CN117887841A - Composition, kit and application for detecting Leber hereditary optic neuropathy by one-step method based on CRISPR/Cas12 - Google Patents

Abstract

The invention discloses a composition, a kit and application for detecting Leber hereditary optic neuropathy based on a CRISPR/Cas12 one-step method. A composition for detecting Leber hereditary optic neuropathy based on CRISPR/Cas12, comprising a crRNA and an RPA amplification primer pair; the crRNA has at least one of the sequences shown in SEQ ID NO. 4 to SEQ ID NO. 12; the RPA amplification primer pair is at least one of the following primer pairs: a primer pair consisting of sequences shown as SEQ ID NO. 23 and SEQ ID NO. 24, a primer pair consisting of sequences shown as SEQ ID NO. 25 and SEQ ID NO. 26 or a primer pair consisting of sequences shown as SEQ ID NO. 27 and SEQ ID NO. 28. The composition and the kit provided by the invention can be used for rapidly diagnosing LHON gene mutation, rapidly detecting LHON pathogenic mutation by a one-step method, accurately detecting whether three primary mutation sites (m.3460G > A, m.11778G > A and m.14484T > C) of LHON pathogenic mutation of a sample exist or not, and have the characteristics of rapid detection, simplicity and convenience in operation (one-step method), specificity, visualization, no need of large-scale expensive instruments and expensive reagents and the like.

Description

Composition, kit and application for detecting Leber hereditary optic neuropathy by one-step method based on CRISPR/Cas12

Technical Field

The invention relates to the field of biotechnology, in particular to a composition, a kit and application for detecting Leber hereditary optic neuropathy based on a CRISPR/Cas12 one-step method.

Background

Leber hereditary optic neuropathy (Leber Hereditary Optic Neuropathy; LHON) is a maternal hereditary optic neurodegenerative disease caused by mutations in mitochondrial DNA. LHON is clinically manifested as a sudden and abrupt decrease in painless vision that occurs in tandem in both eyes. The early fundus manifests as optic disk engorgement edema, optic disk paratelangiectasia, resolution of optic disk edema and telangiectasia in the later stages of the disease, and finally atrophic changes in the temporal side of the optic disk or all. Most LHON patients only have ocular manifestations, and a few can incorporate other systemic symptoms such as mental disorders, epilepsy, hearing disorders, dystonia, etc. LHON is rare, has a prevalence of about 1.18/10000 in Asia and has a pronounced tendency to multiple men, usually occurring at the age of 20-30 years of age, with a proportion of men to women of approximately 5:1.

The primary mutation sites of LHON mitochondria are 11778G > A, 3460G > A and 14484T > C, and about 90% of LHON cases are caused by the three gene mutations. Wherein, the mutation rate of m.11778G > A in China is about 90 percent. Carriers of these genetic mutations may not develop disease, which is a characteristic of LHON exogenesis, but carriers of these females may inherit the mutations to pass on to the next generation, thereby posing a risk to vision of the offspring. Therefore, detection of mutation sites of LHON-related pathogenic genes, genetic counseling and prenatal diagnosis are also highly necessary.

Clinically, diagnosis of LHON can be assisted by Visual Evoked Potential (VEP), fundus Fluorescence Angiography (FFA), optical Coherence Tomography (OCT), visual Field (VF), resonance imaging (MRI), etc., but ultimately diagnosis still needs to be confirmed by detection of the three mutation sites. Currently, common methods for detecting LHON pathogenic mutation include Mulberry sequencing, second generation sequencing, restriction fragment length polymorphism PCR, single-strand conformation polymorphism PCR, allele-specific PCR, reverse transcription quantitative PCR and the like.

The methods have the disadvantages of consuming 12-24 hours to a certain extent, being complicated in operation, requiring expensive instruments such as large sequencers for individual technologies such as sanger sequencing and second generation sequencing, and being unsuitable as a clinically stock rapid detection method.

Disclosure of Invention

In view of this, the invention proposes a composition, a kit and an application for detecting Leber hereditary optic neuropathy based on a one-step method of CRISPR/Cas 12. The one-step method can be used for rapidly detecting LHON pathogenic mutation, and accurately detecting whether three primary mutation sites (m.3460G > A, m.11778G > A and m.14484T > C) of LHON pathogenic mutation of a sample exist or not, and has the characteristics of rapid detection, simplicity and convenience in operation (one-step method), specificity, visualization, no need of large-scale expensive instruments and expensive reagents and the like. The composition and the kit provided by the invention can be used for rapidly diagnosing LHON gene mutation, are suitable for large-scale screening of mutation carriers in crowds, and provide basic support for clinical genetic counseling and prenatal diagnosis.

The technical scheme of the invention is as follows:

the invention provides a composition for detecting Leber hereditary optic neuropathy based on CRISPR/Cas 12.

The composition for detecting Leber hereditary optic neuropathy based on CRISPR/Cas12 provided by the invention comprises crRNA and RPA amplification primer pairs;

The crRNA has at least one of the sequences shown in SEQ ID NO. 4 to SEQ ID NO. 12;

the RPA amplification primer pair is at least one of the following primer pairs: a primer pair consisting of sequences shown as SEQ ID NO. 23 and SEQ ID NO. 24, a primer pair consisting of sequences shown as SEQ ID NO. 25 and SEQ ID NO.26 or a primer pair consisting of sequences shown as SEQ ID NO. 27 and SEQ ID NO. 28.

The Leber hereditary optic neuropathy has at least one of the following three primary mutation sites: m.3460G > A, m.11778G > A and m.14484T > C.

When the m.11778 g > a mutation is detected:

The crRNA has a sequence shown as SEQ ID NO. 8, and the RPA amplification primer pair has a primer pair consisting of sequences shown as SEQ ID NO. 25 and SEQ ID NO. 26.

When the m.3460g > a mutation is detected:

The crRNA has a sequence shown as SEQ ID NO. 6, and the RPA amplification primer pair has a primer pair consisting of sequences shown as SEQ ID NO. 23 and SEQ ID NO. 24.

When detecting m.14484t > c mutation:

the crRNA has a sequence shown as SEQ ID NO. 10, and the RPA amplification primer pair has a primer pair consisting of sequences shown as SEQ ID NO. 27 and SEQ ID NO. 28.

The application of the composition for detecting the Leber hereditary optic neuropathy based on the CRISPR/Cas12 in preparing a kit for detecting the Leber hereditary optic neuropathy also belongs to the protection scope of the invention.

The invention also provides a kit for detecting the Leber hereditary optic neuropathy.

The kit for detecting the Leber hereditary optic neuropathy provided by the invention comprises the composition for detecting the Leber hereditary optic neuropathy based on the CRISPR/Cas 12.

The kit further comprises the following components in independent package: RPA powder, reaction buffer, cas12a protein, and fluorescent probe that can be specifically cleaved by Cas12 a; the fluorescent probe capable of being specifically cut by Cas12a has two ends respectively provided with a luminous group and a quenching group, and the middle is connected by single-stranded DNA.

The application of the kit for detecting the Leber hereditary optic neuropathy based on CRISPR/Cas12 in preparing a reaction system for detecting the primary mutation site of the Leber hereditary optic neuropathy of a clinical sample also belongs to the protection scope of the invention.

The clinical sample is a clinical blood sample;

The primary mutation site of the Leber hereditary optic neuropathy is at least one of the following: m.3460G > A, m.11778G > A, m.14484T > C.

The crRNA of the present invention is selected from crRNA5 of Table 1 for m.11778G > A, crRNA3 of Table 1 for m.3460G > A, and crRNA7 of Table 1 for m.14484T > C. crrnas can be synthesized or transcribed by chemical methods well known in the art, for example: designing and synthesizing a template sequence (table 2); annealing to construct an in vitro transcription template, and carrying out in vitro transcription by using a transcription kit to obtain the crRNA. The Cas12a protein described above can be purchased by commercial companies, such as NEB, and the like.

In order to ensure the specificity of detection, the crRNA and isothermal rapid amplification (RPA) primers provided by the invention are searched by NCBI nucleic acid database, so that the specificity of the sequences is determined, and the sequences are matched with other areas of genome including human, animals, plants and the like in a non-highly homologous manner. In order to improve the specificity of crRNA to a target sequence, a mismatched base can be artificially introduced into the crRNA, so that the targeting specificity of the crRNA to the target sequence is enhanced.

In the reaction, powders of RPA, buffers and specific primers are also included. The RPA powder and buffer described above were purchased from TwistDx. The RPA primer is designed and synthesized by ordering from Kingshi corporation, and comprises a primer pair 1 for amplifying fragments near the m.3460G > A site, a primer pair 2 for amplifying fragments near the m.11778G > A site and a primer pair 3 for amplifying fragments near the m.14484T > C site.

In a specific embodiment, the reaction further comprises a fluorescent probe (ssDNA-FQ) which is independently cleavable by Cas12a and is characterized in that the two ends of the probe are respectively a luminescent group 6-FAM and a quenching group BQH, the middle is connected by single-stranded DNA (ssDNA), and the connecting sequence used in the invention is TTATT. Normally, the fluorescent probe does not excite fluorescence due to the action of the quenching group, and only under specific conditions, ssDNA breaks to release the fluorescent group, fluorescence reading and identification can be performed. The fluorescent probe is commercially available from Shanghai Bioengineering. The detection result can be interpreted by using an enzyme-labeled instrument to carry out fluorescence reading, and can also be continuously detected by using a real-time quantitative PCR instrument.

When detecting m.11778G > A mutation, a reaction is performed using crRNA5 shown in Table 1 and PRA primer pair 2 shown in Table 3; when detecting m.3460G > A mutation, a reaction is performed using crRNA3 shown in Table 1 and PRA primer pair 1 shown in Table 3; when detecting the m.14484T > C mutation, a reaction was performed using crRNA7 shown in Table 1 and PRA primer pair 3 shown in Table 3. Each reaction requires the addition of a fluorescent probe as an indicator to make a decision as to the result.

The detection basic principle of the invention is as follows: the rapid nucleic acid detection system developed based on CRISPR (Clustered regularly interspaced short palindromic repeats) system is widely used in the infectious disease fields such as COVID-19 and zaka Virus, and CRISPR/Cas12a can recognize specific target sequences under the guidance of crRNA and activate endonuclease activity to perform nonspecific cleavage on nearby double-stranded DNA and single-stranded DNA. Based on the above, the inventor of the invention researches and designs a one-step method for rapidly and accurately detecting m.3460G > A, m.11778G > A and m.14484T > C gene mutation in clinical samples. When the CRISPR/Cas12a and the crRNA complex are specifically combined with a target sequence, the endoenzyme activity of the Cas12a can be activated, ssDNA of a peripheral fluorescent probe is cut, so that a fluorescent group is exposed, fluorescence can be detected and quantified by using an enzyme-labeled instrument or a real-time quantitative PCR instrument, and a positive result can be judged according to a fluorescence value; otherwise, if the mutation is not present in the detection sample, the substrate fluorescence is almost the same as that of the negative control, and the detection sample can be judged as negative. FIG. 1 is a flow chart of the one-step method for detecting LHON pathogenic gene mutation.

Compared with the prior art, the invention has the following advantages and beneficial effects:

The composition for detecting Leber hereditary optic neuropathy based on CRISPR/Cas12 provided by the invention can detect any one of three common primary mutations of LHON: m.3460G > A or m.11778G > A or m.14484T > C.

The target sequences containing the Cas12A recognition sequence (PAM, 5'… TTTN …') are respectively searched within 10bp before and after the m.3460G > A, m.11778G > A and m.14484T > C loci, so that the mutation loci are positioned within 6bp of the CRRNA TARGET region, and the highest targeting effect can be obtained.

When detecting m.3460g > a mutations, suitable PAM structures were found near the m.3460g > a site.

When detecting m.11778G > A or m.14484T > C mutations, m.11778G > A and m.14484T > C sites are not present in the vicinity of the appropriate PAM sequence, and therefore the inventors designed specific RPA primers, and directly introduce PAM (5 '… TTTN …') on the RPA primers, so that mutation sites can be present at different positions in the CRRNA TARGET region by the variation of the RPA primer positions, to screen crRNAs with highest activity. To reduce the background signal of WT, a mismatched base was redesigned to be introduced near the mutation site (within 6bp of each other) on the crRNA.

The invention also provides a kit for detecting the Leber hereditary optic neuropathy, which comprises the composition for detecting the Leber hereditary optic neuropathy based on CRISPR/Cas12, and also comprises the following components which are independently packaged: the fluorescent probe is characterized in that a luminous group and a quenching group are respectively arranged at two ends of the fluorescent probe, the middle of the fluorescent probe is connected by single-stranded DNA (ssDNA), under normal conditions, the fluorescent probe does not excite fluorescence due to the effect of the quenching group, and under specific conditions, the ssDNA breaks and releases the fluorescent group, so that fluorescence reading and identification can be performed. And the fluorescent value is read by using an RT-PCR instrument, and other large instruments are not needed, so that the method is convenient and quick.

As a comparison, the present invention selects the resolution of the crRNA transcription template (Table 7) and the RPA primer pair (Table 8) mentioned in the patent application (CN 114774530A) to detect the mutation sites of m.3460G > A, m.11778G > A and m.14484T > C respectively in a one-step system. Compared with the invention, the system used by CN 114774530A can not well distinguish mutant DNA from wild DNA in one-step detection, and has a larger difference from the system provided by the invention in the distinguishing effect.

Drawings

For purposes of illustration and not limitation, the invention will now be described in accordance with its preferred embodiments, particularly with reference to the accompanying drawings, in which:

FIG. 1 is a flow chart of one-step detection of LHON pathogenic gene mutation.

FIG. 2 is a graph of the results of m.11778G > A site crRNA screening; wherein, crRNA5 vs Mut-PCR: detection results of crRNA5 on mutant PCR products; crRNA5 vs WT-PCR: detection results of crRNA5 on wild-type PCR products; crRNA5 NTC: crRNA5 vs no template control results.

FIG. 3 is a graph showing the results of crRNA screening at the m.3460G > A site. Fig. 3 a: detection results of crRNA1 on mutant PCR products (Mut-PCR), wild-type PCR products (WT-PCR) and No Template Control (NTC); b in fig. 3: detection results of crRNA2 on mutant PCR products (Mut-PCR), wild-type PCR products (WT-PCR) and No Template Control (NTC); FIG. 3C shows the results of crRNA3 detection of mutant PCR products (Mut-PCR), wild-type PCR products (WT-PCR) and No Template Control (NTC); fig. 3D: crRNA4 detection results for mutant PCR products (Mut-PCR), wild-type PCR products (WT-PCR) and No Template Control (NTC).

FIG. 4 is a graph showing the results of crRNA screening at m.14484T > C site. Fig. 4 a: detection results of crRNA6 on mutant PCR products (Mut-PCR), wild-type PCR products (WT-PCR) and No Template Control (NTC); b in fig. 4: detection results of crRNA7 on mutant PCR products (Mut-PCR), wild-type PCR products (WT-PCR) and No Template Control (NTC); FIG. 4C shows the results of crRNA8 detection of mutant PCR products (Mut-PCR), wild-type PCR products (WT-PCR) and No Template Control (NTC); d in fig. 4: crRNA9 detection results for mutant PCR products (Mut-PCR), wild-type PCR products (WT-PCR) and No Template Control (NTC).

Fig. 5 shows the results of the detection of three crrnas, crRNA3, crRNA5, crRNA7, on respective clinical samples, wherein a in fig. 5: detection of crRNA3 in clinical mutant samples (Mut-DNA), wild-type samples (WT-DNA) and No Template Control (NTC); FIG. 5B shows the detection results of crRNA5 in clinical mutant samples (Mut-DNA), wild-type samples (WT-DNA) and template-free control (NTC); FIG. 5C shows the results of crRNA7 detection in clinical mutant samples (Mut-DNA), wild-type samples (WT-DNA) and in No Template Control (NTC).

FIG. 6 shows the results of the detection of mutations m.3460G > A, m.11778G > A and m.14484T > C in a single step using crRNA and RPA primers selected for CN 114774530A. Fig. 6 a: ND1-patent-crRNA and RPA primers detect results in clinical mutant samples (Mut-DNA), wild-type samples (WT-DNA) and template-free controls (NTC); fig. 6B: ND4-patent-crRNA and RPA primers detect results in clinical mutant samples (Mut-DNA), wild-type samples (WT-DNA) and template-free controls (NTC); c in fig. 6: ND6-patent-crRNA and RPA primers detect results in clinical mutant samples (Mut-DNA), wild-type samples (WT-DNA) and No Template Control (NTC).

Detailed Description

The RPA amplification kit of the invention is purchased from TwistDx company; the synthesis of the crRNA template, the T7 primer and the RPA primer pair used for detection is completed by Nanjing Jinsrui company; DNA was pre-extracted using the Tiangen Biotechnology Co.Ltd. The invention detects three primary common mutation sites (m.3460G > A or m.11778G > A or m.14484T > C) of LHON, and comprises the following two parts: (1) screening and determining RPA primer and crRNA of mutation sites to be detected; (2) One-step assay for detecting the presence of a gene mutation in a clinical sample is shown in FIG. 1: extracting 1ml of blood of an object to be detected, extracting DNA, then respectively selecting and combining the RPA primer and crRNA obtained by screening when detecting each site, preparing a reaction system, reacting for 20-30 min in a real-time quantitative PCR instrument, namely, carrying out one-step detection on the sample to be detected, and reading a fluorescence value after the reaction is completed, and judging the result.

Example 1 screening of crRNA for three primary common mutation sites of LHON (m.3460G > A, m.11778G > A and m.14484T > C)

1.1 Nucleic acid preparation

Plasmids carrying three primary common mutation sites (m.3460, m.11778 and m. 14484) of LHON in this example, wild-type (WT) gene fragment and Mutant (MT) gene fragment were synthesized by Nanjin Style, inc., and these gene fragments were cloned into the backbone plasmid of PUC57 (addgene, # 54338) after synthesis, and independent amplification was performed in vitro. The m.3460G > A, m.11778G > A and m.14484T > C site synthesis gene sequences are as follows:

ND 1WT (m.3460 position underlined bold display) ）：atacccatggccaacctcctactcctcattgtacccattctaatcgcaatggcattcctaatgcttaccgaacgaaaaattctaggctatatacaactacgcaaaggccccaacgttgtaggcccctacgggctactacaacccttcgctgacGccataaaactcttcaccaaagagcccctaaaacccgccacatctaccatcaccctctacatcaccgccccgaccttagctctcaccatcgctcttctactatgaacccccctccccatacccaaccccctggtcaacctcaacctaggcctcctatttattctagccac （SEQ ID NO:1）.

ND4 WT (m.11778 position underlined bold):

cccttccttgtactatccctatgaggcataattataacaagctccatctgcctacgacaaacagacctaaaatcgctcattgcatactcttcaatcagccacatagccctcgtagtaacagccattctcatccaaaccccctgaagcttcaccggcgcagtcattctcataatcgcccacgggcttacatcctcattactattctgcctagcaaactcaaactacgaacgcactcacagtcGcatcataatcctctctcaaggacttcaaactctactcccactaatagctttttgatgacttctagcaagcctcgctaacctcgccttaccccccactattaacctactgggagaactctctgtgctagtaaccacgttctcctga（SEQ ID NO:2）.

ND6 WT (m.14484 position underlined bold):

cacccacagcaccaatcctacctccatcgctaaccccactaaaacactcaccaagacctcaacccctgacccccatgcctcaggatactcctcaatagccatcgctgtagtatatccaaagacaaccaTcattccccctaaataaattaaaaaaactattaaacccatataacctcccccaaaattcagaataataacacacccgaccacaccgctaacaatcaatactaaacccccataaataggagaaggct（SEQ ID NO:3）.

The above gene sequences are all WT, and are synthesized and recombined to form the PUC57-ND1-WT, PUC57-ND4-WT and PUC57-ND6-WT plasmids respectively. The three-site mutant sequence is that G (the base shown in bold in the above-mentioned dash line) at the ND1 m.3460 site is mutated to A, G (the base shown in bold in the above-mentioned dash line) at the ND4 m.11778 site is mutated to A and T (the base shown in bold in the above-mentioned dash line) at the ND6 m.14484 site is mutated to C, and the remaining sequences are not changed before and after the mutation to synthesize and recombine into mutant plasmids PUC57-ND1-MT, PUC57-ND4-MT and PUC57-ND6-MT, respectively.

1.2 Design and preparation of crRNA at three primary common mutation sites (m.3460G > A, m.11778G > A and m.14484T > C) of LHON

The target sequences containing the Cas12A recognition sequence (PAM, 5'… TTTN …') are respectively searched within 10bp before and after the m.3460G > A, m.11778G > A and m.14484T > C loci, so that the mutation loci are positioned within 6bp of the CRRNA TARGET region, and the highest targeting effect can be obtained. It was found by searching that the appropriate PAM structure was found only near the m.3460g > A site, whereas the appropriate PAM sequence was not present near the m.11778g > A and m.14484t > C sites, so that A specific RPA primer was designed to introduce PAM directly onto the RPA primer (5 '… TTTN …'), so that mutation sites were present at different positions in the CRRNA TARGET region by variation of the RPA primer positions, to screen crrnas with highest activity. To reduce the background signal of WT, a mismatched base is redesigned to be introduced near the mutation site (within 6bp before and after) on crRNA; after the crRNA design is completed, the crRNA in-vitro transcription template is formed through conversion and is directly synthesized by Nanjing Jinsri company.

Embodiments of the invention provide crRNA sequences (table 1) and in vitro transcription templates thereof (table 2). After in vitro transcription template synthesis, the template was dissolved with ddH ₂ O to a final concentration of 100 μm in working solution, while the T7 promoter template was dissolved. Mu.l of crRNA transcription template and 1. Mu. l T7 of promoter template were taken separately, 8. Mu.l of ddH ₂ O was added for a strand annealing reaction (starting at 95℃and decreasing at a rate of 6℃per minute to 20 ℃) to form the final in vitro transcription template, crRNA was produced and purified using NEB company in vitro transcription kit and purification kit, and the final concentration was adjusted to 400 ng/. Mu.l.

TABLE 1 crRNA sequences used in the present invention

TABLE 2 transcription template sequences required for the invention

1.3 RPA primer design and Synthesis

The PAM structure existing near the m.3460G > A site can be directly utilized, so that the amplified fragment of the designed RPA primer is about 150bp, the m.3460 site is positioned in the middle of the amplified fragment, and the upstream and downstream primers of the RPA primer are respectively 30 bases long; since the m.11778G > A and m.14484T > C loci are not provided with A ready-made PAM structure, PAM (5 '… TTTN …') is directly introduced into one section of RPA primer, so that A mutation locus is positioned at A specific position, and therefore, the position of one primer is relatively fixed and needs to be matched with crRNA; the specific RPA primer sequences are shown in Table 3, and can be directly synthesized by Kirschner company after the design is completed.

TABLE 3 RPA primer sequences required for the invention

1.4 Screening crRNA by PCR product one-step method

The wild type and mutant plasmids of different sites were amplified by using the primer sequences listed in the PCR primer Table 4, and the products were purified by using a PCR product purification kit of Tiangen biological Co., ltd, to obtain DNA templates, the concentration of the products was diluted to 10 ng/. Mu.l, and 1. Mu.l was used as a detection sample for one-step detection.

TABLE 4 general PCR primer sequences and templates for use in the invention

The one-step assay system used in the present invention was 50. Mu.l, and the specific composition settings are shown in Table 5 below.

TABLE 5 one-step System

The reaction is carried out in a fluorescent PCR instrument, the reaction temperature is set to be 42 ℃, the reaction time is 30min, and fluorescent quantitative detection is carried out every 30s during the reaction.

The detection shows that:

For the detection of m.11778G > A, crRNA5 has higher specificity (figure 2), and figure 2 is a diagram of the crRNA screening result of m.11778G > A site; wherein, mut-PCR: mutant PCR products; WT-PCR wild-type PCR product; NTC: no template control was added. As can be seen from FIG. 2, crRNA5 can distinguish between mutant (Mut) and wild-type (WT) PCR products well, and can be used for screening and detection of subsequent clinical samples.

For detection of m.3460G > A, crRNA3 has higher specificity (FIG. 3). FIG. 3 is a graph showing the results of crRNA screening at the m.3460G > A site. Fig. 3 a: detection results of crRNA1 on mutant PCR products (Mut-PCR), wild-type PCR products (WT-PCR) and No Template Control (NTC); b in fig. 3: detection results of crRNA2 on mutant PCR products (Mut-PCR), wild-type PCR products (WT-PCR) and No Template Control (NTC); FIG. 3C shows the results of crRNA3 detection of mutant PCR products (Mut-PCR), wild-type PCR products (WT-PCR) and No Template Control (NTC); fig. 3D: crRNA4 detection results for mutant PCR products (Mut-PCR), wild-type PCR products (WT-PCR) and No Template Control (NTC). As can be seen from FIG. 3, crRNA3 is able to distinguish between mutant (Mut) and wild-type (WT) PCR products with the highest sensitivity in the m.3460G > A site crRNA screen. Whereby crRNA3 can be selected for screening assays for subsequent clinical specimens.

For detection of m.14484T > C, crRNA7 has higher specificity (FIG. 4). FIG. 4 is a graph showing the results of crRNA screening at m.14484T > C site. Fig. 4 a: crRNA6 detection results for mutant PCR products (Mut-PCR), wild-type PCR products (WT-PCR) and No Template (NTC) controls; b in fig. 4: detection results of crRNA7 on mutant PCR products (Mut-PCR), wild-type PCR products (WT-PCR) and No Template Control (NTC); FIG. 4C shows the results of crRNA8 detection of mutant PCR products (Mut-PCR), wild-type PCR products (WT-PCR) and No Template Control (NTC); d in fig. 4: crRNA9 detection results for mutant PCR products (Mut-PCR), wild-type PCR products (WT-PCR) and No Template Control (NTC). As can be seen from FIG. 4, crRNA7 is able to distinguish between mutant (Mut) and wild-type (WT) PCR products with the highest sensitivity in the m.14484T > C site crRNA screen. Whereby crRNA7 can be selected for screening assays for subsequent clinical specimens.

And subsequently, detecting clinical samples, and respectively selecting crRNA3, crRNA5 and crRNA7 to detect the corresponding clinical samples.

Example 2 detection of clinical samples Using one-step method

Clinical samples: according to clinical diagnostic records, one example of blood of patients with m.11778G > A mutant, m.3460G > A mutant and m.14484T > C mutant and one example of blood of normal person are respectively taken as controls.

The detection method comprises the following steps: DNA was extracted from the patient's blood using a DNA extraction kit (manufactured by Tiangen Biotechnology Co., ltd.) and diluted to an appropriate concentration (10 ng/. Mu.l) for use, and then three different mutations were detected using the one-step method described above and shown in FIG. 1, respectively. When detecting m.11778G > A mutation, the reaction is performed using crRNA5 shown in Table 1 and PRA primer set 2 shown in Table 3; when detecting m.3460G > A mutation, a reaction is performed using crRNA3 shown in Table 1 and PRA primer pair 1 shown in Table 3; when detecting the m.14484T > C mutation, a reaction was performed using crRNA7 shown in Table 1 and PRA primer pair 3 shown in Table 3. The amounts of the reaction system and other required reagents added are shown in Table 6 below.

TABLE 6 reaction System for one-step detection of clinical samples

As described above, RPA buffers and powders are available from TwistDx, ssDNA-FQ is available from bioengineering limited, cas12a is available from NEB, mg ²⁺ is carried by the RPA powders and buffer reagents. Mg ²⁺ needs to be added last. After the construction of the reaction system is completed, the reaction system is immediately and evenly vibrated, and after short centrifugation, the reaction system is quickly placed in a real-time quantitative PCR instrument for reaction. The reaction set up was: and (3) performing fluorescence quantitative detection every 30s at 42 ℃ for 30min, and judging the result after the reaction is completed.

The results are shown in FIG. 5, and FIG. 5 is a graph of the detection results of three crRNAs, namely crRNA3, crRNA5, and crRNA7, at the respective mutation sites, wherein A in FIG. 5: detection results of crRNA3 in clinical mutant samples and wild type samples; FIG. 5B shows the results of crRNA5 detection in clinical mutant and normal samples; FIG. 5C shows the results of crRNA7 detection in clinical mutant samples and normal samples. WT-DNA: normal human DNA samples; mut-DNA: a clinical mutant DNA sample; NTC: no template control. As can be seen from FIG. 5, the one-step detection system provided by the present invention can rapidly and accurately detect mutations in respective clinical samples.

The crRNA and RPA systems selected in comparative example 1, patent application (CN 114774530A) were tested on the same clinical samples in a one-step assay system.

The detection method used in CN 114774530A is a two-step method detection, which is significantly different from the present invention in terms of steps. The one-step detection of the invention is simpler and more convenient in implementation steps. In design, the invention detects m.11778G > A and m.14484T > C sites by introducing PAM on the RPA primer, thus the mutation site is located in a 6bp seed region away from the PAM, so that the crRNA has higher specificity.

And selecting crRNA and RPA primers for detecting m.3460G > A, crRNA and RPA primers for detecting m.11778G > A and crRNA and RPA primers for detecting m.14484T > C described in CN 114774530A to verify the detection effect in a one-step system. crRNA preparation procedure reference is made to the preparation method in example 1: in the preparation of crRNA for detecting m.3460G > A site, the corresponding template shown in Table 7 (SEQ ID NO: 35) is selected; in preparing crRNA for detecting m.11778G > A site, selecting corresponding template (SEQ ID NO: 36) shown in Table 7; in preparing crRNA for detecting m.14484T > C site, the corresponding template shown in Table 7 (SEQ ID NO: 37) was selected. The one-step method for detecting clinical samples was performed as described in example 2 above, and the clinical Mut-DNA and WT-DNA selected for detecting the m.3460G > A site were identical to the samples detected for crRNA3 in example 2 above; the clinical Mut-DNA and WT-DNA selected when detecting the m.11778G > A site were identical to the samples detected for crRNA5 in example 2 above; the clinical Mut-DNA and WT-DNA selected for detection of the m.14484T > C site were identical to the samples detected for crRNA7 in example 2 above. Selecting RPA primer pair 7 shown in Table 8 for reaction when detecting m.3460G > A locus; selecting RPA primer pair 8 shown in Table 8 for reaction when detecting m.11778G > A locus; the RPA primer pair 9 shown in Table 8 was selected for reaction when detecting the m.14484T > C site.

The results are shown in FIG. 6, and FIG. 6 shows the results of the one-step detection of mutations in m.3460G > A, m.11778G > A and m.14484T > C using crRNA and RPA primers selected in CN 114774530A. Fig. 6 a: ND1-patent-crRNA and RPA systems detect results in clinical mutant samples (Mut-DNA), wild-type samples (WT-DNA) and template-free controls (NTC); fig. 6B: ND4-patent-crRNA and RPA systems detect results in clinical mutant samples (Mut-DNA), wild-type samples (WT-DNA) and template-free controls (NTC); c in fig. 6: ND6-patent-crRNA and RPA systems detect results in clinical mutant samples (Mut-DNA), wild-type samples (WT-DNA) and template-free controls (NTC). As can be seen from the results in FIG. 6, in the one-step reaction system, the ND1 system and ND6 system selected by CN 114774530A cannot well distinguish Mut-DNA from WT-DNA, and there is a larger difference in resolution compared with the corresponding systems (A and C in FIG. 5) provided by the present invention. Although ND4 system can distinguish Mut-DNA and WT-DNA at the beginning of reaction, as the reaction proceeds, the fluorescence intensity in WT-DNA is continuously enhanced to a degree similar to that of Mut-DNA, and a certain interference exists at the later stage, which has a larger difference from ND4 detection system (B in FIG. 5) provided by the invention in the final effect. In summary, the detection system of m.3460G > A, m.11778G > A and m.14484T > C provided by the invention has higher detection resolution and effect in one-step detection compared with other patents.

Table 7 corresponding transcription templates of crRNA selected for CN 114774530A

Table 8 RPA primer selected from CN 114774530A

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. The invention provides a one-step method for detecting three common primary mutations (m.3460G > A or m.11778G > A or m.14484T > C) of LHON, which has the characteristics of sensitivity, specificity and rapidness in implementation and shortens the reaction time to half an hour.

2. The rapid detection technology related by the invention uses the RT-PCR instrument to read the fluorescence value, does not need other large-scale instruments, is convenient and rapid, and is suitable for large-scale screening in general hospitals and laboratories.

3. The reagent related by the invention has been commercialized or synthesized by commercial companies, the cost of large-scale screening reagent is reduced, and the economic and time costs are greatly saved.

The above embodiments do not limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications, combinations, sub-combinations and alternatives can occur depending upon design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of the present invention.

Claims (10)

1. A composition for detecting Leber hereditary optic neuropathy based on CRISPR/Cas12, characterized in that: the composition comprises a crRNA and an RPA amplification primer pair;

The crRNA has at least one of the sequences shown in SEQ ID NO. 4 to SEQ ID NO. 12;

The RPA amplification primer pair is at least one of the following primer pairs: a primer pair consisting of sequences shown as SEQ ID NO. 23 and SEQ ID NO. 24, a primer pair consisting of sequences shown as SEQ ID NO. 25 and SEQ ID NO. 26 or a primer pair consisting of sequences shown as SEQ ID NO. 27 and SEQ ID NO. 28.

2. The CRISPR/Cas 12-based composition for detecting Leber hereditary optic neuropathy according to claim 1, wherein: the Leber hereditary optic neuropathy has at least one of the following three primary mutation sites: m.3460G > A, m.11778G > A and m.14484T > C.

3. The CRISPR/Cas 12-based composition for detecting Leber hereditary optic neuropathy according to claim 2, wherein when detecting m.11778 g > a mutation:

The crRNA has a sequence shown as SEQ ID NO. 8, and the RPA amplification primer pair has a primer pair consisting of sequences shown as SEQ ID NO. 25 and SEQ ID NO. 26.

4. The CRISPR/Cas 12-based composition for detecting Leber hereditary optic neuropathy according to claim 2, wherein when detecting m.3460g > a mutation:

The crRNA has a sequence shown as SEQ ID NO. 6, and the RPA amplification primer pair has a primer pair consisting of sequences shown as SEQ ID NO. 23 and SEQ ID NO. 24.

5. The CRISPR/Cas 12-based composition for detecting Leber hereditary optic neuropathy according to claim 2, wherein when detecting m.14484t > c mutation:

The crRNA has a sequence shown as SEQ ID NO. 10, and the RPA amplification primer pair has a primer pair consisting of sequences shown as SEQ ID NO. 27 and SEQ ID NO. 28.

6. Use of the CRISPR/Cas 12-based composition for detecting Leber hereditary optic neuropathy according to any one of claims 1 to 5 in the preparation of a kit for detecting Leber hereditary optic neuropathy.

7. A kit for detecting Leber hereditary optic neuropathy, which is characterized in that: the kit comprises the CRISPR/Cas 12-based composition for detecting Leber hereditary optic neuropathy according to any one of claims 1 to 5.

8. The kit for detecting Leber hereditary optic neuropathy according to claim 7, wherein: the kit further comprises the following components in independent package: RPA powder, reaction buffer, cas12a protein, and fluorescent probe that can be specifically cleaved by Cas12 a; the fluorescent probe capable of being specifically cut by Cas12a has two ends respectively provided with a luminous group and a quenching group, and the middle is connected by single-stranded DNA.

9. Use of the CRISPR/Cas 12-based kit for detecting Leber hereditary optic neuropathy according to any one of claims 7 or 8 in preparing a reaction system for detecting primary mutation sites of Leber hereditary optic neuropathy in clinical samples.

10. The use according to claim 9, characterized in that:

the clinical sample is a clinical blood sample;

The primary mutation site of the Leber hereditary optic neuropathy is at least one of the following: m.3460G > A, m.11778G > A, m.14484T > C.