CN113201586B

CN113201586B - Cas protein-based detection method

Info

Publication number: CN113201586B
Application number: CN202110437897.4A
Authority: CN
Inventors: 梁德生; 张春华; 周妙金; 邬玲仟
Original assignee: Suzhou Ganjiang Biotechnology Co ltd
Current assignee: Suzhou Ganjiang Biotechnology Co ltd
Priority date: 2021-04-22
Filing date: 2021-04-22
Publication date: 2024-03-26
Anticipated expiration: 2041-04-22
Also published as: CN113201586A

Abstract

The invention relates to a nucleic acid detection method based on Cas protein, which comprises the following steps: s1, designing and synthesizing corresponding connection probes according to sequences of a target gene and an internal reference gene, and designing and synthesizing PCR primers for amplifying the connection probes and crRNA for identifying the connection probes according to the connection probes; s2, respectively hybridizing the target gene and the reference gene with corresponding connecting probes, connecting the hybridized connecting probes into single-stranded DNA by DNA ligase, and carrying out PCR amplification on the single-stranded DNA by adopting the PCR primer in the step S1 to obtain an amplification product; detecting an amplification product by using the Cas protein, the crRNA in the step S1 and a fluorescent probe system, and quantitatively detecting a target gene; the ligation probe comprises PAM sequences and crRNA recognition region sequences required for Cas protein detection. The invention establishes a quantitative detection method and expands the application of the Cas12a nucleic acid detection technology in genetic disease detection.

Description

Cas protein-based detection method

Technical Field

The invention relates to a method for quantitatively detecting target nucleic acid molecules by using Cas proteins.

Background

Clustered regularly interspaced short palindromic repeats (clustered regularly interspaced short palindrome repeats, CRISPR) and CRISPR-associated nucleases (CRISPR associated proteins, cas) ubiquitous in bacteria and archaebacteria are the defenses of bacteria against exogenous plasmid or phage infection, and modern scientists have successively discovered a variety of Cas proteins and their mechanisms of operation. Cas12a specifically recognizes and is activated under the guidance of CRISPR RNA (crRNA) target double stranded DNA (dsDNA), and activated Cas12a can specifically cleave both target dsDNA molecules and non-specifically cleave unrelated single stranded DNA (ssDNA), i.e., with an accompanying cleavage activity.

Detection of target dsDNA molecules using the incidental cleavage activity of Cas12a, prior art protocol principle: (1) designing a primer according to the sequence of the target dsDNA molecule to be detected, amplifying the target dsDNA molecule by a PCR or isothermal amplification method, if the target dsDNA molecule exists, designing and synthesizing crRNA according to the sequence of the target dsDNA molecule to be detected, guiding Cas12a to recognize the target dsDNA molecule, (3) taking the amplified product, incubating with Cas12a, crRNA and probe ssDNA (fluorescent probe), and (4) detecting whether the Cas12a is activated or not by an instrument after the incubation is finished, thereby qualitatively detecting the target dsDNA molecule.

Chen et al combine Cas12a with recombinase polymerase isothermal amplification (recombinase polymerase amplification, RPA) technology, li et al combine Cas12a with PCR technology, and respectively develop nucleic acid detection technologies DETECTER and HOLMES based on Cas12a, the basic principle is: the crRNA is designed according to the target dsDNA, the amplified target dsDNA molecule can be specifically identified by the Cas12a-crRNA complex, simultaneously Cas12a is activated, the activated Cas12a is utilized for cutting the ssDNA probe, the fluorescent group and the quenching group are respectively marked at the two ends of the probe, the fluorescent signal is released after the probe is cut, and the target dsDNA is detected by reading the detection signal released after the probe is cut (figure 1) ^[1,2] 。

However, cas12 a-based nucleic acid detection techniques have the following limitations:

1. restriction of protospacer adjacent motifs (protospacer adjacent motif, PAM)

The first precondition for Cas12a to recognize target dsDNA is: the target dsDNA must be present with the PAM sequence (TTTN or TTN) required for Cas12a. Only in the presence of PAM sequences it is possible to design crrnas 3' downstream of PAM sequences and detect them (fig. 2). If no PAM sequence is present in the target sequence, then crRNA cannot be designed and therefore cannot be detected with Cas12a. PAM sequences greatly limit the range of choice of target sequences and flexibility in crRNA design.

Restriction of crRNA

A second precondition for Cas12a to recognize target dsDNA is: the target dsDNA must have the appropriate crRNA recognition region sequence present. crRNA is a key molecule that directs Cas12a to recognize and determine detection specificity. Even if a PAM sequence is present in the target sequence to be detected, if a crRNA with high specificity cannot be designed downstream of the PAM sequence (fig. 2), detection will not be performed or the specificity of the detection will be poor.

3. Limited to qualitative detection

Most of the existing Cas12a nucleic acid detection technologies are qualitative detection, and the qualitative detection can only judge whether the target nucleic acid molecule exists or not, but cannot judge the copy number of the target nucleic acid molecule. Only a few studies have conceptually achieved quantitative detection ^[3,4] However, in practical applications, there is often a concentration difference between samples, and in order to detect and compare the copy number of target nucleic acid molecules in different samples and their differences, internal references are also required to be established to eliminate the interference of the sample concentration difference on the quantitative detection result.

4. Limited to pathogen detection

The pathogen genome sequence is relatively large, so that a proper PAM sequence is hopeful to be found and crRNA with higher specificity is designed, and therefore, a plurality of documents report that Cas12a nucleic acid detection technology is applied to Zika virus and dengue virus at present ^[5] Novel coronavirus pneumovirus ^[6 _, ^7] And Mycobacterium tuberculosis ^[8] Qualitative detection of a variety of pathogens, etc.

In theory, cas12a nucleic acid detection technology can be used for human genetic disease detection, but due to the limitations described above, existing Cas12a nucleic acid detection technology faces 2-way difficulties in human genetic disease detection. First, point mutations or small indels, because of their small range of variation, limited to 1 or a few bases, often lack PAM sequences in their vicinity or make it difficult to design appropriate crrnas, cannot be detected using current Cas12a nucleic acid detection techniques. Second, for large fragment insertions or deletions, quantitative determination of the copy number of the insertion or deletion sequence is often required to distinguish normal individuals, carriers and patients.

As is well known to those skilled in the art, normal persons typically have 2 copies of the SMN1 gene No.7 exon, the SMN1 gene No.7 exon of SMA carrier is heterozygous deleted (copy number=1), the SMN1 gene No.7 exon of SMA patient is homozygous deleted (copy number=0), and the existing Cas12a nucleic acid detection technology can distinguish SMA patient from non-SMA patient (normal person or SMA carrier) by qualitative detection, but cannot further distinguish normal person from SMA carrier, so the existing Cas12a nucleic acid detection technology has many defects.

Reference to the literature

[1]CHEN J S,MA E,HARRINGTON L B,et al.,CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity[J].Science,2018,360:436-439.

[2]LI S Y,CHENG Q X,WANG J M,et al.,CRISPR-Cas12a-assisted nucleic acid detection[J].Cell discovery,2018,4:20.

[3]LI L,LI S,WU N,et al.,HOLMESv2:A CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation Quantitation[J].ACS synthetic biology,2019,8:2228-2237.

[4]LI H,LI M,YANG Y,et al.,Aptamer-Linked CRISPR/Cas12a-Based Immunoassay[J].Anal.Chem.,2021,93:3209-3216.

[5]CURTI L A,PEREYRA-BONNET F,REPIZO G D,et al.,CRISPR-based platform for carbapenemases and emerging viruses detection using Cas12a(Cpf1)effector nuclease[J].Emerging microbes&infections,2020,9:1140-1148.

[6]BROUGHTON J P,DENG X,YU G,et al.,CRISPR-Cas12-based detection of SARS-CoV-2[J].Nat.Biotechnol.,2020.

[7]KELLNER M J,KOOB J G,GOOTENBERG J S,et al.,SHERLOCK:nucleic acid detection with CRISPR nucleases[J].Nat.Protoc.,2019,14:2986-3012.

[8]AI J W,ZHOU X,XU T,et al.,CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis[J].Emerging microbes&infections,2019,8:1361-1369.

Disclosure of Invention

The invention aims to artificially introduce PAM sequences and crRNA recognition regions through a probe-ligation strategy, and the PAM sequences and the crRNA recognition regions on target dsDNA are not relied on any more, so that the target dsDNA can be detected even if the target dsDNA has no PAM sequences or specific crRNA is difficult to design. Based on probe connection, the copy number of the target gene is quantitatively detected by introducing an internal reference gene.

In order to solve the technical problems, the technical scheme of the invention is as follows:

a Cas protein-based detection method, comprising the steps of:

s1, designing and synthesizing corresponding connection probes according to sequences of a target gene and an internal reference gene, and designing and synthesizing PCR primers for amplifying the connection probes and crRNA for identifying the connection probes according to the connection probes;

s2, respectively hybridizing the target gene and the reference gene with corresponding connecting probes, connecting the hybridized connecting probes into single-stranded DNA by DNA ligase, and carrying out PCR amplification on the single-stranded DNA by adopting the PCR primer in the step S1 to obtain an amplification product; detecting an amplified product by using the Cas protein, the crRNA and a fluorescent probe system in the step S1, and further quantitatively detecting a target gene;

the ligation probe comprises a PAM sequence and a crRNA recognition region sequence.

The ligation probes designed and synthesized according to the target gene or reference gene are usually two DNA sequences, but may be plural, for example, 4 ligation probes, and the ligation probes after hybridization formed after hybridization with the target gene are usually two or plural.

The design of the synthetic ligation probe requires verification, verification of effectiveness (whether ligation is successful), specificity (whether target sequences can be specifically identified), and finally the effective and specific ligation probe is used.

Preferably, the Cas protein is Cas12a, cas13, cas12b or Cas14.

Preferably, the Cas protein is Cas12a.

Cas12a and Cas12b can directly recognize dsDNA, cas13 recognizes that the target nucleic acid molecule is single-stranded RNA (ssDNA, and Cas14 recognizes that the target nucleic acid molecule is ssDNA in the application example is dsDNA, and Cas12a or Cas12b can be used for direct recognition, if Cas13 is used, a detection step is added to transcribe ssRNA molecule from the target dsDNA molecule, and if Cas14 is used, a detection step is also added, 1 strand of the target dsDNA molecule needs to be degraded, and ssDNA molecule can be formed before detection.

Preferably, the hybridized ligation probes (F and R) are ligated into ssDNA using a DNA ligase in step S2.

Preferably, the DNA ligase is a thermostable DNA ligase.

In the probe ligation reaction, firstly, the dsDNA to be detected needs to be incubated at 95 ℃ to enable the dsDNA to be melted into 2 ssDNA, then, the temperature is gradually reduced, and the ligation probes (F and R) can be hybridized with the ssDNA. After hybridization, ligation probes (F and R) were ligated by ligase to form 1 ssDNA. If the selected DNA ligase is not heat-resistant, in order to avoid the inactivation of the DNA ligase in the melting process of 95 ℃, the ligase cannot be directly added when preparing a connection reaction system, and the ligase can be added after the melting and cooling hybridization of 95 ℃, thereby increasing the operation steps and the risk of cross contamination. The heat-resistant DNA ligase has thermal stability, can not be inactivated at 95 ℃, can be directly added when preparing a ligation reaction system, does not need to be added in the reaction process, and completes ligation reaction once.

Preferably, the thermostable DNA ligase is HiFi Taq DNA Ligase.

HiFi Taq DNA Ligase is not only thermostable, but also recognizes mismatched bases between the ligation probe and the target ssDNA, especially when the base mismatch between the ligation probe and the target ssDNA is located at the 3 'end of the upstream probe or the 5' end of the downstream probe, even if the upstream and downstream ligation probes have hybridized with the target ssDNA, they are not ligated. The sensitivity of HiFi Taq DNA Ligase to base mismatches can be used to improve the specificity of the assay.

Preferably, in the step S2, the ligation reaction between the target gene and the reference gene and the corresponding ligation probes is performed in one system.

The connection reaction of the target gene and the reference gene is completed in one system, so that the operation steps can be reduced, and the detection cost can be reduced.

The invention also claims a ligation probe whose sequence comprises a PAM sequence and a crRNA recognition region sequence.

Preferably, the PAM sequence is TTTN (SEQ ID No. 16) or TTN (SEQ ID No. 17).

Preferably, the crRNA recognition region sequence comprises 18-23 bases.

The invention also claims a kit comprising a ligation probe as described above, PCR primers for amplifying the ligation probe as described above and crRNA for recognizing the ligation probe as described above.

The invention also claims the application of the kit in preparing a reagent for detecting the No.7 exon of the SMN1 gene.

Preferably, the target gene is exon 7 of SMN1 gene; the reference gene is ALB.

The ALB gene has a copy number of 2 in the human genome and is less likely to be deleted or duplicated.

Preferably, the sequence of the connecting probe is shown as SEQ ID NO. 1-4; the sequence of PCR primer of the connection probe amplified as shown in SEQ ID NO.1-4 is shown in SEQ ID NO. 5-6; the sequence of crRNA that recognizes the ligation probe shown as SEQ ID NO.1-4 is shown as SEQ ID NO.7-8 and Table 1.

TABLE 1 PCR primer, ligation probe and crRNA sequence information

Preferably, in the step S2, the target gene and the reference gene are hybridized with the ligation probes shown as SEQ ID NO.1-4, PCR amplification is performed by using the PCR primers shown as SEQ ID NO.5-6, and PCR amplification is performed by detecting crRNA shown as SEQ ID NO. 7-8.

The invention also provides application of the connection probe in preparation of a reagent for detecting the No.7 exon of the SMN1 gene.

A PCR primer for amplifying the connection probe shown as SEQ ID NO.1-4 has the sequence shown as SEQ ID NO. 5-6.

The invention also provides application of the PCR primer for amplifying the connection probe shown as SEQ ID NO.1-4 in preparing a reagent for detecting the No.7 exon of the SMN1 gene.

The crRNA of the ligation probe with the recognition sequence shown in SEQ ID NO.1-4 is identified, and the sequence of the crRNA of the ligation probe with the recognition sequence shown in SEQ ID NO.1-4 is shown in SEQ ID NO. 7-8.

The invention also provides application of the crRNA for identifying the connection probe shown as SEQ ID NO.1-4 in preparing a reagent for detecting the No.7 exon of the SMN1 gene.

A kit comprising a ligation probe as shown in SEQ ID No.1-4, PCR primers for amplifying the ligation probe as shown in SEQ ID No.1-4, and crRNA for recognizing the ligation probe as shown in SEQ ID No. 1-4.

Preferably, the kit comprises a ligation probe as shown in SEQ ID NO.1-4, a PCR primer as shown in SEQ ID NO.5-6 and a crRNA as shown in SEQ ID NO. 7-8.

The invention also provides application of the kit in preparation of a reagent for detecting the No.7 exon of the SMN1 gene.

Preferably, the connection reaction system is:

preferably, the connection reaction conditions are as shown in fig. 4, and the connection product is obtained through the reaction.

Preferably, the PCR amplification system is as follows:

preferably, the fluorescence detection of the target gene and the reference gene is performed in two systems, and the SMN1 gene and the ALB gene are detected separately.

Cas12a is activated upon recognition of the target sequence under the guidance of crRNA, and activated Cas12a will release the detection signal from the non-specific cleavage probe molecule. The ligation and amplification reactions described above were completed in 1 system, and the ligation amplification products contained both the ligation amplification products of the target gene and the ligation amplification products of the reference gene. The crRNA recognition region sequences of the two products are different and can be recognized by Cas12a under the guidance of different crrnas. When 1 individual system has only 1crRNA, the ligation amplification products of the target gene and the internal reference gene can be detected respectively. When there are two crrnas in 1 system, the ligation amplification products of the target gene and the reference gene cannot be detected specifically.

The invention is further explained below:

the copy numbers of the target dsDNA in different samples can be different due to different concentrations, in order to avoid the interference of the sample concentration on the detection result, the reference genes in the samples are selected as references, and the influence of the concentration difference on the detection result is eliminated, so that the relative quantitative detection of the target dsDNA is realized, and the comparison can be carried out among different samples.

If the synthetic PCR primers are designed based only on the target dsDNA and the internal reference dsDNA sequences, amplification bias may occur due to differences in amplification efficiency between the target dsDNA and the internal reference dsDNA due to factors such as primer sequences, amplified fragment lengths, etc., ultimately affecting the accuracy of the relative quantitative results. The same PCR amplified primer binding region can be introduced through the probe connection strategy, and the difference of the amplified fragment length can be controlled, so that the amplification efficiency of the target dsDNA is similar to that of the internal reference dsDNA, and the detection accuracy is improved. More importantly, the quantitative detection effect can be achieved through the probe connection strategy.

The detection principle of the invention:

(1) the SMN1 gene No.7 exon is used as a target gene, and a human Albumin gene (Albumin, ALB) is used as an internal reference gene.

(2) The connection recognition region of the SMN1 connection probe F/R is completely complementary with the No.7 exon of the SMN1 gene, annealing is carried out after melting at 95 ℃, the SMN1 connection probe F/R can be hybridized with the No.7 exon of the SMN1 gene, the connection probe F/R of the SMN1 is connected into 1 single chain under the action of ligase, the 2 end of the single chain is the binding region of the PCR primer F/R, a double-chain connection amplification product is generated after PCR amplification, and the double-chain connection amplification product comprises a PAM sequence and a crRNA recognition region sequence. The 3' -terminal base of the SMN1 ligation probe F has 1 mismatch with the exon 7 of the SMN2 gene, and even if the SMN1 ligation probe F/R hybridizes with the exon 7 of the SMN2 gene, the ligation probe F/R cannot be ligated into a single strand by a ligase, and a ligation amplification product cannot be generated by PCR amplification. The ligation principle of the ALB ligation probe was the same as that of SMN1, and the ligation amplification product of the ALB ligation probe contained the same binding region, PAM sequence and crRNA recognition region sequence of the PCR primer F/R as that of the SMN1 ligation amplification product (FIG. 3 a).

(3) Normal people carry 2 copies of the 7-exon of the SMN1 gene and 2 copies of the ALB internal reference gene, after connection and amplification, the copy numbers of the 2 gene connection amplification products are approximately the same, and finally the ratio of the SMN1 to the ALB fluorescent signal is close to 1; heterozygous deletion of the No.7 exon of the SMN1 gene of the SMA carrier occurs, the copy number of the connection amplification product of the No.7 exon of the SMN1 gene is about 1/2 of that of the connection amplification product of the ALB of the internal reference gene, and the ratio of the SMN1 to the ALB fluorescent signal is close to 1/2; the SMA patient had homozygous deletion of exon 7 of the SMN1 gene, no ligation amplification product of exon 7 of the SMN1 gene, and normal production of ligation amplification product of the reference gene ALB, with a final SMN1 to ALB fluorescent signal ratio of approximately 0 (FIG. 3 b).

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

(1) the limitations of PAM sequences and crRNA are broken through, and even under the condition that no PAM sequence exists near the mutation site to be detected or proper crRNA cannot be designed, the PAM sequences and crRNA can be manually introduced through the probe and detected.

(2) The quantitative detection method is established, the copy number of the mutation site can be quantitatively detected, and the application of the Cas12a nucleic acid detection technology in quantitative detection is expanded.

(3) Based on the quantitative detection of the Cas12a connected with the probe, the connection and amplification reaction of the target gene and the reference gene can be completed in 1 reaction system, thus reflecting the capability of multiple detection, being beneficial to reducing operation steps and reducing detection cost.

Drawings

FIG. 1 is a schematic diagram of a nucleic acid detection technique based on Cas12a in the prior art;

FIG. 2 is a schematic representation of PAM sequences and crRNA recognition regions;

fig. 3 is a schematic diagram of the principle of Cas12a quantitative detection based on probe ligation according to the present invention;

FIG. 4 is a schematic diagram of the probe ligation reaction conditions according to the invention;

FIG. 5 shows PCR conditions according to the present invention;

FIG. 6 is a graph depicting the ability of the present invention to verify the quantitative detection method of SMA-Cas12a based on probe ligation to distinguish normal humans from SMA patients;

FIG. 7 is a graph of fluorescence values for normal person, carrier, and SMA patient (1 case each) tested in accordance with the present invention;

FIG. 8 is a graph showing the ratio of fluorescence values for normal person, SMA carrier, and SMA patient for detection in accordance with the present invention;

FIG. 9 is the ability of the prior art SMA-Cas12a fluorescence detection method to distinguish between normal and SMA patients under different SMN1crRNA conditions;

FIG. 10 shows the results of screening SMN1 ligation probes according to the present invention;

FIG. 11 shows the results of screening ALB-ligated probes according to the present invention.

Detailed Description

The present invention will be described in detail with reference to examples. It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.

Example 1

Spinal muscular atrophy (spinal muscular atrophy, SMA) is an autosomal recessive genetic disease, progressive muscle weakness and muscular atrophy of skeletal muscle are caused by degeneration of spinal cord anterior horn motor neurons, the disease is gradually aggravated, dysphagia and spontaneous breathing are caused, and finally death is caused by paralysis of respiratory muscles, and the incidence rate is 1/6000-1/10000.SMA belongs to a severe fatal and disabling genetic disease, and the carrying rate of pathogenic genes in the crowd is as high as 1/40-1/50, which brings heavy burden to families and society of patients, and constitutes a serious public health problem.

The causative gene of SMA is the SMN1 gene encoding motor neuron survivin (survival motor neuron, SMN). Although there is a copy of the SMN2 gene in the human genome that is highly homologous to the SMN1 gene, the SMN1 and SMN2 genes differ in the 7 th exon by c.840c > T, resulting in the SMN2 gene not being able to compensate for the function of the SMN1 gene. The deletion of exon 7 of SMN1 gene is a hot spot mutation that results in SMA, and homozygous deletion of exon 7 of SMN1 gene is present in 95% of SMA patients.

SMA-Cas12a fluorescence quantitative detection method based on probe connection

1. Method of

(1) The ligation probe, ligation probe PCR primer and probe crRNA were synthesized according to the design of exon 7 and reference ALB gene of SMN1 gene as shown in Table 1:

TABLE 1 PCR primer, ligation probe and crRNA sequence information

(2) SMA-Cas12a detection method based on probe connection identifies specificity of No.7 exon of SMN1 gene

(1) And selecting 1 gDNA sample and blank (water) of normal person and SMA patient, checking copy number of 7 exons of SMN1 gene by MLPA detection, wherein the normal person is 2 copies, and the SMA patient is 0 copies.

(2) The probe ligation reaction system for SMN1 was as follows:

(3) after the reaction system is prepared, the materials are reversed and mixed for 30 times, centrifuged briefly and placed in a PCR instrument for probe connection.

The probe ligation reaction is shown in FIG. 4.

(4) PCR amplification was performed using 4. Mu.L of the ligation reaction product as a template.

The PCR amplification system was as follows:

the PCR reaction conditions are shown in FIG. 5.

(5) Taking PCR products to detect SMA-Cas12a fluorescent probes, arranging 3 compound holes in each sample, preparing a mixed system according to 10 parts, wherein the concentration of Lba Cas12a (NEB, M0653T) used in detection is 100 mu M, and the mixed system is as follows:

after mixing, 10 tubes were packed in 18. Mu.L/tube, 2. Mu.L of PCR amplification product was added to each tube, and after capping, mixing was performed, after a short centrifugation, incubation was performed at 37℃and the reaction was started to read the initial fluorescence value, after which 1 fluorescence value was read every 1 min. Delta fluorescence value = endpoint fluorescence value (30 min) -initial fluorescence value (0 min). The difference in fluorescence values between normal, SMA patient and blank (water) was compared.

The results were: under the conditions of 5 amplification 30 cycles, the fluorescence signal of normal was significantly higher than that of SMA patients and blank (water), and the fluorescence signal of normal was 35.39 times that of SMA patients (fig. 6). The results show that: (1) The normal human gDNA sample contains the SMN1 gene No.7 exon, the probe recognizes the SMN1 gene No.7 exon to hybridize, the hybridized DNA is connected into single-stranded DNA by DNA ligase, and the single-stranded DNA is amplified by PCR to generate a large amount of connected amplified products. The ligation amplification product contains artificially introduced PAM and crRNA recognition region sequences, the PAM and crRNA recognition region sequences are recognized by the Cas12a/crRNA, the Cas12a is activated, and the activated Cas12a non-specific cleavage fluorescent probe generates an obvious fluorescent signal. (2) The SMA patient and the blank (water) have no exon 7 of the SMN1 gene, the probe cannot be connected into a single chain, a connection amplification product cannot be generated through PCR amplification, and no obvious fluorescent signal is generated. (3) The SMA-Cas12a detection method based on probe connection has higher specificity for distinguishing normal people from SMA patients, and the fluorescence value difference between the normal people and the SMA patients is 35.39 times.

(3) SMA-Cas12a quantitative detection method based on probe connection establishment and verification

(1) And selecting 20 parts (60 parts in total) of gDNA samples of a normal person, an SMA carrier and an SMA patient, wherein all the samples are subjected to MLPA detection to verify the copy number of the 7-exon of the SMN1 gene, the normal person is 2 copies, the SMA carrier is 1 copy, and the SMA patient is 0 copy.

(2) The initial concentration of DNA in the sample was measured, gDNA was taken at 10. Mu.L, diluted with an appropriate amount of TE solution, and the final concentration was in the range of 5-10 ng/. Mu.L. The diluted gDNA samples were tested in batches. 10 samples were tested per batch, 9 of the gDNA samples and 1 of the blank (water).

(3) The probe ligation reaction of SMN1 and ALB was performed in 1 system, and a probe ligation mixed system of SMN1 and ALB was prepared in 10 parts.

SMN1 and ALB probe connection mixed system

After the preparation of the mixed system is completed, the mixed system is inverted and evenly mixed, and the mixture is centrifuged for a short time. The probe-ligated mixture of SMN1 and ALB was dispensed into 10 PCR tubes at 22.5. Mu.L/tube.

And adding the diluted gDNA sample into the split-packed probe connection mixed system according to 2.5 mu L/pipe, mixing the mixed system for 30 times in a reversed way, centrifuging the mixed system briefly, and placing the mixed system in a PCR instrument for probe connection.

The probe ligation reaction conditions are shown in FIG. 4.

(4) The PCR amplification was performed in 1 system, and a mixed PCR amplification system of SMN1 and ALB was prepared in 10 parts.

PCR amplification mixed system

After the preparation of the mixed system is completed, the mixed system is evenly mixed and centrifuged for a short time. The PCR amplification mix was split into 10 PCR tubes at 16. Mu.L/tube. 4. Mu.L of the probe-ligated product was added to the PCR amplification mix, and the total volume of each tube was 20. Mu.L. After being evenly mixed, the mixture is centrifuged for a short time and is amplified by a PCR instrument.

The PCR reaction conditions are shown in FIG. 5.

(5) The SMA-Cas12a fluorescent probe detection is carried out by a system 2, the SMN1 and ALB genes are detected respectively, and 3 compound holes are arranged on an SMN1 pipe and an ALB pipe respectively. Since the SMN1 gene was detected using the probe crRNA1 and the ALB gene was detected using the probe crRNA2, a mixed system was prepared at 30 parts. The Lba Cas12a (NEB, M0653T) concentration used for detection was 100. Mu.M.

The SMN1 mixed system is as follows:

the ALB mixing system is as follows:

after the preparation of the mixed system is completed, the mixed system is reversed and evenly mixed, and the mixture is centrifuged for a short time.

Each gDNA sample was tested for fluorescence values of SMN1 and ALB, respectively, and SMN1 and ALB were each provided with 3 wells, 60 wells in total. The 8-piece tube was placed in a pre-chilled PCR ice box and the mixture was dispensed to 60 wells at 18. Mu.L/well. Ligation amplification products of the same gDNA sample were ligated into 3 multiplex wells (total volume 20. Mu.L) corresponding to 2. Mu.L/Kong Jiazhi. After capping, centrifugation was performed briefly, incubation was performed at 37℃and the reaction was started to read the initial fluorescence value, after which 1 fluorescence value was read every 1 min. Delta fluorescence value = endpoint fluorescence value (15 min) -initial fluorescence value (0 min). And determining the copy number of the No.7 exon of the SMN1 gene by calculating the ratio of the SMN1 delta fluorescence value to the ALB delta fluorescence value.

The results were as follows:

(1) the fluorescence value curves of the normal SMN1 gene, exon 7, and the reference ALB gene almost overlap, suggesting that the copy numbers of the SMN1 gene, exon 7, and the reference ALB gene are identical (FIG. 7-a). The fluorescence value curve of the SMN1 gene number 7 exon was consistently lower than that of the reference ALB gene for the SMA carrier, suggesting that the copy number of the SMN1 gene number 7 exon was less than that of the reference ALB gene (FIG. 7-b). The fluorescence value curve of exon 7 of the SMN1 gene was near baseline, whereas the fluorescence value curve of the reference ALB gene was normal, suggesting homozygous deletion of exon 7 of the SMN1 gene (FIG. 7-c). The results show that: the detection principle of quantitative detection of SMA-Cas12a based on probe connection is feasible; the introduction of the internal reference ALB gene is favorable for carrying out relative quantification on the copy number of the No.7 exon of the SMN1 gene on one hand, and can be used as a quality control factor on the other hand so as to eliminate false positives caused by connection, amplification and fluorescence detection failure.

(2) Verification results of 60 samples

There was still a significant difference in the fluorescence value ratio between the normal, carrier and SMA patient 3 groups, with the average and standard deviation of the fluorescence value ratio of the normal group being 1.02±0.09 (theoretical value: 1), the average and standard deviation of the fluorescence value ratio of the SMA carrier group being 0.67±0.06 (theoretical value: 0.5), and the average and standard deviation of the fluorescence value ratio of the SMA patient group being 0.06±0.02 (theoretical value: 0) (fig. 8). The results show that: the SMA-Cas12a fluorescence quantitative detection based on probe connection can carry out relative quantitative detection on the copy number of the No.7 exon of the SMN1 gene, and can distinguish normal people, SMA carriers and SMA patients.

Example 2

Comparative experiment method-detection of SMA Using existing Cas12a detection methods and principles

(1) Amplification primers and crRNA were designed based on exon 7 of SMN1 gene.

The existing Cas12a detection method needs to search a PAM sequence from a target gene sequence and design crrnas. Because the human genome has a SMN2 gene that is highly homologous to the SMN1 gene, to avoid interference of SMN2 gene with detection of exon 7 of SMN1 gene, the differential base (C-T) of SMN1 and SMN2 gene c.840 has been included within the recognition region of crRNA to specifically recognize exon 7 of SMN1 gene. To further improve the specificity of the detection, the crRNA is optimized and verified by introducing mismatched bases, changing the length of the crRNA recognition region (18 nt and 23 nt) and other 2 ways, and finally, the SMN1crRNA 1, the SMN1crRNA 2, the SMN1crRNA3 and the SMN1crRNA 4 4 crRNAs are designed and synthesized.

TABLE 2 primer, probe and crRNA sequence information for the Cas12a detection method

(2) PCR amplification

(1) And selecting 1 gDNA sample and blank (water) of normal person and SMA patient, checking copy number of 7 exons of SMN1 gene by MLPA detection, wherein the normal person is 2 copies, and the SMA patient is 0 copies. PCR amplification was performed using SMN1 PCR primers. The amplification reaction system is as follows:

the PCR reaction conditions are shown in FIG. 5.

(3) Cas12a fluorescent probe detection

(1) Because 4 crrnas were designed to be synthesized, the assays were performed in 4 groups. The conditions in each group were identical except that the crrnas used were different.

(2) PCR products of amplification products of normal persons, SMA patients and blank control (water) are detected by using a Cas12a fluorescent probe (the used samples are the same as those used for the quantitative detection of SMA-Cas12a fluorescence based on probe connection), 3 compound holes are formed in each sample, the concentration of Lba Cas12a (NEB, M0653T) used for detection is 100 mu M, a mixed system is prepared according to 10 parts, and the mixed system is as follows (the mixed system is divided into 4 groups, and SMN1crRNA 1, SMN1crRNA 2, SMN1crRNA3 and SMN1crRNA 4 are respectively used):

2. Results

(1) Existing Cas12a detection methods detect

First, SMN1 PCR primers F/R PCR amplification was performed on normal and SMA patients, gDNA samples and blank (water), and the pair of primers can amplify both the SMN1 gene and the SMN2 gene. The amplified products were divided into 4 groups and were detected with 4 crrnas for SMA-Cas12a fluorescent probes, respectively. The fluorescence values of normal, SMA patients and blank were all different in the 4 groups, but each group was different in the ability to distinguish between normal and SMA patients. In the SMN1crRNA 1 group, the normal human fluorescence value was 1.93 times that of the SMA patient, in the SMN1crRNA 2 group, the normal human fluorescence value was 3.90 times that of the SMA patient, in the SMN1crRNA3 group, the normal human fluorescence value was 2.51 times that of the SMA patient, and in the SMN1crRNA 4 group, the normal human fluorescence value was 4.11 times that of the SMA patient (fig. 9). The results show that: the natural difference base of the 7 th exon of the SMN1 gene and the SMN2 gene can be used for distinguishing normal people from SMA patients, but the distinguishing specificity is obviously lower than that of the method. The ability to distinguish normal and SMA patients can be further improved by artificially introducing 1 mismatched base based on the natural differential base.

(2) Comparison of SMA-Cas12a fluorescence quantitative detection method based on probe connection and existing SMA-Cas12a fluorescence detection method

(1) In the SMA-Cas12a fluorescence quantitative detection method based on probe connection, the difference of fluorescence values of normal people and SMA patients is 35.39 times, but in the existing Cas12a detection method, the difference is only 4.11 times at most, which shows that the SMA-Cas12a fluorescence quantitative detection method based on probe connection has higher specificity and has stronger capability of distinguishing normal people and SMA patients.

(2) The existing SMA-Cas12a fluorescence detection method needs to design crRNA by relying on the sequence of the No.7 exon of the SMN1 gene, and the flexibility of crRNA design is limited. Even if 4 crrnas are designed by introducing mismatched bases, changing the length of the recognition region, and the like, the specificity of crRNA recognition is improved, but the degree of improvement in specificity is not ideal.

(3) According to the SMA-Cas12a fluorescence quantitative detection method based on probe connection, the PAM sequence and the crRNA recognition region sequence are introduced manually, so that the design flexibility and the design selectivity are improved. Because a highly specific DNA ligase (HiFi Taq DNA Ligase) was used in this example, it was sensitive to base mismatches between the probe and hybridization template. When the probe hybridizes with the SMN1 gene, there is no mismatch between the probe and the template, and the DNA ligase ligates the upstream and downstream probes into a single strand. When the probe hybridizes to the SMN2 gene, there is a mismatch between the probe and the template and DNA ligase cannot ligate the upstream and downstream probes into single strands.

Example 3

(1) Another pair of ligation probes (F2-R2), ligation probe PCR primers and probe crRNA was synthesized according to the SMN1 gene exon 7 and reference ALB gene design, as shown in Table 3:

TABLE 3 PCR primers, ligation probes and crRNA sequence information

(2) Verification of the specificity, ligation and amplification efficiency of ligation probes F2/R2

(2) The probe ligation reaction system for SMN1 was as follows:

The probe ligation reaction is shown in FIG. 4.

The PCR amplification system was as follows:

the PCR reaction conditions are shown in FIG. 5.

The results were: the data of the SMN1 ligation probes F1/R1 and F2/R2 of example 1 were compared as shown in FIG. 10.

Under the same conditions, 2 groups of ligation probes were used to detect 3 samples of normal, SMA patient and blank (water). The difference in fluorescence values between normal and SMA patients in the SMN1 ligation probe F2/R2 group was only 1.09 times, while the difference in fluorescence values between normal and SMA patients in the SMN1 ligation probe F1/R1 group was 35.39 times. The results show that: the ability of the SMN1 ligation probe F1/R1 to distinguish between normal and SMA patients is superior to that of the SMN1 ligation probe F2/R2 (FIG. 10). Preferably SMN1 ligates probe F1/R1.

(3) Verification of the specificity, ligation and amplification efficiency of the ALB ligation Probe F2/R2

(1) 1 part of normal human gDNA and 1 part of blank (water) are selected, and the normal human gDNA sample contains ALB genes, and the blank has no ALB genes. Ligation was performed using ALB ligation probe F2/R2.

(2) The probe ligation reaction system for ALB was as follows:

The probe ligation reaction conditions are shown in FIG. 4.

The PCR amplification system was as follows:

the PCR reaction conditions are shown in FIG. 5.

The results were as follows:

the data of ALB-ligated probes F1/R1 and F2/R2 of example 1 were compared as shown in FIG. 11.

Under the same conditions, normal human samples and blank control (water) were detected with 3 sets of probes such as SMN1 ligation probe F1/R1, ALB ligation probe F1/R1, and ALB ligation probe F2/R2, respectively. In the ALB ligation probe F1/R1 group and the F2/R2 group, the increase of the fluorescence value of the blank control is not obvious, which shows that the specificity of the 2 groups of probes is better. The fluorescence value generated by the normal person after the detection of the ALB ligation probe F1/R1 is higher than that of the ALB ligation probe F2/R2, which shows that the ligation and amplification efficiency of the ALB ligation probe F1/R1 is higher than that of the ALB ligation probe F2/R2. The normal human sample is detected by the ALB connection probe F1/R1 and the SMN1 connection probe F1/R1 respectively, and the fluorescence values generated by the target gene and the internal reference gene are close, which indicates that the connection and amplification efficiency of the 2 groups of probes are close, and the method can be used for quantitative detection of the target gene and the internal reference gene (figure 11). ALB ligation probe F1/R1 is preferred.

The foregoing examples are set forth in order to provide a more thorough description of the present invention, and are not intended to limit the scope of the invention, since modifications of the invention in various equivalent forms will occur to those skilled in the art upon reading the present invention, and are within the scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south-middle school

<120> a detection method based on Cas protein

<130> 8

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<170> PatentIn version 3.5

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Claims

1. A Cas protein-based detection method for non-disease diagnosis or treatment purposes, comprising the steps of:

s2, respectively hybridizing the target gene and the reference gene with corresponding connecting probes, connecting the hybridized connecting probes into single-stranded DNA by DNA ligase, and carrying out PCR amplification on the single-stranded DNA by adopting the PCR primer in the step S1 to obtain an amplification product; detecting an amplification product by using the Cas protein, the crRNA in the step S1 and a fluorescent probe system, and quantitatively detecting a target gene;

the Cas protein is Cas12 a;

the ligation probe comprises a PAM sequence and a crRNA recognition region sequence;

the target gene is an exon 7 of the SMN1 gene; the reference gene is ALB;

the DNA ligase in the step S2 is HiFi Taq DNA Ligase;

the sequence of the connection probe is shown as SEQ ID NO. 1-4; the sequence of PCR primer of the connection probe amplified as shown in SEQ ID NO.1-4 is shown in SEQ ID NO. 5-6; the sequence of crRNA recognizing the ligation probe shown as SEQ ID NO.1-4 is shown as SEQ ID NO. 7-8.

2. The method according to claim 1, wherein in the step S2, the target gene and the reference gene are hybridized with the ligation probes shown in SEQ ID NO.1-4, PCR amplification is performed using PCR primers shown in SEQ ID NO.5-6, and crRNA shown in SEQ ID NO.7-8 is used for detection.

3. A kit is characterized by comprising a ligation probe shown as SEQ ID NO.1-4, a PCR primer shown as SEQ ID NO.5-6 and crRNA shown as SEQ ID NO. 7-8.

4. Use of the kit of claim 3 for the preparation of a reagent for detecting exon 7 of SMN1 gene.