USP26 gene mutation detection kit based on CRISPR-Cas12a

Abstract

The invention relates to a CRISPR / Cas12a-based USP26 gene mutation detection kit. The CRISPR / Cas12a-based USP26 gene mutation detection kit comprises the following components: a primer pair for amplifying a gene segment containing a human USP26 gene mutation site, and crRNA for the human USP26 gene mutation site, the mutation site of the human USP26 gene is c.370 to 371i nsACA / 494Tgt, and the mutation site of the human USP26 gene is c.370 to 371i nsACA / C / 1423Cgt; t is a combination of one or more of T. The kit provided by the invention is a rapid detection tool, can realize convenient and rapid direct result interpretation by naked eyes, provides an accurate, rapid, simple and convenient detection method for primary experiments and clinical front-line, and has the advantages of high sensitivity, strong specificity, short time consumption, high throughput, no dependence on large-scale test equipment and the like by adopting the combination of the primer and the crRNA.

Description

Technical Field

[0001] The present invention relates to the field of disease detection, and more particularly, to a kit for detecting USP26 gene mutations based on CRISPR / Cas12a. Background Art

[0002] Klinefelter syndrome (KS) is defined by a 47,XXY karyotype and is the most common chromosomal aberration in males, with approximately 1 case per 500 - 1000 males. The prevalence of KS in infertile males can reach 3% - 4%, and in azoospermic patients, the prevalence of KS is as high as 12%. Among fertile males, some USP26 variants increase the proportion of XY aneuploid sperm. We found that the fathers of two KS patients carried haplotypes with USP26 mutations. The deletion or mutant haplotypes of USP26 do indeed greatly increase the probability of offspring developing KS, and the mutant haplotype of USP26 (c.370–371insACA / 494T>C / 1423C>T) has the highest proportion of XY aneuploid sperm production. Traditional techniques for detecting gene mutations include Sanger sequencing and quantitative PCR (qPCR). However, these techniques are time-consuming and costly, and thus the disadvantages of these techniques limit their accessibility. Therefore, it is necessary to develop a rapid, economical, stable, sensitive, and specific technique for detecting human USP26 gene mutations.

[0003] Nucleic acid detection techniques based on CRISPR / Cas have shown great advantages due to their high sensitivity, high specificity, simplicity, and time-saving characteristics and have been developed and utilized. CRISPR-Cas (Clustered regularly interspaced short palindromic repeats, CRISPRs) is an adaptive immune system in bacteria, and Cas proteins target and degrade foreign nucleic acids through RNA-guided nucleases. Among them, Cas12a belongs to the second family of Cas enzymes. By recognizing the protospacer adjacent motif (PAM) rich in thymine (T) nucleotides, it catalyzes the maturation of its own guide CRISPR RNA (crRNA) and specifically recognizes and cleaves complementary double-stranded DNA (dsDNA). When the CRISPR / Cas12 protein recognizes and cleaves the target double-stranded DNA in a sequence-specific manner, it can induce a strong non-specific single-stranded DNA (ssDNA) trans-cleavage activity.

[0004] Therefore, developing a nucleic acid detection kit based on CRISPR / Cas12a can detect USP26 mutations sensitively, specifically, and simply. However, there is no such detection kit on the market currently. Summary of the Invention

[0005] To solve the above problems, the present invention provides a CRISPR / Cas12a-based USP26 gene mutation detection kit, comprising the following components: a primer pair for amplifying a gene fragment containing the human USP26 gene mutation site and a crRNA targeting the human USP26 gene mutation site;

[0006] The human USP26 gene mutation site is one or more combinations of c.370–371insACA / 494T>C / 1423C>T.

[0007] In a specific embodiment, the primer pair for amplifying the gene fragment containing c.370–371insACA comprises a forward primer selected from SEQ ID NO:14-16 and a reverse primer selected from SEQ ID NO:17-19;

[0008] The primer pair for amplifying the gene fragment containing c.494T>C comprises a forward primer selected from SEQ ID NO:20-23 and a reverse primer selected from SEQ ID NO:24-26;

[0009] The primer pair for amplifying the gene fragment containing c.1423C>T comprises a forward primer SEQ ID NO:27-32 and a reverse primer selected from SEQ ID NO:33.

[0010] In a specific embodiment, the primer pair for amplifying the gene fragment containing c.370–371insACA comprises a forward primer SEQ ID NO:15 and a reverse primer SEQ ID NO:19;

[0011] The primer pair for amplifying the gene fragment containing c.494T>C comprises a forward primer SEQ ID NO:22 and a reverse primer SEQ ID NO:26;

[0012] The primer pair for amplifying the gene fragment containing c.1423C>T comprises a forward primer SEQ ID NO:30 and a reverse primer SEQ ID NO:33.

[0013] By using the above primer combinations, gene fragments containing the corresponding mutation sites can be amplified more efficiently and specifically.

[0014] In a specific embodiment, the sequence of the crRNA targeting c.370–371i nsACA is selected from SEQ ID NO:1-3;

[0015] The sequence of the crRNA targeting c.494T>C is selected from SEQ ID NO:4-7;

[0016] The sequences of the crRNAs targeting c.1423C>T are selected from SEQ ID NO:8-13.

[0017] In a specific embodiment, the sequence of the crRNA targeting c.370–371insACA is as shown in SEQ ID NO:2;

[0018] The sequence of the crRNA targeting c.494T>C is as shown in SEQ ID NO:6;

[0019] The sequence of the crRNA targeting c.1423C>T is as shown in SEQ ID NO:10.

[0020] The above-mentioned crRNAs can specifically recognize target nucleic acids.

[0021] In a specific embodiment, the kit further comprises a nucleic acid releasing agent.

[0022] In a specific embodiment, the kit further comprises a red blood cell lysate.

[0023] The above-mentioned nucleic acid releasing agent and red blood cell lysate can efficiently release genomic nucleic acids in blood samples, and better achieve the detection of blood samples.

[0024] The kit of the present invention is a rapid detection tool, which can achieve convenient and quick visual direct result interpretation, providing an accurate, rapid and simple detection method for basic experiments and clinical frontlines. The primer and crRNA combination adopted by the present invention has the advantages of high sensitivity, strong specificity, short time consumption, high throughput, and independence from large-scale test equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram for rapid detection of USP26 mutations (c.370–371insACA / 494T>C / 1423C>T) based on Cpf1.

[0026] Figure 2 Effect diagram of nucleic acid specific crRNA detection for USP26 mutations (c.370–371insACA / 494T>C / 1423C>T).

[0027] Figure 3 Optimal isothermal amplification primer screening based on USP26 mutations (c.370–371insACA / 494T>C / 1423C>T).

[0028] Figure 4Sensitivity determination for detecting USP26 mutations (c.370–371insACA / 494T>C / 1423C>T) based on Cpf1.

[0029] Figure 5 Specific detection of background nucleic acids and high-concentration wild-type nucleic acids for detecting USP26 mutations (c.370–371insACA / 494T>C / 1423C>T) based on Cpf1.

[0030] Figure 6 Optimization determination of nucleic acid release conditions for detecting nucleic acids in patient blood samples based on Cpf1.

[0031] Figure 7 、 8 、9 Highly specific detection of USP26 mutations (c.370–371insACA / 494T>C / 1423C>T) in patient blood samples by Cpf1 visual fluorescence method. Detailed implementation mode

[0032] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0033] The USP26 gene mutation detection kit of the present invention is based on the CRISPR / Cas12 technology, and its detection method is as Figure 1 shown, including four steps:

[0034] S1: Release genomic nucleic acids from patient samples;

[0035] S2: Amplify the USP26 gene;

[0036] S3: Cut the probe;

[0037] S4: Read the results.

[0038] 1. Preparation of the USP26 gene mutation detection kit

[0039] In this embodiment, the LbCpf1 gene is codon-optimized and then cloned into the pET-28a plasmid (this step is completed by Nanjing Genscript Corporation), expressed in Escherichia coli, and used for detection experiments after purification.

[0040] 1) Design of specific crRNA

[0041] For three mutation sites of the USP26 gene, target sequences containing the Cpf1 recognition sequence (PAM) TTTN were searched, and multiple 21-bp-long crRNAs were designed and named cr1-3, cr1-4, and cr1-6 for 370-371insACA, 494T>C, and 1423C>T respectively. The sequences are shown in Table 1 respectively.

[0042] Table 1 Three mutation sites of the USP26 gene

[0043]

[0044] 2) Design of isothermal amplification (RPA) primers

[0045] According to the requirements of the isothermal amplification reaction, RPA amplification primers were designed and synthesized for the three mutation sites. The sequences are shown in Table 2.

[0046] Table 2 RPA primer sequences

[0047]

[0048]

[0049]

[0050] 2. Detection steps

[0051] 1) RPA isothermal amplification

[0052] Amplify the sample to be detected: The isothermal amplification is carried out in a 50-μL reaction system. Take 6 μL of nucleic acid sample, 5 μL of ddH2O, 2.5 μL of RPA-F, 2.5 μL of RPA-R, 20 μL of Reaction Buffer, 10 μL of ERA Basic. Additionally, when detecting c.494T>C and c.1423C>T, 2 μL of restriction enzyme (c.494T>C: MluCI, c.1423C>T: BtsCI) needs to be added. Add them to the reaction tube, dissolve and mix well. Finally, add 2 μL of agonist, mix well and react at 37 °C for 25 minutes. The obtained RPA product is subjected to the next detection.

[0053] 2) CRISPR / Cas12 reaction

[0054] 2 μL of 10× Buffer, 0.1 μL of RNase inhibitor (final concentration 40 U / μL), 1 μL of Cas12a (final concentration 200 ng / μL), 1 μL of ssDNA FQ reporter (ssDNA FQ reporter: 6FAM-TTATT-BHQ1 (5’→3’), final concentration 25 pM / μL), 5 μL of RPA product (i.e., the nucleic acid for detection in Table 3), 1 μL of crRNA (final concentration 1 μM / μL), and 7.9 μL of H2O were added successively into the reaction tube. After mixing all components evenly, the reaction was carried out at 37 °C for 20 min.

[0055] 3) Determine the detection activity of the Cas12a detection system using fluorescence detection. A full-wavelength microplate reader was used to measure the fluorescence of the detection reaction, with an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The fluorescence value measured for 30 min of detection was taken as the reaction value.

[0056] 3. Optimization of crRNA and primers in the kit

[0057] 1) Screen for highly efficient crRNA

[0058] For each mutation site, RPA products were obtained by RPA isothermal amplification and used in the CRISPR / Cas12 reaction to detect the effects of each crRNA. MT is the nucleic acid fragment containing the mutation site, and WT is the USP26 wild-type nucleic acid fragment. The results are as Figure 2 shown: 370-371insACA-cr2 targeting the c.370–371insACA mutation of the USP26 gene can specifically target the nucleic acid; 494T>C-cr3 targeting the c.494T>C mutation of the USP26 gene can specifically target the nucleic acid; c.1423C>T-cr3 targeting the c.1423C>T mutation of the USP26 gene can specifically recognize the target nucleic acid.

[0059] Subsequently, 370-371insACA-cr2, 494T>C-cr3, and 1423C>T-cr3 with stronger detection signals were selected for subsequent experiments.

[0060] 2) Screen for highly efficient RPA primers for amplifying the c.370-371insACA, c.494T, and c.1423C regions of the USP26 gene

[0061] The forward primers and reverse primers for each mutation site in Table 2 were paired pairwise to obtain multiple primer pairs for amplifying the corresponding mutation sites.

[0062] Referring to the relevant sequences in the NCBI gene bank, Tianyi Huiyuan Company synthesized gene fragments containing c.370–371insACA / 494T>C / 1423C>T respectively, and cloned them onto plasmids.

[0063] The plasmid samples were diluted in a 10-fold gradient to obtain samples containing 1x10 5 copies of the corresponding plasmids per microliter, which were used as templates to detect the amplification efficiency of each primer pair. MT is the nucleic acid fragment containing the mutation site, and the water sample is the negative control NC.

[0064] The results are as Figure 3 shown. For the c.370–371insACA gene fragment: the primer combination of 370-371insACA-F2 and 370-371insACA-R3 had the best amplification efficiency, so 370-371insACA-F2 and 370-371insACA-R3 were selected as the primers for amplifying the c.370–371insACA gene fragment;

[0065] For the c.494T>C gene fragment: the primer combination of 494T>C-F3 and 494T>C-R3 had the best amplification efficiency, so 494T>C-F3 and 494T>C-R3 were selected as the primers for amplifying the c.494T>C gene fragment;

[0066] For the c.1423C>T gene fragment: the primer combination of 1423C>T-F4 and 1423C>T-R1 had the best amplification efficiency, so 1423C>T-F4 and 1423C>T-R1 were selected as the primers for amplifying the c.1423C>T gene fragment.

[0067] 4. Sensitivity test of the optimized kit

[0068] Stock solutions containing the masses of each mutation site were prepared and serially diluted to obtain test samples containing 1×10 7 、1×10 6 、1×10 5 、1×10 4 、1×10 3 、1×10 2 and 1×10 1 (copy / μL). The water sample was the negative control NC.

[0069] These samples were detected using the optimized kit, and the results are as Figure 4As shown in the figure, for the c.370–371insACA gene fragment, 10 copies of nucleic acid can be effectively detected; for the c.494T>C gene fragment, 10 copies of nucleic acid can be effectively detected; for the c.1423C>T gene fragment, 1000 copies can be effectively detected.

[0070] 5. Specificity test of the optimized kit

[0071] Nucleic acid fragments containing each mutation site were mixed with samples of background nucleic acid from the human HEK293T cell line (1000 ng, 500 ng, 100 ng, 50 ng) as test samples for detection; and separate E gene fragments with or without 5UTR were used as detection samples; the water sample was used as a negative control NC.

[0072] Secondly, nucleic acid fragments containing each mutation site (MT) and USP26 wild-type nucleic acid fragments (WT) were selected and detected at different concentrations (1×10 12 、1×10 11 、1×10 10 (copy / μL)).

[0073] The results are as Figure 5 shown. For the c.370–371insACA gene fragment: the primer combination of 370-371insACA-F2 and 370-371insACA-R3 can highly specifically distinguish background nucleic acid and the mutant nucleic acid fragment 370-371insACA, and there is no cross-reaction with high-concentration wild-type nucleic acid within 1 h;

[0074] For the c.494T>C gene fragment: the primer combination of 494T>C-F3 and 494T>C-R3 can highly specifically distinguish background nucleic acid and the mutant nucleic acid fragment 494T>C, and there is no cross-reaction with high-concentration wild-type nucleic acid within 1 h;

[0075] For the c.1423C>T gene fragment: the primer combination of 1423C>T-F4 and 1423C>T-R1 can highly specifically distinguish background nucleic acid and the mutant nucleic acid fragment c.1423C>T, and there is no cross-reaction with high-concentration wild-type nucleic acid within 1 h.

[0076] 6. Further optimization of the Cas12a fluorescence method for detecting patient blood samples

[0077] To determine the dosage of the nucleic acid release agent and the lysis time for releasing genomic nucleic acid from patient blood samples, the following detections were carried out.

[0078] Add 300 μL of blood sample to 600 μL of red blood cell lysate, react at room temperature for 15 min, then centrifuge and discard the supernatant. Add 200 μL, 100 μL, 50 μL, and 20 μL of nucleic acid release agent to the remaining white blood cells, and incubate at 95 °C for 5 min. Use nucleic acid fragments at 1x10 5 copies / μL as the positive control and H2O as the negative control.

[0079] In addition, add 300 μL of blood sample to 600 μL of red blood cell lysate, react at room temperature for 15 min, then centrifuge and discard the supernatant. Add 20 μL of nucleic acid release agent to the remaining white blood cells, and incubate at 95 °C for 2, 5, 10, 15, 20, 25 min. Use nucleic acid fragments at 1x10 5 copies / μL as the positive control and H2O as the negative control.

[0080] Detect the incubated samples by CRISPR / Cas12a, and the results are as Figure 6 shown. For complex patient blood samples, 20 μL of nucleic acid release agent and 5 min of incubation can efficiently release genomic nucleic acids in the blood samples.

[0081] 7. Detection examples of the optimized kit

[0082] Use the above optimized kit for the detection of actual samples. The samples are patient blood samples (containing or not containing c.370–371insACA / 494T>C / 1423C>T).

[0083] The results are as Figures 7-9 shown. Under a fluorescent lamp, it can be seen with the naked eye that patients 2 and 11 have the USP26 mutation (c.370–371insACA / 494T>C / 1423C>T), while samples 1, 3, 4, 5, 6, 7, 8, 9, and 10 are wild-type without the USP26 mutation (c.370–371insACA / 494T>C / 1423C>T), which is consistent with the actual situation.

[0084] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A kit for detecting USP26 gene mutations based on CRISPR / Cas12a, characterized in that, It includes the following components: a primer pair for amplifying a gene fragment containing the human USP26 gene mutation site and a crRNA targeting the human USP26 gene mutation site; The human USP26 gene mutation site is one or more combinations of c.370–371insACA / 494T>C / 1423C>T.

2. The kit according to claim 1, wherein The primer pair for amplifying a gene fragment containing c.370–371insACA includes a forward primer selected from SEQ ID NO:14-16 and a reverse primer selected from SEQ ID NO:17-19; The primer pair for amplifying a gene fragment containing c.494T>C includes a forward primer selected from SEQ ID NO:20-23 and a reverse primer selected from SEQ ID NO:24-26; The primer pair for amplifying a gene fragment containing c.1423C>T includes a forward primer SEQ ID NO:27-32 and a reverse primer selected from SEQ ID NO:

33.

3. The kit according to claim 2, wherein The primer pair for amplifying a gene fragment containing c.370–371insACA includes a forward primer SEQ ID NO:15 and a reverse primer SEQ ID NO:19; The primer pair for amplifying a gene fragment containing c.494T>C includes a forward primer SEQ ID NO:22 and a reverse primer SEQ ID NO:26; The primer pair for amplifying a gene fragment containing c.1423C>T includes a forward primer SEQ ID NO:30 and a reverse primer SEQ ID NO:

33.

4. The kit according to claim 1, wherein The sequence of the crRNA targeting c.370–371insACA is selected from SEQ ID NO:1-3; The sequence of the crRNA targeting c.494T>C is selected from SEQ ID NO:4-7; The sequence of the crRNA targeting c.1423C>T is selected from SEQ ID NO:8-13.

5. The kit according to claim 4, characterized in that, The sequence of the crRNA targeting c.370–371insACA is as shown in SEQ ID NO:2; The sequence of the crRNA targeting c.494T>C is as shown in SEQ ID NO:6; The sequence of the crRNA targeting c.1423C>T is as shown in SEQ ID NO:

10.

6. The kit according to claim 1, wherein It also includes a nucleic acid releasing agent.

7. The kit according to claim 1, wherein It also includes a red blood cell lysate.

Images