acid detection are widened, good analysis performance is achieved, under the optimized experiment condition, the same crRNA is used, multiple DNA viruses and RNA viruses can be effectively detected, high sensitivity is achieved, and the concentration can be detected to be 5 pM. By taking the HPV-16 pseudovirus as an example, actual sample detection is carried out.

Description

Technical Field

[0001] The present invention relates to a kit, in particular to a kit for performing multiple nucleic acid detections based on CRISPR / Cas12a combined with universal crRNA, belonging to the field of nucleic acid detection. Background Art

[0002] Nucleic acids include DNA and RNA, which are carriers of genetic information and can be used as markers for the diagnosis of a variety of diseases, such as cancer and pathogenic infectious diseases. Nucleic acid molecular diagnosis has the advantages of high sensitivity, good real-time performance, and simple operation. It is of great significance for the early screening and diagnosis of cancer and pathogenic infectious diseases. Currently, the commonly used nucleic acid detection methods mainly include gene sequencing and nucleic acid amplification detection.

[0003] Most existing nucleic acid detection methods have complex operating steps and rely on expensive instruments and equipment. The CRISPR / Cas system, known as the "gene magic scissors", has shown great potential and advantages in nucleic acid detection. Clustered regularly interspaced short palindromic repeats (CRISPR) is an acquired immune system in bacteria, which together with Cas proteins form the CRISPR / Cas system. Among them, Cas12a, Cas13a and Cas14a activate the "trans-cutting" activity after specifically recognizing the target molecule, and will continue to non-specifically cut other single-stranded DNA (ssDNA) or RNA.

[0004] CrRNA is extremely unstable and expensive in the environment. The amount of crRNA in the traditional CRISPR / Cas system is designed according to a certain viral target nucleic acid. A crRNA can only detect a viral target. In the face of different viral targets, different crRNAs need to be designed, which greatly increases the difficulty and cost of detection. The present invention overcomes this difficulty, using the same crRNA, only needs to bind short-chain DNA or RNA targets to long Track chains, and obtain double-stranded DNA after one-step amplification. The double-stranded DNA region of the amplification includes the binding region of universal crRNA, which is then recognized and cut by Cas12a Reporter, generating a signal, so as to achieve the purpose of using a crRNA to detect multiple viral targets. Summary of the invention

[0005] In view of this, the present invention provides a kit for detecting multiple nucleic acids based on CRISPR / Cas12a combined with universal crRNA, which utilizes the trans-cleavage activity of Cas12a and one-step isothermal amplification to achieve the purpose of using one crRNA to identify different targets, thereby detecting multiple viral DNA and RNA.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A kit for performing multiple nucleic acid detections based on CRISPR / Cas12a combined with universal crRNA, including 0.5μL 20μM crRNA (500nM), 0.5μL 10μM Cas12a protein (250nM), 7.5μL buffer 3.1, 0.5μL RNase inhibitor, 1μL 10μM reporter (500nM), 6.25μL buffer 2, 1μL 1μM Track chain (100nM), 0.75μL 8000units / ml Bst DNA polymerase (0.375units / μL) and 1μL 10mM dNTP solution (250μM), measured by the concentration in the system, wherein the concentration in brackets is in the detection system.

[0008] On the basis of the above technical solution, the present invention can also be improved as follows:

[0009] Further, the sequence of the crRNA is: UAA UUU CUA CUC UUG UAG AU GUAA CUA GCA AGAAUA CCA C.

[0010] Furthermore, the sequence of the reporter is: 5'-Texas Red-TGG GAT ATC TTT AAT TTT ATTTTA ACA AGA TAT CCC A-BHQ2-3'.

[0011] Further, the Cas12a protein is a Cpf1 endonuclease.

[0012] Furthermore, the RNase inhibitor is a recombinant obtained from pig liver RNase inhibitor purified from Escherichia coli.

[0013] Further, the Bst DNA polymerase is prepared from an Escherichia coli strain containing a gene fusion of a Lactobacillus acidophilus DNA polymerase gene lacking a 5′→3′ exonuclease structure and a gene encoding an Escherichia coli maltose binding protein (MBP), the fusion protein being purified to near homogeneity, the fused MBP portion being cleaved off in vitro, and the remaining remaining polymerase being purified and free of MBP.

[0014] Furthermore, the dNTP solution is an equimolar solution of ultrapure dATP, dCTP, dGTP and dTTP.

[0015] The method of using the above-mentioned kit of the present invention can be referred to as follows:

[0016] The Track chain, target, and buffer 2 in the universal crRNA nucleic acid detection reagent were added to a centrifuge tube, cooled from 95°C to 25°C for annealing to form a secondary structure, 0.75μL 8000units / ml Bst DNA polymerase and 1μL 10mM dNTP solution were reacted at 37°C for 45min, then heated to 85°C to inactivate the bst polymerase and cooled to room temperature. Subsequently, 0.5μL 20μM crRNA, 0.5μL 10μM Cas12a protein, 7.5μL buffer 3.1, 0.5μL RNase inhibitor, and 1μL 10μM reporter were added, and the resulting mixture was placed in a real-time fluorescence PCR instrument, reacted at 47°C for 8000s, and real-time fluorescence signals were collected.

[0017] The beneficial effects of the present invention are:

[0018] 1. The present invention uses one crRNA for the first time to detect different DNA or RNA viruses, which expands the detection range of CRISPR / Cas nucleic acid and is suitable for POCT product development. It overcomes the previous phenomenon that one crRNA can only detect one viral nucleic acid, reduces costs, and simplifies the design.

[0019] 2. The kit of the present invention can be used for the detection of a variety of DNA and RNA viruses, and has good analytical performance. Taking the new coronavirus, HPV virus nucleic acid and tumor miRNA as examples, the lowest detectable level is 5pM.

[0020] 3. The present invention can effectively detect HPV-16 pseudovirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the results of detecting the COVID-19 S13 gene in Example 2 of the present invention;

[0022] Figure 2 This is a graph showing the concentration of the amplified partial solution and the amplification time and the detection of the COVID-19 S13 gene result in Example 3 of the present invention;

[0023] Figure 3 This is the result diagram of the optimized detection of the COVID-19 S12 gene in Example 4 of the present invention;

[0024] Figure 4 This is a graph showing the results of detecting the HPV-6b gene after optimization in Example 5 of the present invention;

[0025] Figure 5 This is a graph showing the results of detecting the HPV-11b gene after optimization in Example 6 of the present invention;

[0026] Figure 6The results of the optimized detection of HPV-16b gene in Example 7 of the present invention;

[0027] Figure 7 The results of the optimized HPV-31 gene detection in Example 8 of the present invention are as follows;

[0028] Figure 8 The results of the optimized HPV-33 gene detection in Example 9 of the present invention are as follows;

[0029] Fig. 9 The results of detecting HPV-45a gene after optimization in Example 10 of the present invention;

[0030] Fig.10 The results of the HPV-52 gene detection after optimization are shown in Example 11 of the present invention;

[0031] Fig.11 Example 12 of the present invention is the result of detecting HPV-58 gene after optimization;

[0032] Fig.12 Example 13 of the present invention is the result of detecting the HCOV-NL63 gene after optimization;

[0033] Fig.13 Example 14 of the present invention is the result of detecting the HCOV-OC43 gene after optimization;

[0034] Fig.14 Example 15 of the present invention is the result of detecting HRSV gene after optimization;

[0035] Fig.15 Example 16 of the present invention is the result of detecting the HCOV-HKU1 gene after optimization;

[0036] Fig.16 Example 17 of the present invention is the result of detecting the miRNA-141 gene after optimization;

[0037] Fig.17 Example 18 of the present invention is the actual sample detection result after optimization. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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.

[0039] In the following examples, the nucleic acid sequences used are shown in Table 1-2, which were synthesized by Shanghai Sangon Biotechnology Co., Ltd. and purified by HPLC.

[0040] Table 1 Nucleic acid sequences used for DNA and RNA virus detection in the present invention.

[0041]

[0042]

[0043]

[0044] Table 2 Nucleic acid sequences used in actual sample detection in the present invention.

[0045]

[0046]

[0047] Lba Cas12a, buffer 2, buffer 3.1, Bst DNA polymerase, and dNTP solution were purchased from NEB Beijing;

[0048] RNase inhibitor was purchased from Takara, and DEPC-treated water was purchased from Shanghai Shenggong Biological Company;

[0049] Fluorescence signals were collected using a BIO-GENER Q3200 real-time fluorescence PCR instrument;

[0050] Other instruments and reagents not specially described are commonly used in this field.

[0051] Example 1

[0052] A kit for multiple nucleic acid detection based on CRISPR / Cas12a combined with universal crRNA, including 0.5μL 20μM crRNA (500nM), 0.5μL 10μM Cas12a protein (250nM), 7.5μL buffer 3.1, 0.5μL RNase inhibitor, 1μL 10μM reporter (500nM), 6.25μL buffer 2, 1μL 1μM Track chain (100nM), 0.75μL 8000units / ml Bst DNA polymerase (0.375units / μL) and 1μL 10mM dNTP solution (250μM) according to the concentration in the system;

[0053] Among them, the values ​​in brackets are the concentrations in the detection system;

[0054] The sequence of crRNA is: UAA UUU CUA CUC UUG UAG AU G UAA CUA GCA AGA AUA CCA C;

[0055] The reporter sequence is: 5′-Texas Red-TGG GAT ATC TTT AAT TTT ATT TTA ACA AGATAT CCC A-BHQ2-3′;

[0056] The Cas12a protein is a Cpf1 endonuclease;

[0057] The RNase inhibitor is a recombinant product obtained from pig liver RNase inhibitor purified from Escherichia coli;

[0058] The Bst DNA polymerase large fragment is prepared from an E. coli strain containing a gene fusion of the L. acidophilus DNA polymerase gene lacking the 5′→3′ exonuclease structure and the gene encoding the E. coli maltose binding protein (MBP), the fusion protein is purified to near homogeneity, the MBP portion of the fusion is cleaved off in vitro, and the remaining polymerase is purified free of MBP;

[0059] dNTP solution, which is an equimolar solution of ultrapure dATP, dCTP, dGTP, and dTTP;

[0060] The method steps for nucleic acid detection using the above kit are as follows:

[0061] 1μL 1μM Track chain (100nM), 1uL target, and 6.25μL buffer 2 in the universal crRNA nucleic acid detection reagent were added to the centrifuge tube for annealing, and the temperature was cooled from 95°C to 25°C to form a secondary structure. Then 0.75μL 8000units / mL Bst DNA polymerase (0.375units / μL) and 1μL 10mM dNTP solution (250μM) were added, and the reaction was carried out at 37°C for 45min, then heated to 85°C and finally cooled to room temperature. Then 0.5μL 20μM crRNA, 0.5μL 10μM Cas12a protein, 7.5μL buffer 3.1, 0.5μL RNase inhibitor, 1μL 10μM reporter (500nM) were added, and the resulting mixture was placed in a real-time fluorescence PCR instrument, reacted at 47°C for 8000s, and the fluorescence signal was collected.

[0062] Example 2

[0063] The target in Example 1 was replaced with the SARS-CoV-2-S13 (COVID-19 S13) gene, and the target chain was replaced with the COVID-19 S13 genome at different concentrations (5nM, 50pM, 5pM, 0). The detection was performed according to the method in Example 1, and the fluorescence signal was collected.

[0064] The results are as follows Figure 1 , Figure 1Real-time fluorescence intensity curves and bar graphs of different concentrations.

[0065] Depend on Figure 1 It can be seen that as the concentration of the SARS-CoV-2-S13 genome increases, the fluorescence value increases accordingly. The lowest concentration of the target that can be detected is 5pM, but the gradient is not obvious.

[0066] Example 3

[0067] Amplification process optimization experiment

[0068] 1μL 1μM Track chain (100nM), 1μL SARS-CoV-2-S13 genome target (5nM, 500pM, 50pM, 5pM, 0), and 6.25μL buffer 2 were added to a centrifuge tube for annealing, and the temperature was lowered from 95°C to 25°C to form a secondary structure; then (0.75μL, 0.5μL) 8000units / mL Bst DNA polymerase (0.375units / μL, 0.1875units / uL) and (1μL, 2μL, 4μL) 10mM dNTP solution (250μM, 500uM, 1mM) were added respectively, and the reaction was carried out at 37°C (30min, 45min, 60min), then the temperature was raised to 85°C and finally cooled to room temperature; then 0.5μL 20μM crRNA, 0.5μL 10 μM Cas12a protein, 7.5 μL buffer 3.1, 0.5 μL RNase inhibitor, 1 μL 10 μM reporter (500 nM), the resulting mixture was placed in a real-time fluorescence PCR instrument, reacted at 47 ° C for 8000 s, and the fluorescence signal was collected. The results are as follows Figure 2 .

[0069] Depend on Figure 2 It can be seen that during the amplification process, Figure 2 (A) The optimal amplification time is 45 min. Figure 2 (B) The optimal concentration of Bst DNA polymerase is 0.375 units / μL. Figure 2 (C) The optimal concentration of 10mM dNTP solution is 250μM. Figure 2 (D) Under optimal amplification conditions, as the target concentration of the SARS-CoV-2 S13 genome increases, the fluorescence value increases accordingly.

[0070] Example 4

[0071] The target of Example 1 was replaced with the SARS-CoV-2-S12 gene.

[0072] 1 μL 1 μM Track chain (100 nM), 1 uL SARS-CoV-2-S12 target chain (50 nM, 5 nM, 500 pM, 50 pM, 5 pM, 0), and 6.25 μL buffer 2 were added to a centrifuge tube for annealing, cooled from 95 ° C to 25 ° C to form a secondary structure, and then 0.75 μL 8000 units / mL Bst DNA polymerase (0.375 units / μl) and 1 μL 10mM dNTP solution (250 μM) were added, reacted at 37 ° C for 45 min, then heated to 85 ° C and finally cooled to room temperature; then 0.5 μL 20 μM crRNA, 0.5 μL 10 μM Cas12a protein, 7.5 μL buffer 3.1, and 0.5 μL RNAse inhibitor, 1 μL 10 μM reporter (500 nM), the resulting mixture was placed in a real-time fluorescence PCR instrument, reacted at 47°C for 8000 s, and the fluorescence signal was collected.

[0073] The results are as follows Figure 3 , Figure 3 Real-time fluorescence intensity curves and bar graphs of different concentrations.

[0074] Depend on Figure 3 (A) It can be seen that as the concentration of SARS-CoV-2-S12 genome increases, the fluorescence value increases accordingly. Figure 3 (B) It can be seen that there is a significant difference in the fluorescence value of the amplification control and the 5pM SARS-CoV-2-S12 target, so the lowest concentration of target DNA that can be detected is 5pM.

[0075] Example 5

[0076] The target of Example 1 was replaced with the HPV-6b gene, 1 uL HPV-6b target chain (50 nM, 5 nM, 500 pM, 50 pM, 5 pM, 0), and the process of Example 4 was followed to collect the fluorescence signal.

[0077] The results are as follows Figure 4 , Figure 4 Real-time fluorescence intensity curves and bar graphs of different concentrations.

[0078] Depend on Figure 4 (A) As can be seen, as the concentration of HPV-6b genome increases, the fluorescence value increases accordingly. Figure 4 (B) It can be seen that the fluorescence value of the amplification control is significantly different from that of the 5 pM HPV-6b target, so the lowest concentration of target DNA can be detected is 5 pM.

[0079] Example 6

[0080] The target of Example 1 was replaced with the HPV-11b gene, 1 uL HPV-11b target chain (50 nM, 5 nM, 0), and the process of Example 4 was followed to collect the fluorescence signal.

[0081] The results are as follows Figure 5 , Figure 5 Real-time fluorescence intensity curves and bar graphs of different concentrations.

[0082] Depend on Figure 5 (A) As can be seen, as the concentration of HPV-11b genome increases, the fluorescence value increases accordingly. Figure 5 (B) It can be seen that the fluorescence value of the amplification control is significantly different from that of the 5 nM HPV-11b target, so the lowest concentration of target DNA can be detected is 5 nM.

[0083] Example 7

[0084] The target of Example 1 was replaced with the HPV-16b gene, 1 uL HPV-16b target chain (50 nM, 5 nM, 500 pM, 50 pM, 5 pM, 0), and the process of Example 4 was followed to collect the fluorescence signal.

[0085] The results are as follows Figure 6 , Figure 6 Real-time fluorescence intensity curves and bar graphs of different concentrations.

[0086] Depend on Figure 6 (A) As can be seen, as the concentration of HPV-16b genome increases, the fluorescence value increases accordingly. Figure 6 (B) It can be seen that the fluorescence value of the amplification control is significantly different from that of the 5 pM HPV-16b target, so the lowest concentration of target DNA that can be detected is 5 pM.

[0087] Example 8

[0088] The target in Example 1 was replaced with the HPV-31 gene, 1 uL HPV-31 target chain (50 nM, 5 nM, 500 pM, 50 pM, 5 pM, 0), and the process in Example 4 was followed to collect the fluorescence signal.

[0089] The results are as follows Figure 7 , Figure 7 Real-time fluorescence intensity curves and bar graphs of different concentrations.

[0090] Depend on Figure 7 (A) As can be seen, as the concentration of HPV-31 genome increases, the fluorescence value increases accordingly. Figure 7 (B) It can be seen that the fluorescence value of the amplification control is significantly different from that of the 5 pM HPV-31 target, so the lowest concentration of target DNA can be detected is 5 pM.

[0091] Example 9

[0092] The target in Example 1 was replaced with the HPV-33 gene, 1 uL HPV-33 target chain (50 nM, 5 nM, 500 pM, 50 pM, 5 pM, 0), and the process in Example 4 was followed to collect the fluorescence signal.

[0093] The results are as follows Figure 8 , Figure 8 Real-time fluorescence intensity curves and bar graphs of different concentrations.

[0094] Depend on Figure 8 (A) As can be seen, the fluorescence value increases with the increase of HPV-33 genome concentration. Figure 8 (B) It can be seen that the fluorescence value of the amplification control is significantly different from that of the 5 pM HPV-33 target, so the lowest concentration of target DNA can be detected is 5 pM.

[0095] Example 10

[0096] The target in Example 1 was replaced with the HPV-45a gene, 1 uL of HPV-45a target chain (50 nM, 5 nM, 500 pM, 50 pM, 0), and the process in Example 4 was followed to collect the fluorescence signal.

[0097] The results are as follows Fig. 9 , Fig. 9 Real-time fluorescence intensity curves and bar graphs of different concentrations.

[0098] Depend on Fig. 9 (A) As can be seen, as the concentration of HPV-45a genome increases, the fluorescence value increases accordingly. Fig. 9 (B) It can be seen that the fluorescence value of the amplification control is significantly different from that of the 50 pM HPV-45a target, so a minimum concentration of 50 pM of target DNA can be detected.

[0099] Embodiment 11

[0100] The target of Example 1 was replaced with the HPV-52 gene, 1 uL HPV-52 target chain (50 nM, 5 nM, 500 pM, 50 pM, 5 pM, 0), and the process of Example 4 was followed to collect the fluorescence signal.

[0101] The results are as follows Fig.10 , Fig.10 Real-time fluorescence intensity curves and bar graphs of different concentrations.

[0102] Depend on Fig.10 (A) As can be seen, as the concentration of HPV-52 genome increases, the fluorescence value increases accordingly. Fig.10(B) It can be seen that the fluorescence value of the amplification control is significantly different from that of the 5 pM HPV-52 target, so the lowest concentration of target DNA that can be detected is 5 pM.

[0103] Example 12

[0104] The target in Example 1 was replaced with the HPV-58 gene, and the procedure in Example 4 was followed to collect the fluorescence signal.

[0105] The results are as follows Fig.11 , Fig.11 Real-time fluorescence intensity curves and bar graphs of different concentrations.

[0106] Depend on Fig.11 (A) As the concentration of HPV-58 genome increases, the fluorescence value increases accordingly. Fig.11 (B) It can be seen that the fluorescence value of the amplification control is significantly different from that of the 50 pM HPV-58 target, so a minimum concentration of 50 pM of target DNA can be detected.

[0107] Example 13

[0108] The target of Example 1 was replaced with the HCOV-NL63 gene, 1 uL HCOV-NL63 target chain (50 nM, 5 nM, 50 pM, 5 pM, 0), and the process of Example 4 was followed to collect the fluorescence signal.

[0109] The results are as follows Fig.12 , Fig.12 Real-time fluorescence intensity curves and bar graphs of different concentrations.

[0110] Depend on Fig.12 (A) As the concentration of HCOV-NL63 genome increases, the fluorescence value increases accordingly. Fig.12 (B) It can be seen that the fluorescence value of the amplification control is significantly different from that of the 5 pM HCOV-NL63 target, so the lowest concentration of target DNA that can be detected is 5 pM.

[0111] Embodiment 14

[0112] The target of Example 1 was replaced with the HCOV-OC43 gene, 1 uL HCOV-OC43 target chain (50 nM, 5 nM, 500 pM, 0), and the process of Example 4 was followed to collect the fluorescence signal.

[0113] The results are as follows Fig.13 , Fig.13 Real-time fluorescence intensity curves and bar graphs of different concentrations.

[0114] Depend on Fig.13 (A) As the concentration of HCOV-OC43 genome increases, the fluorescence value increases accordingly. Fig.13 (B) It can be seen that the fluorescence value of the amplification control is significantly different from that of the 5 pM HCOV-OC43 target, so the lowest concentration of target DNA that can be detected is 500 pM.

[0115] Embodiment 15

[0116] The target of Example 1 was replaced with the HRSV gene, 1 uL HRSV target chain (50 nM, 5 nM, 500 pM, 0), and the process of Example 4 was followed to collect the fluorescence signal.

[0117] The results are as follows Fig.14 , Fig.14 Real-time fluorescence intensity curves and bar graphs of different concentrations.

[0118] Depend on Fig.14 (A) As the concentration of HRSV genome increases, the fluorescence value increases accordingly. Fig.14 (B) It can be seen that the fluorescence value of the amplification control is significantly different from that of the 5 pM HRSV target, so the lowest concentration of target DNA that can be detected is 500 pM.

[0119] Example 16

[0120] The target in Example 1 was replaced with the HKU1 gene, 1 uL of HKU1 target chain (50 nM, 5 nM, 500 pM, 50 pM, 5 pM, 0), and the process in Example 4 was followed to collect the fluorescence signal.

[0121] The results are as follows Fig.15 , Fig.15 Real-time fluorescence intensity curves and bar graphs of different concentrations.

[0122] Depend on Fig.15 (A) As the concentration of HKU1 genome increases, the fluorescence value increases accordingly. Fig.15 (B) It can be seen that the fluorescence value of the amplification control is significantly different from that of the 5 pM HKU1 target, so the lowest concentration of target DNA can be detected is 5 pM.

[0123] Example 16

[0124] The target in Example 1 was replaced with the miRNA-141 gene.

[0125] 1 uL miRNA-141 target chain (50 nM, 5 nM, 500 pM, 50 pM, 5 pM, 0) was added and the procedure of Example 4 was followed to collect the fluorescence signal.

[0126] The results are as follows Fig.15 , Fig.15 Real-time fluorescence intensity curves and bar graphs of different concentrations.

[0127] Depend on Fig.15 (A) As the concentration of miRNA-141 increases, the fluorescence value increases accordingly. Fig.15 (B) It can be seen that the fluorescence value of the amplification control is significantly different from that of the 5 pM miRNA target, so the target RNA can be detected at a minimum concentration of 5 pM.

[0128] Embodiment 17

[0129] Actual sample testing.

[0130] HPV-16 pseudovirus was selected, and the target nucleic acid sequence fragment was extracted from virus culture, nucleic acid extraction, and PCR amplification. The kit and method in Example 1 were used for detection. The results are as follows: Fig.16 , with good analytical performance.

[0131] As can be seen from the above examples, the present invention uses Cas12a and universal crRNA to develop a simple and sensitive method for detecting DNA and RNA viruses. The innovation of this study is that a variety of DNA and RNA targets can be detected using one crRNA, which greatly reduces the cost and design difficulty of crRNA, is suitable for POCT detection, and develops nucleic acid detection products with good application value.

Claims

1. A kit for multiple nucleic acid detection based on CRISPR / Cas12a combined with universal crRNA, characterized in that: In terms of concentration in the system, it includes 0.5μL20μM crRNA500nM, 0.5μL10μM Cas12a protein250nM, 7.5μL buffer 3.1, 0.5μL RNase inhibitor, 1μL10μM reporter500nM, 6.25μL buffer 2, 1μL1μM Track chain100nM, 0.75μL8000units / mL BstDNA polymerase0.375units / μL and 1μL10mM dNTP solution250μM.

2. The kit according to claim 1, characterized in that The sequence of the crRNA is: UAAUUUCUACUCUUGUAGAUGUAACUAGCAAGAAUACCAC.

3. The kit according to claim 1, characterized in that The sequence of the reporter is: 5'-TexasRed-TGGGATATCTTTAATTTTATTTTAACAAGATATCCC A-BHQ2-3'.

4. The kit according to claim 1, characterized in that The Cas12a protein is a Cpf1 endonuclease.

5. The kit according to claim 1, characterized in that The RNase inhibitor is a recombinant obtained from pig liver RNase inhibitor purified from Escherichia coli.

6. The kit according to claim 1, characterized in that The BstDNA polymerase is prepared from an Escherichia coli strain containing a gene fusion of a Lactobacillus acidophilus DNA polymerase gene lacking a 5′→3′ exonuclease structure and a gene encoding an Escherichia coli maltose binding protein (MBP). The fusion protein is purified to near uniformity, the fused MBP portion is cleaved in vitro, and the remaining polymerase is purified and does not contain MBP.

7. The kit according to claim 1, characterized in that The dNTP solution is an equimolar solution of ultrapure dATP, dCTP, dGTP and dTTP.

Images