Single-temperature single-tube simultaneous isothermal amplification reaction and crisper / cas12a detection reaction target sequence detection system, method and application thereof

Abstract

The application provides a target sequence detection system and a method and application thereof for simultaneously performing isothermal amplification reaction and CRISPR / Cas12a detection reaction of a single temperature single tube, wherein the target sequence comprises a PAM sequence for Cas12a protein recognition; and the Cas12a signal system comprises a crRNA with the following structure: the 5' end repeat sequence of the crRNA is completely consistent with the repeat sequence recognizable by the used Cas12a protein; the length of the 3' end spacer sequence is 18-24 bp; the 1-16 bp of the 3' end spacer sequence of the crRNA has only one mismatched base with the target sequence; and the sequence at other positions of the 3' end spacer sequence is completely complementary to the target sequence. Through different designs of the crRNA, the amplification rate of the isothermal amplification reaction and the high efficient cutting rate of the Cas system are balanced in the same temperature and the same space, so that the one-step method for high-sensitivity detection of nucleic acid at a single temperature is achieved by using the isothermal amplification combined with the full premixed reagent or full freeze-dried reagent of the Cas.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, specifically to a target sequence detection system, method, and application that simultaneously performs isothermal amplification reaction and CRISPR / Cas12a detection reaction in a single tube at a single temperature. Background Technology

[0002] Isothermal nucleic acid amplification has become a new mainstream nucleic acid detection technology after qPCR due to its simplicity (no temperature fluctuation required throughout the process), short amplification time, and low dependence on equipment. Meanwhile, the CRISPR / Cas system, with its targeted binding and trans-cleavage capabilities, has been increasingly used in molecular detection technologies over the past five years, especially Class 2 Cas12 and Cas13. These not only target nucleic acids for cleavage but also activate trans-cleavage activity after cleaving the target nucleic acid, cleaving surrounding single-stranded DNA or RNA. This characteristic is widely used as a signal indication system in current molecular detection technologies because its trans-cleavage has a "one-to-many" characteristic, thus amplifying the signal and improving amplification sensitivity compared to ordinary "one-to-one" nucleic acid probes. The simple and rapid isothermal binding of the Cas system and its signal amplification have become one of the main modes of exploration for simple, rapid, and highly sensitive nucleic acid amplification in recent years.

[0003] Currently, most application systems combine isothermal amplification with the Cas12a system. However, when the isothermal amplification component and the Cas12a signaling system component are in the same system, the efficient cleavage of the Cas12a system will quickly cut off the original template and the amplified template. It is difficult to achieve a balance between the amplification rate of the isothermal amplification reaction and the efficient cleavage rate of the Cas12a system, making it difficult to form effective target amplification and signal accumulation.

[0004] Therefore, in most current applications combining isothermal and Cas12a systems, a two-step method (amplification followed by Cas signaling response) is commonly used. Some of these two-step methods are purely manual, while others achieve this through physical isolation of the isothermal reaction components from the Cas12a reaction components (tube cap isolation, paraffin isolation, glycerol isolation, light-controlled isolation, etc.). These two-step detection modes present problems such as the risk of contamination from opening the cap and numerous operational steps, and they do not achieve the ideal simplified and highly sensitive mode. Furthermore, they fail to address the reaction equilibrium issue of the isothermal reaction and the Cas12a reaction within the same tube.

[0005] In another part of the exploratory applications, although a one-step isothermal binding Cas12a system was developed, operating simultaneously in a single tube at a single temperature, these methods were mostly derived from process optimization within the experimental system. They did not utilize the optimal PAM sequence recognized by the Cas12a protein in the experiment for crRNA design, nor did they address the reaction equilibrium issue between the isothermal reaction of the target sequence containing the optimal PAM sequence and the Cas12a reaction in the same tube during one-step amplification. These one-step detection systems typically require a significant amount of time for adjustment and optimization of each component during system setup, and the amplification time for these methods is generally 30-60 minutes, which is not user-friendly for home use. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a target sequence detection system for simultaneous isothermal amplification and CRISPR / Cas12a detection in a single tube at a single temperature. The target sequence includes a PAM sequence for Cas12a protein recognition, wherein the PAM sequence is “TTTV”, and V is any one of A, C, or G bases. The Cas12a signaling system includes crRNA with the following structure: (1) The 5' repeat region sequence of the crRNA is completely identical to the repeat region sequence recognizable by the Cas12a protein used. When the 3' spacer region sequence is 18-24 bp in length, there is only one mismatched base in the first 1-16 bp of the 3' spacer region sequence of the crRNA with the target sequence, and the sequences at other positions of the 3' spacer region sequence are completely complementary to the target sequence; or (2) The 5' repeat region sequence of the crRNA, except for the loop structure sequence, is completely identical to the repeat region sequence recognizable by the Cas12a protein. The loop structure sequence has one less base than the loop structure sequence recognizable by the Cas12a protein. The first base of the loop structure sequence is the same as the corresponding base of the loop structure recognizable by the Cas12a protein. The second to fourth bases of the loop structure sequence are different from the corresponding bases of the loop structure recognizable by the Cas12a protein. When the 3' spacer region sequence is 18-24 bp in length, the first to fourth bp of the 3' spacer region sequence of the crRNA has only one mismatched base with the target sequence, and the sequences at other positions of the 3' spacer region sequence are completely complementary to the target sequence. Alternatively, the 15-18 bp of the crRNA spacer region sequence has only one mismatched base with the target sequence, and the sequences at other positions of the 3' spacer region sequence are completely complementary to the target sequence.

[0007] In one embodiment, the target sequence is a DNA sequence or an RNA sequence.

[0008] In one embodiment, the isothermal amplification reaction is a RAA isothermal amplification reaction.

[0009] In one embodiment, the above-described detection system is applied to the detection of single nucleotide polymorphisms.

[0010] In one embodiment, a CRISPR / Cas12a detection method based on isothermal amplification is provided, wherein the isothermal amplification reaction and the CRISPR / Cas12a detection reaction are performed simultaneously in a single tube at a single temperature. The target sequence detected by the CRISPR / Cas12a detection reaction includes a PAM sequence for Cas12a protein recognition, wherein the PAM sequence is “TTTV”, and V is any one of A, C, or G bases. The Cas12a signaling system includes crRNA with the following structure: (1) The 5' repeat region sequence of the crRNA is completely identical to the repeat region sequence recognizable by the Cas12a protein used. When the 3' spacer region sequence is 18-24 bp in length, there is only one mismatched base in the first 1-16 bp of the 3' spacer region sequence of the crRNA with the target sequence, and the sequences at other positions of the 3' spacer region sequence are completely complementary to the target sequence; or (2) The 5' repeat region sequence of the crRNA, except for the loop structure sequence, is completely identical to the repeat region sequence recognizable by the Cas12a protein. The loop structure sequence has one less base than the loop structure sequence recognizable by the Cas12a protein. The first base of the loop structure sequence is the same as the corresponding base of the loop structure recognizable by the Cas12a protein. The second to fourth bases of the loop structure sequence are different from the corresponding bases of the loop structure recognizable by the Cas12a protein. When the 3' spacer region sequence is 18-24 bp in length, the first to fourth bp of the 3' spacer region sequence of the crRNA has only one mismatched base with the target sequence, and the sequences at other positions of the 3' spacer region sequence are completely complementary to the target sequence. Alternatively, the 15-18 bp of the crRNA spacer region sequence has only one mismatched base with the target sequence, and the sequences at other positions of the 3' spacer region sequence are completely complementary to the target sequence.

[0011] In the Cas12a signaling system, the Cas12a protein, crRNA, and target sequence must bind to form an RNP complex before the system can cleave the target sequence. Therefore, the binding efficiency of these three components affects the cleavage efficiency of the Cas12a system. crRNA is crucial for RNP complex formation; altering its sequence can affect the binding efficiency of the RNP complex, thus further impacting the cleavage efficiency of the Cas12a system. The crRNA consists of two regions: 1) a 5' repeat region, which is fixed for a specific Cas12a protein, and the main difference between different Cas12a proteins lies in the "loop region" within this sequence. The repeat region determines the mutual recognition and binding between the crRNA and the chosen Cas12a protein; 2) a 3' spacer region, which is identical to the target sequence. The spacer region determines whether the crRNA can successfully bind to the target sequence. Therefore, by designing different sequences for the 5' repeat region and the 3' spacer region of crRNA, we can reduce the RNP complex formation rate, thereby reducing the cleavage efficiency of Cas12a protein.

[0012] Therefore, in order to solve the problems of "balancing the amplification rate of isothermal amplification reaction with the efficient cleavage rate of the Cas system in the same temperature and space" and "being able to identify the optimal PAM sequence based on the Cas protein", this invention, from the direction of reducing the efficient cleavage rate of the Cas12a system, targets the optimal PaM sequence and achieves a balance between the amplification rate of isothermal amplification reaction and the efficient cleavage rate of the Cas system by designing different crRNAs. This results in a "one-step method that, based on the optimal PAM sequence, achieves high sensitivity detection of nucleic acids at a single temperature within 30 minutes using fully premixed or fully lyophilized reagents that are isothermally amplified and bound to Cas within a single temperature, through simple and diverse rapid design".

[0013] Compared with the existing technology, the present invention has the following advantages: 1) In the Cas12a signaling system components, different crRNA designs can be made based on the optimal PAM sequence "TTTV" format. This allows the optimal PAM "TTTV" position in the target sequence to be designed and applied in a one-step method, achieving equilibrium between the isothermal reaction and the Cas12a signaling system reaction in the one-step method. This results in a detection system where the isothermal amplification reaction and the Cas signaling reaction occur simultaneously in the same physical space and at the same temperature. Overall, this increases the number of design positions for the "one-step detection system of isothermal reaction and Cas12a signaling reaction" targeting the target sequence, making the design and establishment of the detection system easier and faster.

[0014] 2) The operation steps of detection systems such as RAA isothermal reaction combined with Cas signal reaction have been simplified from the original four steps of "isothermal reagent - template addition - isothermal reaction - Cas reaction" or "mixing isothermal reagent and Cas reagent - template addition - isothermal reaction - Cas reaction" to two steps of "adding "template and magnesium ion mixture" to premixed solution or pre-lyophilized reagent - instrument detection", which greatly simplifies the operation.

[0015] 3) The reaction time of the isothermal amplification reaction combined with the Cas signaling system has been shortened from the original 30-120 min to 30 min. These improvements greatly increase the possibility of the detection system in this invention being used for home testing in the field of subsequent nucleic acid detection. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram of the crRNA structure. Detailed Implementation

[0018] To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this application. Example 1

[0019] Based on the optimal PAM sequence TTTV of Cas12a, when the 5' repeat region sequence of the crRNA is completely consistent with the repeat region sequence recognized by the LbCas12a protein and the 3' spacer region sequence length is 18-24 bp, the amplification results of different designed crRNAs in the detection system of this invention are shown below.

[0020] I. Design of Amplification Primers and Reporter Probes We selected a conserved region of the B7R gene of monkeypox virus and designed RAA amplification primers and reporter probes (Seq no.1-Seq no.3, Table 1) to achieve RAA isothermal amplification of the target region fragment and indication of the amplification signal.

[0021] Table 1. Information on RAA amplification primers and reporter probe sequences for the B7R target region in the Cas reaction. SEQ ID NO: 1 MPV-RPA-F TATACAGGCTATTTACAAGATGCACCGTTA SEQ ID NO: 2 MPV-RPA-R CTCCAGTGATCGTACCAATAGTAGTTAGAG SEQ ID NO: 3 Cas-RP 5’FAM-TTATT-3’BHQ1 II. Within the aforementioned RAA amplification region, different crRNAs were designed based on the optimal PAM sequence "TTTV" of Cas12a. The crRNA structures are shown below. Figure 1 As shown, the specific different designs and test results are described below.

[0022] 1. When the length of the 3' end spacer region sequence is 18 bp, the base sequence of the spacer region varies, specifically designed as MPV-crRNA-O1-O24 (Seq no. 4-27, Table 2). The single-tube, single-temperature, one-step detection reaction system formulation and reaction procedure of this invention are shown in Table 3. The recombinant plasmid dilution of the monkeypox B7R gene was detected, and the results are shown in Table 4.

[0023] Table 2. Different designs where the repeat region sequence is completely identical to the sequence identified by LbCas12 and the spacer region sequence length is 18 bp. SEQ ID NO: 4 MPV-crRNA-O1 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGC]]> SEQ ID NO: 5 MPV-crRNA-O2 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGC]]> SEQ ID NO: 6 MPV-crRNA-O3 <![CDATA[UAAUUUCUAC UAAGU GUAGAUAGACGUGAUUCAUUGGC]]> SEQ ID NO: 7 MPV-crRNA-O4 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACGCGUGAUUCAUUUGGC]]> SEQ ID NO: 8 MPV-crRNA-O5 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACAAGUGAUUCAUUUGGC]]> SEQ ID NO: 9 MPV-crRNA-O6 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACCUGAUUCAUUUGGC]]> SEQ ID NO: 10 MPV-crRNA-O7 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGAGAUUCAUUUGGC]]> SEQ ID NO: 11 MPV-crRNA-O8 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUCAUUCAUUUGGC]]> SEQ ID NO: 12 MPV-crRNA-O9 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGUUUCAUUGGC]]> SEQ ID NO: 13 MPV-crRNA-O10 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAAUCAUUUGGC]]> SEQ ID NO: 14 MPV-crRNA-O11 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUGCAUUUGGC]]> SEQ ID NO: 15 MPV-crRNA-O12 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUGAUUGGC]]> SEQ ID NO: 16 MPV-crRNA-O13 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCCUUUGGC]]> SEQ ID NO: 17 MPV-crRNA-O14 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAGUUGGC]]> SEQ ID NO: 18 MPV-crRNA-O15 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUAUGGC]]> SEQ ID NO: 19 MPV-crRNA-O16 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUAGGC]]> SEQ ID NO: 20 MPV-crRNA-O17 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUCGC]]> SEQ ID NO: 21 MPV-crRNA-O18 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGAC]]> SEQ ID NO: 22 MPV-crRNA-O19 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGG]]> SEQ ID NO: 23 MPV-crRNA-O20 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCAGGUGAUUCAUUGGC]]> SEQ ID NO: 24 MPV-crRNA-O21 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUCAUUCAUUGGC<!-- 4 --> ]]> SEQ ID NO: 25 MPV-crRNA-O22 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUAUGGC]]> SEQ ID NO: 26 MPV-crRNA-O23 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGAC]]> SEQ ID NO: 27 MPV-crRNA-O24 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUUGGG]]> Note: Underlined bases indicate cyclic sequences, and red indicates mismatched bases in spacer regions.

[0024] Table 3. Basic formulation and reaction procedure of the single-tube, single-temperature, one-step DNA detection reaction system in this invention. .

[0025] Table 4. Detection results of different designed crRNAs with repeat region sequences completely identical to those identified by LbCas12 and a spacer region sequence length of 18 bp. .

[0026] From Table 4, no positive amplification was formed under the following conditions: (1) when the crRNA spacer sequence completely matched the target sequence (MPV-crRNA-O1); (2) when the 17th and 18th bases of the spacer sequence had one mismatch with the target sequence and the other positions matched the target sequence (MPV-crRNA-O18, O19); (3) when there were two mismatches in the 1-16bp of the crRNA spacer sequence and the other positions matched the target sequence (MPV-crRNA-O20-22); and (4) when there was one mismatch in the 1-16bp of the crRNA spacer sequence and one mismatch in the 17-18bp of the target sequence (MPV-crRNA-O23, O24). No positive amplification was formed in any of these cases. Positive amplification was only formed when there was one mismatch in the 1-16bp of the crRNA spacer sequence and the other positions matched the target sequence (MPV-crRNA-O2-O17), and the amplification sensitivity was up to 10 copies / µL.

[0027] 2. When the length of the 3' end spacer region sequence is 19 bp, the base sequence of the spacer region varies, specifically designed as MPV-crRNA-O25-O50 (Seq no. 28-53, Table 5). The single-tube, single-temperature, one-step detection reaction system formulation and reaction procedure of this invention are shown in Table 3. The recombinant plasmid dilution of the monkeypox B7R gene was detected, and the results are shown in Table 6.

[0028] Table 5. Different designs where the repeat region sequence is completely identical to the sequence identified by LbCas12 and the spacer region sequence length is 19 bp. SEQ ID NO: 28 MPV-crRNA-O25 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGCA]]> SEQ ID NO: 29 MPV-crRNA-O26 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCA]]> SEQ ID NO: 30 MPV-crRNA-O27 <![CDATA[UAAUUUCUAC UAAGU GUAGAUAGACGUGAUUCAUUGGCA]]> SEQ ID NO: 31 MPV-crRNA-O28 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACGCGUGAUUCAUUUGGCA]]> SEQ ID NO: 32 MPV-crRNA-O29 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACAAGUGAUUCAUUGGCA]]> SEQ ID NO: 33 MPV-crRNA-O30 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACCUGAUUCAUUUGGCA]]> SEQ ID NO: 34 MPV-crRNA-O31 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGAGAUUCAUUUGGCA]]> SEQ ID NO: 35 MPV-crRNA-O32 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUCAUUCAUUUGGCA]]> SEQ ID NO: 36 MPV-crRNA-O33 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGUUUCAUUGGCA]]> Seq no.37 MPV-crRNA-O34 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAAUCAUUUGGCA]]> Seq no.38 MPV-crRNA-O35 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUGCAUUUGGCA]]> Seq no.39 MPV-crRNA-O36 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUGAUUUGGCA]]> Seq no.40 MPV-crRNA-O37 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCCUUUGGCA]]> Seq no.41 MPV-crRNA-O38 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACAGUGAUCAGUUGGCA]]> Seq no.42 MPV-crRNA-O39 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUAUGGCA]]> Seq no.43 MPV-crRNA-O40 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUAGGCA]]> Seq no.44 MPV-crRNA-O41 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUCGCA]]> Seq no.45 MPV-crRNA-O42 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUGACA]]> Seq no.46 MPV-crRNA-O43 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGGA]]> Seq no.47 MPV-crRNA-O44 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGCU]]> Seq no.48 MPV-crRNA-O45 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCAGGUGAUUCAUUGGCA]]> Seq no.49 MPV-crRNA-O46 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUCAUUCAUUGGCA]]> Seq no.50 MPV-crRNA-O47 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUAUGGCA]]> Seq no.51 MPV-crRNA-O48 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGACA]]> Seq no.52 MPV-crRNA-O49 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUUGGG]]> Seq no.53 MPV-crRNA-O50 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCU]]> .

[0029] Table 6. Detection results of different designed crRNAs with repeat region sequences completely identical to those identified by LbCas12 and spacer region sequences of 19 bp. .

[0030] From Table 6, we can see that no positive amplification was formed under the following conditions: (1) when the crRNA spacer sequence completely matched the target sequence (MPV-crRNA-O25); (2) when there was one mismatch between the 17th and 19th bases of the spacer sequence and the target sequence, and the other positions matched the target sequence (MPV-crRNA-O42-44); (3) when there were two mismatches between the 1st and 16th bp of the crRNA spacer sequence and the other positions matched the target sequence (MPV-crRNA-O45-47); (4) when there was one mismatch between the 1st and 16th bp of the crRNA spacer sequence and one mismatch between the 17th and 19th bp of the target sequence (MPV-crRNA-O48-50). Only when there was one mismatch between the 1st and 16th bp of the crRNA spacer sequence and the other positions matched the target sequence (MPV-crRNA-O26-O41) was a positive amplification formed, and the amplification sensitivity was up to 10 copies / µL.

[0031] 3. When the length of the terminal spacer region sequence is 20 bp, the base sequence of the spacer region is different for each region. The specific design is MPV-crRNA-O51-O78 (Seq no. 54-81, Table 7). The single-tube, single-temperature, one-step detection reaction system formulation and reaction procedure of this invention are shown in Table 3. The recombinant plasmid dilution of the monkeypox B7R gene was detected, and the results are shown in Table 8.

[0032] Table 7. Different designs where the repeat region sequence is completely identical to the sequence identified by LbCas12 and the spacer region sequence length is 20 bp. Seq no.54 MPV-crRNA-O51 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGCAU]]> Seq no.55 MPV-crRNA-O52 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCAU]]> Seq no.56 MPV-crRNA-O53 <![CDATA[UAAUUUCUAC UAAGU GUAGAUAGACGUGAUCAUUUGGCAU]]> Seq no.57 MPV-crRNA-O54 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACGCGUGAUUCAUUUGGCAU]]> Seq no.58 MPV-crRNA-O55 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACAAGUGAUUCAUUUGGCAU]]> Seq no.59 MPV-crRNA-O56 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACCUGAUUCAUUUGGCAU]]> Seq no.60 MPV-crRNA-O57 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGAGAUUCAUUUGGCAU]]> Seq no.61 MPV-crRNA-O58 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUCAUUCAUUUGGCAU]]> Seq no.62 MPV-crRNA-O59 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGUUUCAUUGGCAU]]> Seq no.63 MPV-crRNA-O60 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAAUCAUUUGGCAU]]> Seq no.64 MPV-crRNA-O61 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUGCAUUUGGCAU]]> Seq no.65 MPV-crRNA-O62 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUGAUUUGGCAU]]> Seq no.66 MPV-crRNA-O63 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCCUUUGGCAU]]> Seq no.67 MPV-crRNA-O64 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAGUUGGCAU]]> Seq no.68 MPV-crRNA-O65 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUAUGGCAU]]> Seq no.69 MPV-crRNA-O66 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUAGGCAU]]> Seq no.70 MPV-crRNA-O67 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUCAUUUCGCAU]]> Seq no.71 MPV-crRNA-O68 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGACAU]]> Seq no.72 MPV-crRNA-O69 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGGAU]]> Seq no.73 MPV-crRNA-O70 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUGGCUU]]> Seq no.74 MPV-crRNA-O71 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGCAC]]> Seq no.75 MPV-crRNA-O72 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCAGGUGAUUCAUUGGCAU]]> Seq no.76 MPV-crRNA-O73 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUCAUUCAUUGGCAU]]> Seq no.77 MPV-crRNA-O74 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUAUGGCAU]]> Seq no.78 MPV-crRNA-O75 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGACAU]]> Seq no.79 MPV-crRNA-O76 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUUGGGAU]]> Seq no.80 MPV-crRNA-O77 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCUU<!-- 9 --> ]]> Seq no.81 MPV-crRNA-O78 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCAC]]> .

[0033] Table 8. Detection results of different designed crRNAs with repeat region sequences completely identical to those identified by LbCas12 and spacer region sequences of 20 bp. .

[0034] From Table 8, we can see that no positive amplification was formed in the following situations: (1) when the crRNA spacer sequence completely matched the target sequence (MPV-crRNA-O51); (2) when there was one mismatch between the 17th and 20th bases of the spacer sequence and the target sequence, and the other positions matched the target sequence (MPV-crRNA-O68-71); (3) when there were two mismatches between the 1st and 16th bp of the crRNA spacer sequence and the other positions matched the target sequence (MPV-crRNA-O72-74); (4) when there was one mismatch between the 1st and 16th bp of the crRNA spacer sequence and the target sequence between the 17th and 20th bp (MPV-crRNA-O75-78). Positive amplification was only formed when there was one mismatch between the 1st and 16th bp of the crRNA spacer sequence and the other positions matched the target sequence (MPV-crRNA-O52-O67), and the amplification sensitivity was up to 10 copies / µL.

[0035] 4. When the length of the 3' end spacer region sequence is 21 bp, the base sequence of the spacer region varies, specifically designed as MPV-crRNA-O79-O108 (Seq no. 82-111, Table 9). The single-tube, single-temperature, one-step detection reaction system formulation and reaction procedure of this invention are shown in Table 3. The recombinant plasmid dilution of the monkeypox B7R gene was detected, and the results are shown in Table 10.

[0036] Table 9. Different designs where the repeat region sequence is completely identical to the sequence identified by LbCas12 and the spacer region sequence length is 21 bp. Seq no.82 MPV-crRNA-O79 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGCAUA]]> Seq no.83 MPV-crRNA-O80 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCAUA]]> Seq no.84 MPV-crRNA-O81 <![CDATA[UAAUUUCUAC UAAGU GUAGAUAGACGUGAUUCAUUGGCAUA]]> Seq no.85 MPV-crRNA-O82 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACGCGUGAUUCAUUUGGCAUA]]> Seq no.86 MPV-crRNA-O83 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACAAGUGAUUCAUUUGGCAUA]]> Seq no.87 MPV-crRNA-O84 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACCUGAUUCAUUUGGCAUA]]> Seq no.88 MPV-crRNA-O85 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGAGAUUCAUUUGGCAUA]]> Seq no.89 MPV-crRNA-O86 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUCAUUCAUUUGGCAUA]]> Seq no.90 MPV-crRNA-O87 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGUUUCAUUGGCAUA]]> Seq no.91 MPV-crRNA-O88 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAAUCAUUUGGCAUA]]> Seq no.92 MPV-crRNA-O89 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUGCAUUUGGCAUA]]> Seq no.93 MPV-crRNA-O90 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUGAUUUGGCAUA]]> Seq no.94 MPV-crRNA-O91 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCCUUUGGCAUA]]> Seq no.95 MPV-crRNA-O92 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACAGUGAUCAGUUGGCAUA]]> Seq no.96 MPV-crRNA-O93 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUAUGGCAUA]]> Seq no.97 MPV-crRNA-O94 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUAGGCAUA]]> Seq no.98 MPV-crRNA-O95 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUCGCAUA]]> Seq no.99 MPV-crRNA-O96 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGACAUA]]> Seq no.100 MPV-crRNA-O97 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGGAUA]]> Seq no.101 MPV-crRNA-O98 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACAGUGAUUCAUUGGCUUA]]> Seq no.102 MPV-crRNA-O99 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGCACA]]> Seq no.103 MPV-crRNA-O100 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUGGCAUU]]> Seq no.104 MPV-crRNA-O101 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCAGGUGAUUCAUUGGCAUA]]> Seq no.105 MPV-crRNA-O102 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUCAUUCAUUUGGCAUA]]> Seq no.106 MPV-crRNA-O103 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUAUGGCAUA]]> Seq no.107 MPV-crRNA-O104 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGACAUA]]> Seq no.108 MPV-crRNA-O105 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGGAUA]]> Seq no.109 MPV-crRNA-O106 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCUUA]]> Seq no.110 MPV-crRNA-O107 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCACA<!-- 12 --> ]]> Seq no.111 MPV-crRNA-O108 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCAUU]]> .

[0037] Table 10. Detection results of different designed crRNAs with repeat region sequences completely identical to those identified by LbCas12 and a spacer region sequence length of 21 bp. .

[0038] From Table 10, we can see that no positive amplification occurred in the following situations: (1) when the crRNA spacer sequence completely matched the target sequence (MPV-crRNA-O79); (2) when there was one mismatch between the 17th and 21st bases of the spacer sequence and the target sequence, and the other positions matched the target sequence (MPV-crRNA-O96-100); (3) when there were two mismatches between the 1st and 16th bp of the crRNA spacer sequence and the other positions matched the target sequence (MPV-crRNA-O101-103); (4) when there was one mismatch between the 1st and 16th bp of the crRNA spacer sequence and one mismatch between the 17th and 21st bp of the target sequence (MPV-crRNA-O104-108). Only when there was one mismatch between the 1st and 16th bp of the crRNA spacer sequence and the other positions matched the target sequence (MPV-crRNA-O80-O95) did a positive amplification occur, and the amplification sensitivity was up to 10 copies / uL.

[0039] 5. When the length of the 3' end spacer region sequence is 22 bp, the base sequence of the spacer region varies, specifically designed as MPV-crRNA-O109-O140 (Seq no. 112-143, Table 11). The single-tube, single-temperature, one-step detection reaction system formulation and reaction procedure of this invention are shown in Table 3. The recombinant plasmid dilution of the monkeypox B7R gene was detected, and the results are shown in Table 12.

[0040] Table 11. Different designs where the repeat region sequence is completely identical to the sequence identified by LbCas12 and the spacer region sequence length is 22 bp. Seq no.112 MPV-crRNA-O109 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGCAUAG]]> Seq no.113 MPV-crRNA-O110 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCAUAG]]> Seq no.114 MPV-crRNA-O111 <![CDATA[UAAUUUCUAC UAAGU GUAGAUAGACGUGAUUCAUUGGCAUAG]]> Seq no.115 MPV-crRNA-O112 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACGCGUGAUUCAUUUGGCAUAG]]> Seq no.116 MPV-crRNA-O113 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACAAGUGAUUCAUUUGGCAUAG]]> Seq no.117 MPV-crRNA-O114 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACCUGAUUCAUUUGGCAUAG]]> Seq no.118 MPV-crRNA-O115 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGAGAUUCAUUUGGCAUAG]]> Seq no.119 MPV-crRNA-O116 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUCAUUCAUUUGGCAUAG]]> Seq no.120 MPV-crRNA-O117 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGUUUCAUUGGCAUAG]]> Seq no.121 MPV-crRNA-O118 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAAUCAUUUGGCAUAG]]> Seq no.122 MPV-crRNA-O119 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUGCAUUUGGCAUAG]]> Seq no.123 MPV-crRNA-O120 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUGAUUUGGCAUAG]]> Seq no.124 MPV-crRNA-O121 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCCUUUGGCAUAG]]> Seq no.125 MPV-crRNA-O122 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACAGUGAUUCAGUUGGCAUAG]]> Seq no.126 MPV-crRNA-O123 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUAUGGCAUAG]]> Seq no.127 MPV-crRNA-O124 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUAGGCAUAG]]> Seq no.128 MPV-crRNA-O125 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUCGCAUAG]]> Seq no.129 MPV-crRNA-O126 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUGACAUAG]]> Seq no.130 MPV-crRNA-O127 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGGAUAG]]> Seq no.131 MPV-crRNA-O128 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUGGCUUAG]]> Seq no.132 MPV-crRNA-O129 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGCACAG]]> Seq no.133 MPV-crRNA-O130 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUGGCAUUG]]> Seq no.134 MPV-crRNA-O131 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGCAUAC]]> Seq no.135 MPV-crRNA-O132 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCAGGUGAUUCAUUGGCAUAG]]> Seq no.136 MPV-crRNA-O133 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUCAUUCAUUUGGCAUAG]]> Seq no.137 MPV-crRNA-O134 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUAUGGCAUAG]]> Seq no.138 MPV-crRNA-O135 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGACAUAG]]> Seq no.139 MPV-crRNA-O136 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUUGGGAUAG]]> Seq no.140 MPV-crRNA-O137 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCUUAG<!-- 15 --> ]]> Seq no.141 MPV-crRNA-O138 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCACAG]]> Seq no.142 MPV-crRNA-O139 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCAUUG]]> Seq no.143 MPV-crRNA-O140 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCAUAC]]> .

[0041] Table 12. Detection results of different designed crRNAs with repeat region sequences completely identical to those identified by LbCas12 and spacer region sequences of 22 bp. .

[0042] From Table 12, we can see that no positive amplification occurred in the following situations: (1) when the crRNA spacer sequence completely matches the target sequence (MPV-crRNA-O109); (2) when there is one mismatch between the 17th and 22nd bases of the spacer sequence and the target sequence, and the other positions match the target sequence (MPV-crRNA-O126-131); (3) when there are two mismatches between the 1st and 16th bp of the crRNA spacer sequence and the other positions match the target sequence (MPV-crRNA-O132-134); (4) when there is one mismatch between the 1st and 16th bp of the crRNA spacer sequence and one mismatch between the 17th and 22nd bp of the target sequence (MPV-crRNA-O135-140). Positive amplification is only achieved when there is one mismatch in the first 16 bp of the crRNA spacer sequence and the other positions match the target sequence (MPV-crRNA-O110-O125), and the amplification sensitivity can reach 10 copies / uL.

[0043] 6. When the length of the 3' end spacer region sequence is 23 bp, the base sequence of the spacer region varies, specifically designed as MPV-crRNA-O141-O174 (Seq no. 144-177, Table 13). The single-tube, single-temperature, one-step detection reaction system formulation and reaction procedure of this invention are shown in Table 3. The recombinant plasmid dilution of the monkeypox B7R gene was detected, and the results are shown in Table 14.

[0044] Table 13. Different designs where the repeat region sequence is completely identical to the sequence identified by LbCas12 and the spacer region sequence length is 23 bp. Seq no.144 MPV-crRNA-O141 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUGGCAUAGU]]> Seq no.145 MPV-crRNA-O142 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCAUAGU]]> Seq no.146 MPV-crRNA-O143 <![CDATA[UAAUUUCUAC UAAGU GUAGAUAGACGUGAUUCAUUGGCAUAGU]]> Seq no.147 MPV-crRNA-O144 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACGCGUGUAUUCAUUUGGCAUAGU]]> Seq no.148 MPV-crRNA-O145 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACAAGUGAUUCAUUUGGCAUAGU]]> Seq no.149 MPV-crRNA-O146 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACCUGAUUCAUUUGGCAUAGU]]> Seq no.150 MPV-crRNA-O147 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGAGAUUCAUUUGGCAUAGU]]> Seq no.151 MPV-crRNA-O148 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUCAUUCAUUUGGCAUAGU]]> Seq no.152 MPV-crRNA-O149 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACAGGUUUUCAUUGGCAUAGU]]> Seq no.153 MPV-crRNA-O150 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAAUCAUUUGGCAUAGU]]> Seq no.154 MPV-crRNA-O151 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUGCAUUUGGCAUAGU]]> Seq no.155 MPV-crRNA-O152 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUGAUUUGGCAUAGU]]> Seq no.156 MPV-crRNA-O153 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACAGUGAUUCCUUUGGCAUAUGU]]> Seq no.157 MPV-crRNA-O154 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACAGUGAUUCAGUGGCAUAGU]]> Seq no.158 MPV-crRNA-O155 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUAUGGCAUAGU]]> Seq no.159 MPV-crRNA-O156 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUAGGCAUAGU]]> Seq no.160 MPV-crRNA-O157 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUCGCAUAGU]]> Seq no.161 MPV-crRNA-O158 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACAGUGAUUCAUUGACAUAGU]]> Seq no.162 MPV-crRNA-O159 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGGAUAGU]]> Seq no.163 MPV-crRNA-O160 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACAGUGAUUCAUUGGCUUAGU]]> Seq no.164 MPV-crRNA-O161 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUGGCACAG]]> Seq no.165 MPV-crRNA-O162 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUGU]]> Seq no.166 MPV-crRNA-O163 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGCAUACU]]> Seq no.167 MPV-crRNA-O164 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUGGCAUAGA]]> Seq no.168 MPV-crRNA-O165 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCAGGUGAUUCAUUGGCAUAGU]]> Seq no.169 MPV-crRNA-O166 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUCAUUCAUUUGGCAUAGU]]> Seq no.170 MPV-crRNA-O167 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUAUGGCAUAGU]]> Seq no.171 MPV-crRNA-O168 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGACAUAGU]]> Seq no.172 MPV-crRNA-O169 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGGAUAGU<!-- 18 --> ]]> Seq no.173 MPV-crRNA-O170 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCUUAGU]]> Seq no.174 MPV-crRNA-O171 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCACAG]]> Seq no.175 MPV-crRNA-O172 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGUG]]> Seq no.176 MPV-crRNA-O173 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCAUAU]]> Seq no.177 MPV-crRNA-O174 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCAUAGA]]> .

[0045] Table 14. Detection results of different designed crRNAs with repeat region sequences completely identical to those identified by LbCas12 and a spacer region sequence length of 23 bp.

[0046] From Table 14, we can see that the following situations did not result in positive amplification: (1) when the crRNA spacer sequence completely matches the target sequence (MPV-crRNA-O141); (2) when there is one mismatch between the 17th and 23rd bases of the spacer sequence and the target sequence, and the other positions match the target sequence (MPV-crRNA-O158-164); (3) when there are two mismatches between the 1st and 16th bp of the crRNA spacer sequence and the other positions match the target sequence (MPV-crRNA-O165-167); (4) when there is one mismatch between the 1st and 16th bp of the crRNA spacer sequence and one mismatch between the 17th and 23rd bp of the target sequence (MPV-crRNA-O168-174). Positive amplification is only achieved when there is one mismatch in the first 16 bp of the crRNA spacer sequence and the other positions match the target sequence (MPV-crRNA-O142-O157), and the amplification sensitivity can reach 10 copies / uL.

[0047] 7. When the length of the 3' end spacer region sequence is 24 bp, the base sequence of the spacer region varies, specifically designed as MPV-crRNA-O175-O210 (Seq no. 178-213, Table 15). The single-tube, single-temperature, one-step detection reaction system formulation and reaction procedure of this invention are shown in Table 3. The recombinant plasmid dilution of the monkeypox B7R gene was detected, and the results are shown in Table 16.

[0048] Table 15. Different designs where the repeat region sequence is completely identical to the sequence identified by LbCas12 and the spacer region sequence length is 24 bp. Seq no.178 MPV-crRNA-O175 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGCAUAGUA]]> Seq no.179 MPV-crRNA-O176 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCAUAGUA]]> Seq no.180 MPV-crRNA-O177 <![CDATA[UAAUUUCUAC UAAGU GUAGAUAGACGUGAUUCAUUUGGCAUAGUA]]> Seq no.181 MPV-crRNA-O178 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACGCGUGAUUCAUUUGGCAUAGUA]]> Seq no.182 MPV-crRNA-O179 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACAAGUGAUUCAUUUGGCAUAGUA]]> Seq no.183 MPV-crRNA-O180 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACCUGAUUCAUUUGGCAUAGUA]]> Seq no.184 MPV-crRNA-O181 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGAGAUUCAUUUGGCAUAGUA]]> Seq no.185 MPV-crRNA-O182 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUCAUUCAUUUGGCAUAGUA]]> Seq no.186 MPV-crRNA-O183 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGUUUCAUUUGGCAUAGUA]]> Seq no.187 MPV-crRNA-O184 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAAUCAUUUGGCAUAGUA]]> Seq no.188 MPV-crRNA-O185 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUGCAUUUGGCAUAGUA]]> Seq no.189 MPV-crRNA-O186 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUGAUUUGGCAUAGUA]]> Seq no.190 MPV-crRNA-O187 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCCUUUGGCAUAGUA]]> Seq no.191 MPV-crRNA-O188 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAGUUGGCAUAGUA]]> Seq no.192 MPV-crRNA-O189 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUAUGGCAUAGUA]]> Seq no.193 MPV-crRNA-O190 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUAGGCAUAGUA]]> Seq no.194 MPV-crRNA-O191 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUCGCAUAGUA]]> Seq no.195 MPV-crRNA-O192 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGACAUAGUA]]> Seq no.196 MPV-crRNA-O193 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGGAUAGUA]]> Seq no.197 MPV-crRNA-O194 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGCUUAGUA]]> Seq no.198 MPV-crRNA-O195 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGCACAGUA]]> Seq no.199 MPV-crRNA-O196 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUGGCAUUGUA]]> Seq no.200 MPV-crRNA-O197 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUUGGCAUACUA]]> Seq no.201 MPV-crRNA-O198 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUGGCAUAGAA]]> Seq no.202 MPV-crRNA-O199 <![CDATA[UAAUUUCUAC UAAGU GUAGAUACACGUGAUUCAUUGGCAUAGUU]]> Seq no.203 MPV-crRNA-O200 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCAGGUGAUUCAUUGGCAUAGUA]]> Seq no.204 MPV-crRNA-O201 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUCAUUCAUUUGGCAUAGUA]]> Seq no.205 MPV-crRNA-O202 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUAUGGCAUAGUA]]> Seq no.206 MPV-crRNA-O203 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGAAGUA<!-- 21 --> ]]> Seq no.207 MPV-crRNA-O204 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGGAUAGUA]]> Seq no.208 MPV-crRNA-O205 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCUUAGUA]]> Seq no.209 MPV-crRNA-O206 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCACAGUA]]> Seq no.210 MPV-crRNA-O207 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCAUUGUA]]> Seq no.211 MPV-crRNA-O208 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCAUACUA]]> Seq no.212 MPV-crRNA-O209 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCAUAGAA]]> Seq no. 213 MPV-crRNA-O210 <![CDATA[UAAUUUCUAC UAAGU GUAGAUUCACGUGAUUCAUUGGCAUAGUU]]> .

[0049] Table 16. Detection results of different designed crRNAs with repeat region sequences completely identical to those identified by LbCas12 and a spacer region sequence length of 24 bp.

[0050] From Table 16, we can see that no positive amplification occurred in the following situations: (1) when the crRNA spacer sequence completely matched the target sequence (MPV-crRNA-O175); (2) when there was one mismatch between the 17th and 24th bases of the spacer sequence and the target sequence, and the other positions matched the target sequence (MPV-crRNA-O192-199); (3) when there were two mismatches between the 1st and 16th bp of the crRNA spacer sequence and the other positions matched the target sequence (MPV-crRNA-O200-202); (4) when there was one mismatch between the 1st and 16th bp of the crRNA spacer sequence and one mismatch between the 17th and 24th bp of the target sequence (MPV-crRNA-O203-210). Only when there was one mismatch between the 1st and 16th bp of the crRNA spacer sequence and the other positions matched the target sequence (MPV-crRNA-O176-O191) did a positive amplification occur, and the amplification sensitivity was up to 10 copies / uL.

[0051] Example 2 Based on the optimal PAM sequence TTTV format of Cas12a, when the sequences other than the loop structure in the repeat region at the 5' end of the crRNA are consistent with the repeat region sequence recognized by the LbCas12a protein, but the loop structure sequence in the repeat region is one base shorter than the loop structure sequence in the repeat region recognized by the LbCas12a protein, and the base sequence from the 2nd to the 4th position is completely inconsistent, and the spacer sequence of the crRNA is 18-24 bp, the amplification results of different designed crRNAs in the detection system of this invention are shown below.

[0052] Amplification primers were designed by selecting conserved regions of the M gene of influenza A virus and designing RAA amplification primers (Seqno. 214, 215, Table 17) to achieve RAA isothermal amplification of the target region fragment.

[0053] Table 17. Primer sequence information for RAA amplification of the target region of the H1N1 influenza virus M gene. Seq no. 213 IFA-RPA-F CGTCCCATCAGGCCCTCTCAAAGCCGAAAT Seq no. 214 IFA-RPA-R CATGTTATTTGGATCCCCATTCCCATTGAGG .

[0054] 2.2 Within the aforementioned RAA amplification region, different crRNAs were designed based on the optimal PAM sequence "TTTV" of Cas12a. The crRNA structures are shown in Example 1. Figure 1 As shown in Table 18, the comparison between the repeat region sequence of crRNA and the repeat region sequence recognizable by the LbCas12a protein used in the detection system in this embodiment is shown below.

[0055] Table 18. Sequence information showing the consistency between the repeat region sequence of crRNA and the repeat region recognized by the LbCas12a protein in this embodiment. The detection system uses a sequence consistent with the crRNA repeat region recognized by the LbCas12a protein. <![CDATA[UAAUUUCUAC UAAGU GUAGAU]]> The crRNA repeat region designed in this embodiment <![CDATA[UAAUUUCUAC UCUU GUAGAU]]> Note: Underlined bases indicate the cyclic sequence.

[0056] 1. When the length of the 3' spacer region sequence is 18 bp, the base sequence of the spacer region varies, specifically designed as IFA-crRNA-O1-O22 (Seq no. 215-236, Table 19). The single-tube, single-temperature, one-step RNA detection reaction system formulation and reaction procedure of this invention are shown in Table 20. RNA extracted from the dilution buffer of inactivated influenza A virus culture was detected, and the results are shown in Table 21. These RNAs were simultaneously detected by qPCR using the Influenza A / B virus nucleic acid detection kit (National Medical Device Registration Certificate 20253401119) from Jiangsu Hongweites Pharmaceutical Technology Co., Ltd., according to the instructions.

[0057] Table 19. Different designs where the repeat region sequence is not completely identical to the sequence recognized by the LbCas12 protein and the spacer region sequence is 18 bp in length. Seq no. 215 IFA-crRNA-O1 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUG]]> Seq no. 216 IFA-crRNA-O2 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGCUG]]> Seq no. 217 IFA-crRNA-O3 <![CDATA[UAAUUUCUAC UCUU GUAGAUGUCAAAGCGUCUACGCUG]]> Seq no. 218 IFA-crRNA-O4 <![CDATA[UAAUUUCUAC UCUU GUAGAUGAGAAAGCGUCUACGCUG<!-- 24 --> ]]> Seq no. 219 IFA-crRNA-O5 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACUAAGCGUCUACGCUG]]> Seq no. 220 IFA-crRNA-O6 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAUAGCGUCUACGCUG]]> Seq no. 221 IFA-crRNA-O7 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAUGCGUCUACGCUG]]> Seq no. 222 IFA-crRNA-O8 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAACCGUCUACGCUG]]> Seq no. 223 IFA-crRNA-O9 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGUGUCUACGCUG]]> Seq no. 224 IFA-crRNA-O10 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCCUCUACGCUG]]> Seq no. 225 IFA-crRNA-O11 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGACUACGCUG]]> Seq no. 226 IFA-crRNA-O12 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUGUACGCUG]]> Seq no. 227 IFA-crRNA-O13 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCAACGCUG]]> Seq no. 228 IFA-crRNA-O14 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUUCGCUG]]> Seq no. 229 IFA-crRNA-O15 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUAGGCUG]]> Seq no. 230 IFA-crRNA-O16 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACCCUG]]> Seq no. 231 IFA-crRNA-O17 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUG]]> Seq no. 232 IFA-crRNA-O18 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCAG]]> Seq no. 233 IFA-crRNA-O19 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUC]]> Seq no. 234 IFA-crRNA-O20 <![CDATA[UAAUUUCUAC UCUU GUAGAUCAGAAAGCGUCUACGCUG]]> Seq no. 235 IFA-crRNA-O21 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGCUC]]> Seq no. 236 IFA-crRNA-O22 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUC]]> Note: Underlined bases indicate cyclic sequences, and red indicates mismatched bases in spacer regions.

[0058] Table 20. Basic formulation and reaction procedure of the single-tube, single-temperature, one-step RNA detection reaction system in this invention. .

[0059] Table 21. Detection results of different designed crRNAs with repeat region sequences that do not completely match the sequence recognized by LbCas12 protein and have a spacer region sequence length of 18 bp. .

[0060] From Table 21, we can see that the following situations did not result in positive amplification: (1) when the crRNA spacer sequence completely matches the target sequence (IFA-crRNA-O1); (2) when there is one mismatch between the 5th and 14th bases of the spacer sequence and the target sequence, and the other positions match the target sequence (IFA-crRNA-O6-15); (3) when there are two mismatches in the 1st to 4th bp of the crRNA spacer sequence and the other positions match the target sequence (IFA-crRNA-O20); (4) when there is one mismatch in the 1st to 4th bp of the crRNA spacer sequence and one mismatch between the 15th to 18th bp of the target sequence (IFA-crRNA-O21); (5) when there are two mismatches in the 15th to 18th bp of the crRNA spacer sequence and the other positions match the target sequence (IFA-crRNA-O22). Positive amplifications were only achieved when there was one mismatch in the first 4 bp of the crRNA spacer sequence and the other positions matched the target sequence (IFA-crRNA-O2-O5), and when there was one mismatch in the first 15-18 bp of the crRNA spacer sequence and the other positions matched the target sequence (IFA-crRNA-O16-O19). Both of these conditions can effectively detect RNA nucleic acids with a Ct value between 31 and 32 detected by qPCR.

[0061] 2. When the length of the 3' spacer region sequence is 19 bp, the base sequence of the spacer region varies, specifically designed as IFA-crRNA-O23-O47 (Seq no. 237-261, Table 22). The single-tube, single-temperature, one-step RNA detection reaction system formulation and reaction procedure of this invention are shown in Table 20. RNA extracted from the dilution of inactivated influenza A virus culture was detected, and the results are shown in Table 23. These RNAs were simultaneously detected by qPCR using the Influenza A / B virus nucleic acid detection kit (National Medical Device Registration Certificate 20253401119) from Jiangsu Hongweites Pharmaceutical Technology Co., Ltd., according to the instructions.

[0062] Table 22. Different designs with repeat region sequences that are not completely identical to the sequence recognized by the LbCas12 protein and with a spacer region sequence length of 19 bp. Seq no. 237 IFA-crRNA-O23 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGC]]> Seq no. 238 IFA-crRNA-O24 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGCUGC]]> Seq no. 239 IFA-crRNA-O25 <![CDATA[UAAUUUCUAC UCUU GUAGAUGUCAAAGCGUCUACGCUGC]]> Seq no. 240 IFA-crRNA-O26 <![CDATA[UAAUUUCUAC UCUU GUAGAUGAGAAAGCGUCUACGCUGC]]> Seq no. 241 IFA-crRNA-O27 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACUAAGCGUCUACGCUGC]]> Seq no. 242 IFA-crRNA-O28 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAUAGCGUCUACGCGUGC]]> Seq no. 243 IFA-crRNA-O29 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAUGCGUCUACGCUGC]]> Seq no. 244 IFA-crRNA-O30 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAACCGUCUACGCUGC]]> Seq no.245 IFA-crRNA-O31 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGUGUCUACGCUGC]]> Seq no.246 IFA-crRNA-O32 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCCUCUACGCUGC]]> Seq no.247 IFA-crRNA-O33 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGACUACGCUGC]]> Seq no.248 IFA-crRNA-O34 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUGUACGCCUGC]]> Seq no.249 IFA-crRNA-O35 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCAACGCUGC]]> Seq no.250 IFA-crRNA-O36 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUUCGCUGC]]> Seq no.251 IFA-crRNA-O37 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUAGGCUGC]]> Seq no.252 IFA-crRNA-O38 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACCCUGC]]> Seq no.253 IFA-crRNA-O39 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUGC]]> Seq no.254 IFA-crRNA-O40 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCAGC]]> Seq no.255 IFA-crRNA-O41 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUCC]]> Seq no.256 IFA-crRNA-O42 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGG]]> Seq no.257 IFA-crRNA-O43 <![CDATA[UAAUUUCUAC UCUU GUAGAUCAGAAAGCGUCUACGCUGC]]> Seq no.258 IFA-crRNA-O44 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGAUGC]]> Seq no.259 IFA-crRNA-O45 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGCUGG <!-- 27 -->]]> Seq no.260 IFA-crRNA-O46 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUCC]]> Seq no.261 IFA-crRNA-O47 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUGG]]> .

[0063] Table 23. Detection results of different designed crRNAs with repeat region sequences that do not completely match the sequence recognized by LbCas12 protein and have a spacer region sequence length of 19 bp. .

[0064] From Table 23, we can see that: when the crRNA spacer sequence perfectly matches the target sequence (IFA-crRNA-O23); when the 5th-14th bases of the spacer sequence each have one mismatch with the target sequence and the other positions match the target sequence (IFA-crRNA-O28-37); when the 19th bp of the crRNA spacer sequence has a mismatched base and the other sequences perfectly match the target sequence (IFA-crRNA-O42); when the 1st-4th bp of the crRNA spacer sequence has two mismatches and the other positions match the target sequence (IFA-crRNA-O43 ...23); when the 19th bp of the crRNA spacer sequence has a mismatched base and the other sequences perfectly match the target sequence (IFA-crRNA-O28-37); when the 19th bp of the crRNA spacer sequence has a mismatched base and the other sequences perfectly match the target sequence (IFA-crRNA-O42); when the 19th bp of the crRNA spacer sequence has a mismatched base and the other sequences perfectly match the target sequence (IFA-crRNA-O43); when the 19th bp of the crRNA spacer sequence has a mismatched base and the other sequences perfectly match the target sequence (IFA-crRNA-O28-37); when the 19th bp of the crRNA spacer sequence has a mismatched base and the other sequences perfectly match the target sequence (IFA-crRNA-O28-37); when the 19th bp of the cr No positive amplification was formed when there was one mismatch in 1-4 bp and one mismatch between 15-18 bp and the target sequence (IFA-crRNA-O44); when there was one mismatch in 1-4 bp and one mismatch between 19 bp and the target sequence (IFA-crRNA-O45); when there were two mismatches in 15-18 bp and other positions matched the target sequence (IFA-crRNA-O46); and when there was one mismatch in 15-18 bp and one mismatch between 19 bp and the target sequence (IFA-crRNA-O47). Positive amplifications were only achieved when there was one mismatch in the first 4 bp of the crRNA spacer sequence and the other positions matched the target sequence (IFA-crRNA-O24-O27), and when there was one mismatch in the first 15-18 bp of the crRNA spacer sequence and the other positions matched the target sequence (IFA-crRNA-O38-O41). Both of these conditions can effectively detect RNA nucleic acids with a Ct value between 31 and 32 detected by qPCR.

[0065] 3. When the length of the 3' spacer region sequence is 20 bp, the base sequence of the spacer region varies, specifically designed as IFA-crRNA-O48-O73 (Seq no. 262-287, Table 24). The single-tube, single-temperature, one-step RNA detection reaction system formulation and reaction procedure of this invention are shown in Table 20. RNA extracted from the dilution of inactivated influenza A virus culture was detected, and the results are shown in Table 25. These RNAs were simultaneously detected by qPCR using the Influenza A / B virus nucleic acid detection kit (National Medical Device Registration Certificate 20253401119) from Jiangsu Hongweites Pharmaceutical Technology Co., Ltd., according to the instructions.

[0066] Table 24. Different designs where the repeat region sequence is not completely identical to the sequence recognized by the LbCas12 protein and the spacer region sequence length is 20 bp. Seq no.262 IFA-crRNA-O48 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCA]]> Seq no.263 IFA-crRNA-O49 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGCUGCA]]> Seq no.264 IFA-crRNA-O50 <![CDATA[UAAUUUCUAC UCUU GUAGAUGUCAAAGCGUCUACGCUGCA]]> Seq no.265 IFA-crRNA-O51 <![CDATA[UAAUUUCUAC UCUU GUAGAUGAGAAAGCGUCUACGCUGCA]]> Seq no.266 IFA-crRNA-O52 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACUAAGCGUCUACGCUGCA]]> Seq no.267 IFA-crRNA-O53 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAUAGCGUCUACGCUGCA]]> Seq no.268 IFA-crRNA-O54 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAUGCGUCUACGCUGCA]]> Seq no.269 IFA-crRNA-O55 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAACCGUCUACGCUGCA]]> Seq no.270 IFA-crRNA-O56 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGUGUCUACGCUGCA]]> Seq no.271 IFA-crRNA-O57 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCCUCUACGCUGCA]]> Seq no.272 IFA-crRNA-O58 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGACUACGCUGCA]]> Seq no.273 IFA-crRNA-O59 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUGUACGCUGCA]]> Seq no.274 IFA-crRNA-O60 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCAACGCUGCA]]> Seq no.275 IFA-crRNA-O61 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUUCGCUGCA]]> Seq no.276 IFA-crRNA-O62 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUAGGCUGCA]]> Seq no.277 IFA-crRNA-O63 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACCCUGCA]]> Seq no.278 IFA-crRNA-O64 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUGCA]]> Seq no.279 IFA-crRNA-O65 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCAGCA]]> Seq no.280 IFA-crRNA-O66 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUCCA]]> Seq no.281 IFA-crRNA-O67 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGGA]]> Seq no.282 IFA-crRNA-O68 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCU<!-- 30 --> ]]> Seq no.283 IFA-crRNA-O69 <![CDATA[UAAUUUCUAC UCUU GUAGAUCAGAAAGCGUCUACGCUGCA]]> Seq no.284 IFA-crRNA-O70 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGAUGCA]]> Seq no.285 IFA-crRNA-O71 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGCUGCU]]> Seq no.286 IFA-crRNA-O72 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUCCA]]> Seq no.287 IFA-crRNA-O73 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUGCU]]> .

[0067] Table 25. Detection results of different designed crRNAs with repeat region sequences that do not completely match the sequence recognized by LbCas12 protein and have a spacer region sequence length of 20 bp. .

[0068] From Table 25, we can see that no positive amplification occurred in the following situations: (1) when the crRNA spacer sequence completely matched the target sequence (IFA-crRNA-O48); (2) when there was one mismatch between the 5th and 14th bases of the spacer sequence and the target sequence, and the other positions matched the target sequence (IFA-crRNA-O53-62); (3) when there was one mismatch between the 19th and 20th bp of the crRNA spacer sequence and the other sequences completely matched the target sequence (IFA-crRNA-O67, 68); (4) when there were two mismatches between the 1st and 4th bp of the crRNA spacer sequence and the other positions matched the target sequence (IFA-crRNA-O69). (5) There is one mismatch in the first 4 bp of the crRNA spacer sequence and one mismatch in the first 15-18 bp of the target sequence (IFA-crRNA-O70); (6) There is one mismatch in the first 4 bp of the crRNA spacer sequence and one mismatch in the first 19-20 bp of the target sequence (IFA-crRNA-O71); (7) There are two mismatches in the first 15-18 bp of the crRNA spacer sequence and the other positions match the target sequence (IFA-crRNA-O72); (8) There is one mismatch in the first 15-18 bp of the crRNA spacer sequence and one mismatch in the first 19-20 bp of the target sequence (IFA-crRNA-O73). Positive amplifications were only achieved when there was one mismatch in the first 4 bp of the crRNA spacer sequence and the other positions matched the target sequence (IFA-crRNA-O49-O52), and when there was one mismatch in the first 15-18 bp of the crRNA spacer sequence and the other positions matched the target sequence (IFA-crRNA-O63-O66). Both of these conditions can effectively detect RNA nucleic acids with a Ct value between 31 and 32 as detected by qPCR.

[0069] 4. When the length of the 3' spacer region sequence is 21 bp, the base sequence of the spacer region varies, specifically designed as IFA-crRNA-O74-O100 (Seq no. 288-314, Table 26). The single-tube, single-temperature, one-step RNA detection reaction system formulation and reaction procedure of this invention are shown in Table 20. RNA extracted from the dilution buffer of inactivated influenza A virus culture was detected, and the results are shown in Table 27. These RNAs were simultaneously detected by qPCR using the Influenza A / B virus nucleic acid detection kit (National Medical Device Registration Certificate 20253401119) from Jiangsu Hongweites Pharmaceutical Technology Co., Ltd., according to the instructions.

[0070] Table 26. Different designs with repeat region sequences that are not completely identical to the sequence recognized by the LbCas12 protein and with a spacer region sequence length of 21 bp. Seq no.288 IFA-crRNA-O74 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCAG]]> Seq no.289 IFA-crRNA-O75 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGCUGCAG]]> Seq no.290 IFA-crRNA-O76 <![CDATA[UAAUUUCUAC UCUU GUAGAUGUCAAAGCGUCUACGCUGCAG]]> Seq no.291 IFA-crRNA-O77 <![CDATA[UAAUUUCUAC UCUU GUAGAUGAGAAAGCGUCUACGCUGCAG]]> Seq no.292 IFA-crRNA-O78 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACUAAGCGUCUACGCUGCAG]]> Seq no.293 IFA-crRNA-O79 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAUAGCGUCUACGCUGCAG]]> Seq no.294 IFA-crRNA-O80 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAUGCGUCUACGCUGCAG]]> Seq no.295 IFA-crRNA-O81 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAACCGUCUACGCUGCAG]]> Seq no.296 IFA-crRNA-O82 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGUGUCUACGCUGCAG]]> Seq no.297 IFA-crRNA-O83 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCCUCUACGCUGCAG]]> Seq no.298 IFA-crRNA-O84 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGACUACGCUGCAG]]> Seq no.299 IFA-crRNA-O85 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUGUACGCUGCAG]]> Seq no.300 IFA-crRNA-O86 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCAACGCUGCAG]]> Seq no.301 IFA-crRNA-O87 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUUCGCUGCAG]]> Seq no.302 IFA-crRNA-O88 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUAGGCUGCAG]]> Seq no.303 IFA-crRNA-O89 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACCCUGCAG]]> Seq no.304 IFA-crRNA-O90 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUGCAG]]> Seq no.305 IFA-crRNA-O91 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCAGCAG]]> Seq no.306 IFA-crRNA-O92 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUCCAG]]> Seq no.307 IFA-crRNA-O93 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGGAG]]> Seq no.308 IFA-crRNA-O94 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCUG <!-- 33 -->]]> Seq no.309 IFA-crRNA-O95 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCAC]]> Seq no.310 IFA-crRNA-O96 <![CDATA[UAAUUUCUAC UCUU GUAGAUCAGAAAGCGUCUACGCUGCAG]]> Seq no.311 IFA-crRNA-O97 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGAUGCAG]]> Seq no.312 IFA-crRNA-O98 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGCUGCAC]]> Seq no.313 IFA-crRNA-O99 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUCCAG]]> Seq no.314 IFA-crRNA-O100 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUGCAC]]> .

[0071] Table 27. Detection results of different designed crRNAs with repeat region sequences that do not completely match the sequence recognized by LbCas12 protein and have a spacer region sequence length of 21 bp.

[0072] From Table 27, we can see that the following situations did not result in positive amplification: (1) when the crRNA spacer sequence completely matches the target sequence (IFA-crRNA-O74); (2) when there is one mismatch between the 5th and 14th bases of the spacer sequence and the target sequence, and the other positions match the target sequence (IFA-crRNA-O79-88); (3) when there is one mismatch between the 19th and 21st bp of the crRNA spacer sequence and the other sequences completely match the target sequence (IFA-crRNA-O93-95); (4) when there are two mismatches between the 1st and 4th bp of the crRNA spacer sequence and the other positions match the target sequence (IFA-crRNA-O96); (4) There is one mismatch in the first 4 bp of the crRNA spacer sequence and one mismatch in the first 15-18 bp with the target sequence (IFA-crRNA-O97); (5) There is one mismatch in the first 4 bp of the crRNA spacer sequence and one mismatch in the first 19-21 bp with the target sequence (IFA-crRNA-O98); (6) There are two mismatches in the first 15-18 bp of the crRNA spacer sequence and the other positions match the target sequence (IFA-crRNA-O99); (7) There is one mismatch in the first 15-18 bp of the crRNA spacer sequence and one mismatch in the first 19-21 bp with the target sequence (IFA-crRNA-O100). Positive amplifications were only achieved when there was one mismatch in the first 4 bp of the crRNA spacer sequence and the other positions matched the target sequence (IFA-crRNA-O75-O78), and when there was one mismatch in the first 15-18 bp of the crRNA spacer sequence and the other positions matched the target sequence (IFA-crRNA-O63-O66). Both of these conditions can effectively detect RNA nucleic acids with a Ct value between 31 and 32 as detected by qPCR.

[0073] 5. When the length of the 3' spacer region sequence is 22 bp, the base sequence of the spacer region varies, specifically designed as IFA-crRNA-O101-O128 (Seq no. 315-342, Table 28). The single-tube, single-temperature, one-step RNA detection reaction system formulation and reaction procedure of this invention are shown in Table 20. RNA extracted from the dilution buffer of inactivated influenza A virus culture was detected, and the results are shown in Table 29. These RNAs were simultaneously detected by qPCR using the Influenza A / B virus nucleic acid detection kit (National Medical Device Registration Certificate 20253401119) from Jiangsu Hongweites Pharmaceutical Technology Co., Ltd., according to the instructions.

[0074] Table 28. Different designs where the repeat region sequence is not completely identical to the sequence recognized by the LbCas12 protein and the spacer region sequence length is 22 bp. Seq no.315 IFA-crRNA-O101 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCAGAU]]> Seq no.316 IFA-crRNA-O102 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGCUGCAGU]]> Seq no.317 IFA-crRNA-O103 <![CDATA[UAAUUUCUAC UCUU GUAGAUGUCAAAGCGUCUACGCUGCAGAU]]> Seq no.318 IFA-crRNA-O104 <![CDATA[UAAUUUCUAC UCUU GUAGAUGAGAAAGCGUCUACGCUGCAGU]]> Seq no.319 IFA-crRNA-O105 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACUAAGCGUCUACGCUGCAGU]]> Seq no.320 IFA-crRNA-O106 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAUAGCGUCUACGCUGCAGU]]> Seq no.321 IFA-crRNA-O107 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAUGCGUCUACGCUGCAGU]]> Seq no.322 IFA-crRNA-O108 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAACCGUCUACGCUGCAGU]]> Seq no.323 IFA-crRNA-O109 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGUGUCUACGCUGCAG]]> Seq no.324 IFA-crRNA-O110 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCCUCUACGCUGCAG]]> Seq no.325 IFA-crRNA-O111 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGACUAACGCUGCAGU]]> Seq no.326 IFA-crRNA-O112 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUGUACGCUGCAG]]> Seq no.327 IFA-crRNA-O113 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCAACGCUGCAGU]]> Seq no.328 IFA-crRNA-O114 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUUCGCUGCAGU]]> Seq no.329 IFA-crRNA-O115 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUAGGCUGCAGU]]> Seq no.330 IFA-crRNA-O116 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACCCUGCAG]]> Seq no.331 IFA-crRNA-O117 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUGCAGU]]> Seq no.332 IFA-crRNA-O118 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCAGCAGU]]> Seq no.333 IFA-crRNA-O119 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGUCCAGU]]> Seq no.334 IFA-crRNA-O120 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGUGGAGU]]> Seq no.335 IFA-crRNA-O121 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCUGU<!-- 36 --> ]]> Seq no.336 IFA-crRNA-O122 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCACU]]> Seq no.337 IFA-crRNA-O123 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCAGCAGA]]> Seq no.338 IFA-crRNA-O124 <![CDATA[UAAUUUCUAC UCUU GUAGAUCAGAAAGCGUCUACGCUGCAGU]]> Seq no.339 IFA-crRNA-O125 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGAUGCAGU]]> Seq no.340 IFA-crRNA-O126 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGCUGCAGA]]> Seq no.341 IFA-crRNA-O127 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUCCAGU]]> Seq no.342 IFA-crRNA-O128 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUGCAGA]]> .

[0075] Table 29. Detection results of different designed crRNAs with repeat region sequences that do not completely match the sequence recognized by LbCas12 protein and have a spacer region sequence length of 22 bp.

[0076] From Table 29, we can see that no positive amplification occurred in the following situations: (1) when the crRNA spacer sequence completely matched the target sequence (IFA-crRNA-O101); (2) when there was one mismatch between the 5th and 14th bases of the spacer sequence and the target sequence, and the other positions matched the target sequence (IFA-crRNA-O106-115); (3) when there was one mismatch between the 19th and 22nd bp of the crRNA spacer sequence and the other sequences completely matched the target sequence (IFA-crRNA-O120-123); (4) when there were two mismatches between the 1st and 4th bp of the crRNA spacer sequence and the other positions matched the target sequence (IFA-crRNA-O124). (5) There is one mismatch in the first 4 bp of the crRNA spacer sequence and one mismatch in the first 15-18 bp with the target sequence (IFA-crRNA-O125); (6) There is one mismatch in the first 4 bp of the crRNA spacer sequence and one mismatch in the first 19-22 bp with the target sequence (IFA-crRNA-O126); (7) There are two mismatches in the first 15-18 bp of the crRNA spacer sequence and the other positions match the target sequence (IFA-crRNA-O127); (8) There is one mismatch in the first 15-18 bp of the crRNA spacer sequence and one mismatch in the first 19-22 bp with the target sequence (IFA-crRNA-O128). Positive amplification was only achieved when there was one mismatch in the first 4 bp of the crRNA spacer sequence and the other positions matched the target sequence (IFA-crRNA-O102-O105), and when there was one mismatch in the first 15-18 bp of the crRNA spacer sequence and the other positions matched the target sequence (IFA-crRNA-O116-O119). Both of these conditions can effectively detect RNA nucleic acids with a Ct value between 31 and 32 detected by qPCR.

[0077] 6. When the length of the 3' spacer region sequence is 23 bp, the base sequence of the spacer region is different for each region. The specific design is IFA-crRNA-O129-O157 (Seq no. 343-371, Table 30). The single-tube, single-temperature, one-step RNA detection reaction system formula and reaction procedure of this invention are shown in Table 20. RNA extracted from the dilution of inactivated influenza A virus culture was detected, and the results are shown in Table 31. These RNAs were simultaneously detected by qPCR using the Influenza A / B virus nucleic acid detection kit (National Medical Device Registration Certificate 20253401119) from Jiangsu Hongweites Pharmaceutical Technology Co., Ltd., according to the instructions.

[0078] Table 30. Different designs where the repeat region sequence is not completely identical to the sequence recognized by the LbCas12 protein and the spacer region sequence length is 23 bp. Seq no.343 IFA-crRNA-O129 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCAGUC]]> Seq no.344 IFA-crRNA-O130 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGCUGCAGUC]]> Seq no.345 IFA-crRNA-O131 <![CDATA[UAAUUUCUAC UCUU GUAGAUGUCAAAGCGUCUACGCUGCAGUC]]> Seq no.346 IFA-crRNA-O132 <![CDATA[UAAUUUCUAC UCUU GUAGAUGAGAAAGCGUCUACGCUGCAGUC]]> Seq no.347 IFA-crRNA-O133 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACUAAGCGUCUACGCUGCAGUC]]> Seq no.348 IFA-crRNA-O134 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAUAGCGUCUACGCUGCAGUC]]> Seq no.349 IFA-crRNA-O135 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAUGCGUCUACGCUGCAGUC]]> Seq no.350 IFA-crRNA-O136 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAACCGUCUACGCUGCAGUC]]> Seq no.351 IFA-crRNA-O137 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGUGUCUACGCUGCAGUC]]> Seq no.352 IFA-crRNA-O138 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCCUCUACGCUGCAGUC]]> Seq no.353 IFA-crRNA-O139 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGACUACGCUGCAGUC]]> Seq no.354 IFA-crRNA-O140 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUGUACGCUGCAGUC]]> Seq no.355 IFA-crRNA-O141 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCAACGCUGCAGUC]]> Seq no.356 IFA-crRNA-O142 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUUCGCUGCAGUC]]> Seq no.357 IFA-crRNA-O143 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUAGGCUGCAGUC]]> Seq no.358 IFA-crRNA-O144 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACCCUGCAGUC]]> Seq no.359 IFA-crRNA-O145 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUGCAGUC]]> Seq no.360 IFA-crRNA-O146 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCAGCAGCAGUC]]> Seq no.361 IFA-crRNA-O147 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUCCAGUC]]> Seq no.362 IFA-crRNA-O148 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCGUGGAGUC]]> Seq no.363 IFA-crRNA-O149 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCUGUC <!-- 39 -->]]> Seq no.364 IFA-crRNA-O150 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCACUC]]> Seq no.365 IFA-crRNA-O151 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCAGAC]]> Seq no.366 IFA-crRNA-O152 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCAGUG]]> Seq no.367 IFA-crRNA-O153 <![CDATA[UAAUUUCUAC UCUU GUAGAUCAGAAAGCGUCUACGCUGCAGUC]]> Seq no.368 IFA-crRNA-O154 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGAUGCAGUC]]> Seq no.369 IFA-crRNA-O155 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGCUGCAGUG]]> Seq no.370 IFA-crRNA-O156 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUCCAGUC]]> Seq no.371 IFA-crRNA-O157 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUGCAGUG]]> .

[0079] Table 31. Detection results of different designed crRNAs with repeat region sequences that do not completely match the sequence recognized by LbCas12 protein and have a spacer region sequence length of 23 bp.

[0080] From Table 31, we can see that no positive amplification occurred in the following situations: (1) when the crRNA spacer sequence completely matched the target sequence (IFA-crRNA-O129); (2) when there was one mismatch between the 5th and 14th bases of the spacer sequence and the target sequence, and the other positions matched the target sequence (IFA-crRNA-O134-143); (3) when there was one mismatch between the 19th and 23rd bp of the crRNA spacer sequence and the other sequences completely matched the target sequence (IFA-crRNA-O148-152); (4) when there were two mismatches between the 1st and 4th bp of the crRNA spacer sequence and the other positions matched the target sequence (IFA-crRNA-O153). (5) There is one mismatch in the first 4 bp of the crRNA spacer sequence and one mismatch in the first 15-18 bp with the target sequence (IFA-crRNA-O154); (6) There is one mismatch in the first 4 bp of the crRNA spacer sequence and one mismatch in the first 19-23 bp with the target sequence (IFA-crRNA-O155); (7) There are two mismatches in the first 15-18 bp of the crRNA spacer sequence and the other positions match the target sequence (IFA-crRNA-O156); (8) There is one mismatch in the first 15-18 bp of the crRNA spacer sequence and one mismatch in the first 19-23 bp with the target sequence (IFA-crRNA-O157). Positive amplifications were only achieved when there was one mismatch in the first 4 bp of the crRNA spacer sequence and the other positions matched the target sequence (IFA-crRNA-O130-O133), and when there was one mismatch in the first 15-18 bp of the crRNA spacer sequence and the other positions matched the target sequence (IFA-crRNA-O144-O147). Both of these conditions can effectively detect RNA nucleic acids with a Ct value between 31 and 32 as detected by qPCR.

[0081] 7. When the length of the 3' spacer region sequence is 24 bp, the base sequence of the spacer region is different for each region. The specific design is IFA-crRNA-O158-O187 (Seq no. 372-401, Table 32). The single-tube, single-temperature, one-step RNA detection reaction system formula and reaction procedure of this invention are shown in Table 20. RNA extracted from the dilution of inactivated influenza A virus culture was detected, and the results are shown in Table 33. These RNAs were simultaneously detected by qPCR using the Influenza A / B virus nucleic acid detection kit (National Medical Device Registration Certificate 20253401119) from Jiangsu Hongweites Pharmaceutical Technology Co., Ltd., according to the instructions.

[0082] Table 32 shows different designs where the repeat region sequence is not completely identical to the sequence recognized by the LbCas12 protein, and the spacer region sequence is 24 bp in length. Seq no.372 IFA-crRNA-O158 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCAGUCC]]> Seq no.373 IFA-crRNA-O159 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGCUGCAGUCC]]> Seq no.374 IFA-crRNA-O160 <![CDATA[UAAUUUCUAC UCUU GUAGAUGUCAAAGCGUCUACGCUGCAGUCC]]> Seq no.375 IFA-crRNA-O161 <![CDATA[UAAUUUCUAC UCUU GUAGAUGAGAAAGCGUCUACGCUGCAGUCC]]> Seq no.376 IFA-crRNA-O162 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACUAAGCGUCUACGCUGCAGUCC]]> Seq no.377 IFA-crRNA-O163 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAUAGCGUCUACGCGUGCCAGUCC]]> Seq no.378 IFA-crRNA-O164 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAUGCGUCUACGCUGCAGUCC]]> Seq no.379 IFA-crRNA-O165 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAACCGUCUACGCUGCAGUCC]]> Seq no.380 IFA-crRNA-O166 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGUGUCUACGCUGCAGUCC]]> Seq no.381 IFA-crRNA-O167 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCCUCUACGCUGCAGUCC]]> Seq no.382 IFA-crRNA-O168 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGACUACGCUGCAGUCC]]> Seq no.383 IFA-crRNA-O169 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUGUACGCUGCAGUCC]]> Seq no.384 IFA-crRNA-O170 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCAACGCUGCAGUCC]]> Seq no.385 IFA-crRNA-O171 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUUCGCUGCAGUCC]]> Seq no.386 IFA-crRNA-O172 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUAGGCUGCAGUCC]]> Seq no.387 IFA-crRNA-O173 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACCCUGCAGUCC]]> Seq no.388 IFA-crRNA-O174 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUGCAGUCC]]> Seq no. 389 IFA-crRNA-O175 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCAGCAGUCC]]> Seq no. 390 IFA-crRNA-O176 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGUCCAGUCC]]> Seq no. 391 IFA-crRNA-O177 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGGAGUCC]]> Seq no. 392 IFA-crRNA-O178 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCUGUCC <!-- 42 -->]]> Seq no. 393 IFA-crRNA-O179 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCACUCC]]> Seq no. 394 IFA-crRNA-O180 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCAGACC]]> Seq no. 395 IFA-crRNA-O181 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCAGUGC]]> Seq no. 396 IFA-crRNA-O182 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGCUGCAGUCG]]> Seq no. 397 IFA-crRNA-O183 <![CDATA[UAAUUUCUAC UCUU GUAGAUCAGAAAGCGUCUACGCUGCAGUCC]]> Seq no. 398 IFA-crRNA-O184 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGAUGCAGUCC]]> Seq no. 399 IFA-crRNA-O185 <![CDATA[UAAUUUCUAC UCUU GUAGAUCACAAAGCGUCUACGCUGCAGUCG]]> Seq no. 400 IFA-crRNA-O186 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUCCAGUCC]]> Seq no. 401 IFA-crRNA-O187 <![CDATA[UAAUUUCUAC UCUU GUAGAUGACAAAGCGUCUACGAUGCAGUCG]]> .

[0083] Table 33. Detection results of different designed crRNAs with repeat region sequences that do not completely match the sequence recognized by LbCas12 protein and have a spacer region sequence length of 24 bp. .

[0084] From Table 33, we can see that no positive amplification occurred in the following situations: (1) when the crRNA spacer sequence completely matched the target sequence (IFA-crRNA-O158); (2) when there was one mismatch between the 5th and 14th bases of the spacer sequence and the target sequence, and the other positions matched the target sequence (IFA-crRNA-O163-172); (3) when there was one mismatch between the 19th and 24th bp of the crRNA spacer sequence and the other sequences completely matched the target sequence (IFA-crRNA-O177-182); (4) when there were two mismatches between the 1st and 4th bp of the crRNA spacer sequence and the other positions matched the target sequence (IFA-crRNA-O183). (5) There is one mismatch in the first 4 bp of the crRNA spacer sequence and one mismatch in the first 15-18 bp with the target sequence (IFA-crRNA-O184); (6) There is one mismatch in the first 4 bp of the crRNA spacer sequence and one mismatch in the first 19-24 bp with the target sequence (IFA-crRNA-O185); (7) There are two mismatches in the first 15-18 bp of the crRNA spacer sequence and the other positions match the target sequence (IFA-crRNA-O186); (8) There is one mismatch in the first 15-18 bp of the crRNA spacer sequence and one mismatch in the first 19-24 bp with the target sequence (IFA-crRNA-O187). Positive amplification was only achieved when there was one mismatch in the first 4 bp of the crRNA spacer sequence and the other positions matched the target sequence (IFA-crRNA-O159-O162), and when there was one mismatch in the first 15-18 bp of the crRNA spacer sequence and the other positions matched the target sequence (IFA-crRNA-O173-O176). Both of these conditions can effectively detect RNA nucleic acids with a Ct value between 31 and 32 detected by qPCR.

[0085] Example 3 RAA amplification primers, wild-type crRNA, and mutant crRNA (Seq no. 402-405, Table 34) were designed targeting the rs671 SNP site of the human ALDH2 gene. The two crRNAs were designed based on the optimal PAM sequence “TTTV” format, with the repeat region sequence perfectly matching the repeat sequence of the LbCas12a protein used, and the spacer sequence length being 20 bp. The 3rd bp of the spacer sequence in the wild-type crRNA formed a mismatch with the target sequence, while the other bases perfectly matched the wild-type target sequence; similarly, the 3rd bp of the spacer sequence in the mutant crRNA formed a mismatch with the target sequence, while the other bases perfectly matched the mutant target sequence. The method described in this invention was used to detect 20 human genomic DNA samples with known genotypes. For each sample, wild-type crRNA and mutant crRNA reaction solutions were used separately. The detection formulas and reaction procedures are shown in Tables 35 and 36, and the detection results are shown in Table 37.

[0086] Table 34: RAA amplification primers and crRNA for the rs671 SNP site Seq no. 402 671-RPA-F tggctacaagatgtcggggagtggccgggag Seq no. 403 671-RPA-R aggtcctgaacttccagcaggccctgagtcc Seq no. 404 671-crRNA-W <![CDATA[UAAUUUCUAC UAAGU GUAGAUAcAUcagUgUaUgccUgcag]]> Seq no. 405 671-crRNA-T <![CDATA[UAAUUUCUAC UAAGU GUAGAUAcAUUagUgUaUgccUgcag]]> .

[0087] Table 35. Formulation and reaction procedure for rs671 wild-type reaction solution .

[0088] Table 36. Formulation and reaction procedure for the rs671 mutant reaction solution. .

[0089] Table 37. Statistical analysis of the results of 20 human genomic DNA samples detected by rs671 wild-type and mutant reaction solutions. hg1 wild type + - hg2 wild type + - hg3 wild type + - hg4 wild type + - hg5 wild type + - hg6 wild type + - hg7 wild type + - hg8 wild type + - hg9 mutant - + hg10 wild type + - hg11 wild type + - hg12 wild type + - hg13 wild type + - hg14 wild type + - hg15 Hybrid + + hg16 wild type + - hg17 wild type + - hg18 wild type + - hg19 wild type + - hg20 wild type + - As shown in Table 37, of the 20 samples, hg1-8, hg10-14, and hg16-20 (18 samples in total) were wild-type samples. These 18 samples showed positive amplification only in the wild-type reaction solution and no amplification in the mutant reaction solution, thus correctly identified as wild-type. hg9 was a mutant sample; this sample showed no amplification in the wild-type reaction solution but positive amplification in the mutant reaction solution, thus correctly identified as mutant. hg15 was a heterozygous sample; this sample showed amplification in both the wild-type and mutant reaction solutions, thus correctly identified as heterozygous. In other words, the method of this invention can correctly identify the SNP type in nucleic acid mutations within 30 minutes.

[0090] Those skilled in the art will also recognize, or be able to identify, many equivalents of the specific embodiments of the invention described herein using no more than conventional experiments. These equivalents are also included in the appended claims.

Claims

1. A target sequence detection system that simultaneously performs isothermal amplification and CRISPR / Cas12a detection reactions in a single tube at a single temperature, characterized in that, The target sequence includes a PAM sequence for Cas12a protein recognition, wherein the PAM sequence is "TTTV", and V is any one of A, C, or G bases; the Cas12a signaling system includes crRNAs with the following structures: (1) The 5' repeat region sequence of the crRNA is completely consistent with the repeat region sequence that can be recognized by the Cas12a protein. When the length of the 3' spacer region sequence is 18-24 bp, there is only one mismatched base in the first 1-16 bp of the 3' spacer region sequence of the crRNA and the target sequence, and the sequences at other positions of the 3' spacer region sequence are completely complementary to the target sequence. or (2) The 5' repeat region sequence of the crRNA, except for the loop structure sequence, is completely identical to the repeat region sequence recognizable by the Cas12a protein. The loop structure sequence has one less base than the loop structure sequence recognizable by the Cas12a protein, and the first base of the loop structure sequence is the same as the corresponding base of the loop structure recognizable by the Cas12a protein. The second to fourth bases of the loop structure sequence are different from the corresponding bases of the loop structure recognizable by the Cas12a protein. When the 3' spacer region sequence is 18-24 bp in length, the first to fourth bp of the 3' spacer region sequence of the crRNA has only one mismatched base with the target sequence, and the other positions of the 3' spacer region sequence are completely complementary to the target sequence. Alternatively, the 15-18 bp of the 3' spacer region sequence of the crRNA has only one mismatched base with the target sequence, and the other positions of the 3' spacer region sequence are completely complementary to the target sequence.

2. The detection system according to claim 1, characterized in that, The target sequence is a DNA sequence or an RNA sequence.

3. The detection system according to claim 1, characterized in that, The isothermal amplification reaction is the RAA isothermal amplification reaction.

4. The application of the detection system according to any one of claims 1-3 in the detection of single nucleotide polymorphisms.

5. A CRISPR / Cas12a detection method based on isothermal amplification reaction, characterized in that, The isothermal amplification reaction and CRISPR / Cas12a detection reaction are performed simultaneously in a single tube at a single temperature. The target sequence detected by the CRISPR / Cas12a detection reaction includes a PAM sequence for Cas12a protein recognition. The PAM sequence is "TTTV", where V is any one of A, C, or G bases. The Cas12a signaling system includes crRNA with the following structure: (1) The 5' repeat region sequence of the crRNA is completely consistent with the repeat region sequence that can be recognized by the Cas12a protein. When the length of the 3' spacer region sequence is 18-24 bp, there is only one mismatched base in the first 1-16 bp of the 3' spacer region sequence of the crRNA and the target sequence, and the sequences at other positions of the 3' spacer region sequence are completely complementary to the target sequence. or (2) The 5' repeat region sequence of the crRNA, except for the loop structure sequence, is completely identical to the repeat region sequence recognizable by the Cas12a protein. The loop structure sequence has one less base than the loop structure sequence recognizable by the Cas12a protein. The first base of the loop structure sequence is the same as the corresponding base of the loop structure recognizable by the Cas12a protein. The second to fourth bases of the loop structure sequence are different from the corresponding bases of the loop structure recognizable by the Cas12a protein. When the 3' spacer region sequence is 18-24 bp in length, the first to fourth bp of the 3' spacer region sequence of the crRNA has only one mismatched base with the target sequence, and the sequences at other positions of the 3' spacer region sequence are completely complementary to the target sequence. Alternatively, the 15-18 bp of the crRNA spacer region sequence has only one mismatched base with the target sequence, and the sequences at other positions of the 3' spacer region sequence are completely complementary to the target sequence.

Images