CN117925806A - ABO blood group genotyping method based on CRISPR/Cas13a and composition used by same - Google Patents
ABO blood group genotyping method based on CRISPR/Cas13a and composition used by same Download PDFInfo
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Abstract
The invention discloses an ABO blood group genotyping method based on CRISPR/Cas13a and a composition used by the same. The invention belongs to the field of biological detection, and particularly relates to an ABO blood group genotyping method based on CRISPR/Cas13a and a composition used by the same. The invention relates to a composition for blood group genotyping detection, which comprises a primer group A and a primer group B which are specific to nucleic acid to be detected, crRNA-6-261-G2, crRNA-6-261-D3crRNA-7-796-C1, crRNA-7-796-A3, cas13a protein and a fluorescent reporter probe which are specific to the nucleic acid to be detected, wherein the primer group A consists of primers ABO-6-T7-F1 and ABO-6-R1; the primer group B consists of primers ABO-7-T7-F1 and ABO-7-R1.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to an ABO blood group genotyping method based on CRISPR/Cas13a and a composition used by the same.
Background
The ABO blood group proposed by cal-landenstein (KARL LANDSTEINER) at the beginning of the 20 th century was the first blood genetic marker system discovered by humans. The ABO gene comprises 7 exons and 1062 base pair sequences located at chromosome 9 9q34.1-34.2, encoding a glycosyltransferase (tissue blood group ABO system transferase) that catalyzes transfer of carbohydrates to H antigen. ABO blood group antigens are in three allelic forms, A, B and O. The a allele differs from the B allele by nucleotide substitutions, while the O allele differs from the a allele by a guanine deletion at position 261. Genetically, each child acquires one of three alleles from a parent, resulting in six possible genotypes (AA, AO, BB, BO, OO and AB) and four possible phenotypes (A, B, O and AB). The ABO blood group system is important not only in blood transfusion but also in relation to tumors, cardiovascular diseases and the like. In addition, ABO is a genetic marker that plays an important role in association analysis and individual identification.
Diagnostic (CRISPR-Dx) assays based on clustered regularly interspaced short palindromic repeats have drastically changed diagnostic methods in the application of nucleic acids, small molecules, proteins and other targets. The CRISPR-Dx system consists of a CRISPR-associated (Cas) protein and a guide RNA (gRNA) molecule designed to specifically recognize a target. Cas9, cas12 and Cas13 are Cas proteins widely used in CRISPR-Dx. Cas9 relies on complex grnas consisting of transactivation CRISPR RNA (tracrRNA) and CRISPR RNA (crRNA), whereas Cas12 and Cas13 use only a single crRNA. Furthermore, cas12 and Cas13 also exhibit endoenzyme activity on single stranded DNA (ssDNA) and RNA (ssRNA), respectively. Therefore, cas12 and Cas13 have been used to design many detection platforms. Cas 12-based diagnostic systems are limited by in situ adjacent motifs, limiting their potential applications. Thus, cas 13-based diagnostic systems are suitable for ABO typing by detecting SNPs in A, B and O alleles.
There are various methods for typing ABO blood types. Serological blood typing methods are used mainly as gold standard in clinical practice. Wherein, standard anti-A and anti-B serum are used to identify antigens on subject erythrocytes, while standard A and B erythrocytes are used to identify antibodies in the subject serum. Serological typing is simple but requires reliable typing of antisera and blood withdrawal, not suitable for samples from other sources. With the development of molecular biology techniques, more and more techniques are used for ABO typing, including PCR-restriction fragment length polymorphism, PCR-single strand conformational polymorphism, PCR-amplified product length polymorphism, consumption allele-specific primer analysis, mutagenesis separation PCR, real-time quantitative PCR, DNA chip, and sequencing. However, these assays may also take a significant amount of time due to electrophoresis, complex enzymatic cleavage, or precise design of probes, and may require specialized personnel and cumbersome, precise instrumentation. Therefore, it is necessary to reliably determine ABO blood group genotypes based on Single Nucleotide Polymorphisms (SNPs) between A, B and O alleles.
Disclosure of Invention
The technical problem to be solved by the invention is how to rapidly and efficiently identify the ABO blood group genotype.
In order to solve the above problems, the present invention provides a composition for blood typing detection.
The invention provides a composition for blood group genotyping detection, which comprises a primer group A and a primer group B which are specific to nucleic acid to be detected, a crRNA group 1 and a crRNA group 2 which are specific to the nucleic acid to be detected, cas13a protein and a fluorescence report probe; the primer group A consists of a primer ABO-6-T7-F1 and a primer ABO-6-R1; the primer group B consists of a primer ABO-7-T7-F1 and a primer ABO-7-R1; the crRNA group 1 consists of crRNA-6-261-G2 and crRNA-6-261-D3; the crRNA group 2 consists of crRNA-7-796-C1 and crRNA-7-796-A3; the fluorescent reporter probe is single-stranded RNA with one end connected with a fluorescent group and the other end connected with a quenching group;
The primer ABO-6-T7-F1 is (b 1) or (b 2) as follows:
(b1) A single-stranded DNA molecule shown in a sequence 2 of a sequence table;
(b2) A DNA molecule which has the same function as the sequence 2 and is obtained by substituting and/or deleting and/or adding nucleotides in the sequence 2;
The primer ABO-6-R1 is (b 3) or (b 4) as follows:
(b3) A single-stranded DNA molecule shown in a sequence 7 of a sequence table;
(b4) A DNA molecule having the same function as sequence 7 and obtained by subjecting sequence 7 to nucleotide substitution and/or deletion and/or addition;
The primer ABO-7-T7-F1 is as follows (b 5) or (b 6):
(b5) A single-stranded DNA molecule shown in a sequence 19 of a sequence table;
(b6) A DNA molecule having the same function as sequence 19 obtained by subjecting sequence 19 to nucleotide substitution and/or deletion and/or addition;
the primer ABO-7-R1 is (b 7) or (b 8) as follows:
(b7) A single-stranded DNA molecule represented by sequence 27 of the sequence listing;
(b8) A DNA molecule having the same function as sequence 27 and obtained by subjecting sequence 27 to nucleotide substitution and/or deletion and/or addition;
The crRNA-6-261-G2 of the nucleic acid to be detected is (c 1) or (c 2) as follows:
(c1) A single-stranded DNA molecule shown in a sequence 13 of a sequence table;
(c2) A DNA molecule having the same function as sequence 13 obtained by subjecting sequence 13 to nucleotide substitution and/or deletion and/or addition;
The crRNA-6-261-D3 of the nucleic acid to be detected is (c 3) or (c 4) as follows:
(c3) A single-stranded DNA molecule shown in a sequence 17 of a sequence table;
(c4) A DNA molecule having the same function as sequence 17 obtained by subjecting sequence 17 to nucleotide substitution and/or deletion and/or addition;
the crRNA-7-796-C1 of the nucleic acid to be detected is (C5) or (C6) as follows:
(c5) A single-stranded DNA molecule shown in a sequence 35 of a sequence table;
(c6) A DNA molecule having the same function as the sequence 35 by substitution and/or deletion and/or addition of nucleotides to the sequence 35;
the crRNA-7-796-A3 of the nucleic acid to be detected is (c 7) or (c 8) as follows:
(c7) A single-stranded DNA molecule represented by sequence 40 of the sequence listing;
(c8) A DNA molecule having the same function as sequence 40 by substitution and/or deletion and/or addition of nucleotides to sequence 40;
The nucleotide sequence of the single-stranded RNA of the fluorescent reporter probe is the sequence FAM-UUUUU-BHQ1 in the sequence table.
In the above, the composition further comprises reagents for performing nucleic acid amplification.
The invention also provides a kit for genotyping of blood groups, the kit comprising the composition as described hereinbefore.
In a specific embodiment, the components of the kit comprise the following: can consist of only crRNA specific for the nucleic acid to be detected, cas13 protein and fluorescent reporter probes; may also contain at least one of the following: cas13 protein, T7 RNA polymerase, rnase inhibitor, rtp, RNA reporter probe, guide RNA, primer set a, and primer set B, cas a buffer.
(1) Specifically, the Cas13 protein is LwaCas-50 pmol/. Mu.l, preferably 10 pmol/. Mu.l, protein.
(2) Specifically, the T7 RNA polymerase concentration is 10-100U/. Mu.L, preferably 50U/. Mu.L.
(3) Specifically, the rNTP concentration is 5-50mM, preferably 25mM.
(4) Specifically, the fluorescent group of the RNA report probe can be FAM, ROX, HEX, CY, CY5 and the like, and the quenching group can be BHQ1, BHQ2, BHQ3 and the like; the single-stranded RNA probe is prepared according to a conventional method in the field, and the sequence can be 5'-UUUUU-3', and the like; the concentration is 10-200. Mu.M.
In the present invention, the crRNA may be obtained commercially by synthesis or by in vitro transcription.
The CRISPR CRRNA reaction generated by in vitro transcription contained 5-200. Mu.M of a synthetic ssDNA template with an upstream T7 promoter, 5-200. Mu. M T7-3G IVT primer and 1X standard Taq buffer, denatured at 90-95℃for 3-10 min, and then gradient annealed at 4-12℃with a slope of 0.8-0.15 ℃/s in a PCR thermocycler.
CRISPR CRRNA produced by in vitro transcription the transcription product was used overnight at 36-38 ℃ using an RNA synthesis kit.
The CRISPR CRRNA product produced by in vitro transcription was purified after DNase I treatment according to the manufacturer's instructions. The purified crRNA was quantified and the crRNA produced was stored at-20℃to 80 ℃.
The invention also provides the use of a composition as hereinbefore described in any of the following:
a1 Preparing a product for genotyping to identify or identify blood groups;
a2 Blood typing.
The invention also provides the application of the kit in any one of the following:
a1 Preparing a product for genotyping to identify or identify blood groups;
a2 Blood typing.
The invention also provides crRNA set 1 and crRNA set 2 as described hereinbefore.
The invention also provides the primer group A or the primer group B.
The invention also provides a blood group genotyping detection method, which comprises the following steps:
A1 Extracting DNA of a sample to be detected;
A2 Using the cDNA as a template, and performing PCR amplification by using the primer group A and the primer group B to obtain an amplification product;
A3 Using a CRISPR-Cas13a detection system to detect the amplification product; the CRISPR/Cas13a detection system comprises crRNA set 1 and crRNA set 2 in the composition described previously.
In the method, the content ratio of the primer group A to the primer group B is 2:8.
In the above method, the CRISPR-Cas13a detection system further comprises a Cas13a protein, a Cas13a buffer, and a fluorescent reporter probe.
A2 The reaction system for PCR amplification comprises MASTER PCR. Mu.L of mixed solution, 1-5. Mu.L of upstream primer, 1-15. Mu.L of downstream primer 1-5. Mu. L, DNA sample and water without nuclease, and the total amount is 50. Mu.L.
The reaction system of the PCR is carried out under the following conditions: 98℃for 5s to 30s,55℃for 5s to 30s,68℃for 5s to 30s (30 to 40 cycles).
The nucleotide sequence of the PCR amplification product obtained by the amplification of the primer group A is a sequence 1 in a sequence table.
The nucleotide sequence of the PCR amplification product obtained by amplifying the primer group B is a sequence 18 in a sequence table. In sequence 18, m represents A or C.
A3 The CRISPR-Cas13a detection system comprises the following: 1-5. Mu.L of PCR amplification product, 2. Mu.L of 10 XCas 13a buffer, 0.5-1. Mu. L rNTPs (25 mM/seed), 0.1-0.5. Mu.L of ssRNA reporter probe (100. Mu.M), 0.1-0.5. Mu.L of Cas13a nuclease (10 pmol/. Mu.L), 0.5-1. Mu. L T7 RNA polymerase (50U/. Mu.L), 0.2-1. Mu.L of RNase inhibitor (40U/. Mu.L) and 0.1-1. Mu.L of crRNA (1. Mu.M). Wherein 4 crRNAs (6-261-G2, 6-261-D3, 7-796-C1 and 7-796-A3) were added to the above system, respectively, i.e.the PCR amplification products obtained in each step two were subjected to four CRISPR/Cas13a reactions (each reaction corresponds to a different crRNA, and the others were identical).
The Cas13a nuclease, cas13 enzyme buffer, T7 RNA polymerase, rNTPs, RNase inhibitor are commercially available in a concentration of Cas13a nuclease (10 pmol/. Mu.L), cas13 buffer 10X, T7 RNA polymerase (50U/. Mu.L), rNTPs mix (25 mM/seed), RNase inhibitor (40U/. Mu.L).
In a specific embodiment, the CRISPR-Cas13a detection system may specifically be: 1 μ L A2), 2 μL of 10 XCas 13a buffer, 0.8 μ L rNTPs (25 mM/seed), 0.2 μL of ssRNA reporter probe (100 μM), 0.2 μL of Cas13a nuclease (10 pmol/. Mu.L), 0.8 μ L T7 RNA polymerase (50U/. Mu.L), 0.5 μL of RNase inhibitor (40U/. Mu.L), and 0.5 μL of crRNA (1 μM). Wherein 4 crRNAs (6-261-G2, 6-261-D3, 7-796-C1 and 7-796-A3) were added to the above system, respectively, i.e.the PCR amplification products obtained in each step two were subjected to four CRISPR/Cas13a reactions (each reaction corresponds to a different crRNA, and the others were identical).
The CRISPR/Cas13a detection reactions were detected at 36-38 ℃ for 10min-1h using ABI 7500, the fluorescence intensity of each reaction was read automatically during cycling, and the optical data was analyzed using 7500 software v 2.340.
The reporter probe is ssRNA, and the length is 5-15nt, preferably 5nt; a 5' end modified fluorescent group, preferably a 6-FAM group; the 3' end modifies the quenching group, preferably BHQ1.
The result criteria for the above detection method are as follows, in a 4-tube CRISPR/Cas13a reaction containing crRNA-6-261-G2, crRNA-6-261-D3, crRNA-7-796-C1, crRNA-7-796-A3:
1) if the CRISPR/Cas13a (crRNA-6-261-G2) reaction detects a FAM fluorescent signal, the CRISPR/Cas13a (crRNA-6-261-D3) reaction does not detect a FAM fluorescent signal, the CRISPR/Cas13a (crRNA-7-796-C1) reaction detects a FAM fluorescent signal, and the CRISPR/Cas13a (crRNA-7-796-A3) reaction does not detect a FAM fluorescent signal, blood typing is AA;
2) If the CRISPR/Cas13a (crRNA-6-261-G2) reaction detects a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-6-261-D3) reaction detects a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-7-796-C1) reaction detects a FAM fluorescence signal, and the CRISPR/Cas13a (crRNA-7-796-A3) reaction does not detect a FAM fluorescence signal, the blood group genotype is AO;
3) If the CRISPR/Cas13a (crRNA-6-261-G2) reaction detects a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-6-261-D3) reaction does not detect a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-7-796-C1) reaction does not detect a FAM fluorescence signal, and the CRISPR/Cas13a (crRNA-7-796-A3) reaction detects a FAM fluorescence signal, the blood group genotyping is BB;
4) If the CRISPR/Cas13a (crRNA-6-261-G2) reaction detects a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-6-261-D3) reaction detects a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-7-796-C1) reaction detects a FAM fluorescence signal, and the CRISPR/Cas13a (crRNA-7-796-A3) reaction detects a FAM fluorescence signal, the blood group genotype is BO;
5) If the CRISPR/Cas13a (crRNA-6-261-G2) reaction detects a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-6-261-D3) reaction does not detect a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-7-796-C1) reaction detects a FAM fluorescence signal, and the CRISPR/Cas13a (crRNA-7-796-A3) reaction detects a FAM fluorescence signal, the blood group genotype is AB;
6) If the CRISPR/Cas13a (crRNA-6-261-G2) reaction does not detect a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-6-261-D3) reaction detects a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-7-796-C1) reaction detects a FAM fluorescence signal, and the CRISPR/Cas13a (crRNA-7-796-A3) reaction does not detect a FAM fluorescence signal, the blood group genotyping is OO.
The above-described applications or methods are non-disease diagnostic applications or methods. The above applications or methods are not directed to obtaining disease diagnosis results or health status of a living human or animal body.
The invention provides a CRISPR/Cas13a mediated ABO blood typing technology, which is used for detecting SNPs in A, B and O alleles. Six genotypes (AA, AO, BB, BO, OO and AB) were selected for ABO blood group at positions 6-261 (g stands for A/B allele, del stands for O allele) and 7-796 (c stands for A/O allele, a stands for B allele).
The CRISPR/Cas13a system provided by the invention can specifically distinguish SNP loci of exons 6-261 and SNP loci of exons 7-796 of A, B and O alleles within 60 minutes, and complete reliable ABO blood group genotyping with a detection limit of 50 pg/reaction, does not need blood drawing, and does not need professional personnel and heavy and precise instruments. The method provided by the invention is suitable for detecting six genotypes of oral swab samples, the reliability of the oral swab samples is further verified, and the accuracy rate is 100% compared with a serological method and a sequencing method. Therefore, CRISPR/Cas13 a-mediated detection methods show great potential for reliable identification of ABO blood group genotypes.
Drawings
FIG. 1 is a system design of an inspection platform. Wherein a.ABO blood group coding sequence is schematically shown. Exon 6-261 (g represents the A/B allele, del represents the O allele) and exon 7-796 (c represents the A/O allele, a represents the B allele) were selected to distinguish A, B from the O allele; schematic of crispr/Cas13a mediated genotyping of six genotypes of ABO blood group (AA, AO, BB, BO, OO and AB).
FIG. 2 is a screen of 5 pairs of PCR primers for exon 6 locus, wherein ABO-6-T7-F1 and ABO-6-R1, abbreviated as F1R1; ABO-6-T7-F2 and ABO-6-R2, abbreviated as F2R2; ABO-6-T7-F3 and ABO-6-R3, abbreviated as F3R3; ABO-6-T7-F4 and ABO-6-R4, abbreviated as F4R4; ABO-6-T7-F5 and ABO-6-R5, abbreviated as F5R5; in the figure, marker (Beijing Takara, 3428Q) is a combination of DNA fragments with different molecular weights, and bp is a nucleic acid length unit. Of these, F1R1 is the most preferable.
FIG. 3 shows that 8 pairs of PCR primers were selected for exon 7 sites, with F1R1 being the most preferred. ABO-7-T7-F1 and ABO-7-R1, abbreviated as F1R1; ABO-7-T7-F2 and ABO-7-R2, abbreviated as F2R2; ABO-7-T7-F3 and ABO-7-R3, abbreviated as F3R3; ABO-7-T7-F4 and ABO-7-R4, abbreviated as F4R4; ABO-7-T7-F5 and ABO-7-R5, abbreviated as F5R5; ABO-7-T7-F6 and ABO-7-R6, abbreviated as F6R6; ABO-7-T7-F7 and ABO-7-R7, abbreviated as F7R7. In the figure, marker is a combination of DNA fragments (Beijing Takara, 3428Q) with different molecular weights, and bp is a nucleic acid length unit.
FIG. 4 shows multiplex PCR primers and CRISPR RNA screening. Wherein a. The best performing primer pair (shown in bold) and the best performing crRNA pair (shown in different colours) are detailed sequences; b. electrophoresis image of optimized primer concentration for exons 6-261, exons 7-796 (2:8); c. exon 6-261crRNA selection (6-261-G2 and 6-261-D3) 60 min endpoint fluorescence intensity heatmap; d. 60 min endpoint fluorescence intensity heatmap for exon 7-796crRNA selection (7-796-C1 and 7-796-A3).
FIG. 5 is a detection sensitivity study of 261-G, 261-D, 796-C and 796-A using gradient diluted DNA samples (5000, 500, 50 and 5 pg/. Mu.L, and NTC). Multiple comparisons of the different groups were performed by bi-directional analysis of variance and using fluorescence values collected at the 20 minute endpoint, which indicated a detection limit of 50 pg/reaction for this assay. Wherein the sensitivity of the a.261-G site is analyzed; sensitivity analysis of 261-D locus; c, analyzing the sensitivity of 796-C locus; sensitivity analysis of the 796-A site.
FIG. 6 shows the analytical performance of the detection method. Wherein a, six DNA samples of genotypes AA, AO, BB, BO, OO and AB were sequenced by Sanger; b. detecting real-time fluorescence curves of six DNA samples based on CRISPR/Cas13 a; c. 20 minute endpoint fluorescence heatmaps of six DNA samples were detected based on CRISPR/Cas13 a.
Fig. 7 shows an application of the detection method established in the present invention. Wherein phenotype, genotype and sequencing results of the a.26 samples; b. 20 minute endpoint fluorescence heatmap of 26 DNA samples were detected based on CRISPR/Cas13 a.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The quantitative experiments in the following examples were performed in triplicate unless otherwise indicated.
The following examples were run using GRAPHPAD PRISM statistical software and the experimental results were expressed as mean ± standard deviation using Two-way ANOVA test, P < 0.05 (x) indicated significant differences, P < 0.01 (x) indicated very significant differences, and P < 0.001 (x) indicated very significant differences.
Example 1, establishment of CRISPR/Cas13 a-based ABO blood typing method
1. Sample collection and processing
1. Oral swab DNA samples were collected from healthy persons without blood relationship, who signed informed consent. The ABO phenotype of all volunteers was identified by serological means and their genotypes by Sanger sequencing.
The extraction of genomic DNA from DNA samples was performed using a magnetic bead oral swab DNA extraction kit (Beijing Tiangen Biochemical, DP 322) according to the manufacturer's instructions. The extracted DNA was quantified and stored at-80℃for further use.
2. Multiplex PCR
1. Multiplex PCR Primer design Using common Primer design tools such as PRIMER PREMIER V5.0.0 and NCBI's Primer-BLAST tool (https:// BLAST. NCBI. Lm. Nih. Gov/BLAST. Cgi), primer specific information is shown in Table 1.
TABLE 1 PCR primers, amplification products and crRNA sequences used in the present invention
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Note that: m in sequence 18 represents A or C.
2. Nonspecific hybridization of multiplex PCR primers to other genomic regions was examined using the BLAST tool of NCBI.
The PCR amplification reaction system comprises: MASTER PCR. Mu.L of the mixture, 1-5. Mu.L of the upstream primer, 1-5. Mu. L, DNA of the downstream primer, 1-15. Mu.L of the sample and water free of the nuclease, 50. Mu.L of the total, preferably 2. Mu.L of the upstream primer and 1. Mu.L of the downstream primer, 2. Mu. L, DNA of the sample.
Wherein the upstream primers for amplification product-exons 6-261 are: ABO-6-T7-F1, ABO-6-T7-F2, ABO-6-T7-F3, ABO-6-T7-F4 and ABO-6-T7-F5; the downstream primer is: ABO-6-R1, ABO-6-R2, ABO-6-R3, ABO-6-R4 and ABO-6-R5, are shown in Table 1.
Wherein the upstream primers for amplification product-exons 7-796 are: ABO-7-T7-F1, ABO-7-T7-F2, ABO-7-T7-F3, ABO-7-T7-F4, ABO-7-T7-F5, ABO-7-T7-F6, ABO-7-T7-F7 and ABO-7-T7-F8; the downstream primer is: ABO-7-R1, ABO-7-R2, ABO-7-R3, ABO-7-R4, ABO-7-R5, ABO-7-R6, ABO-7-R7 and ABO-7-R8 are shown in Table 1.
The PCR reaction system was carried out under the following conditions: 98℃10s,55℃10s,68℃10s (35 cycles).
5. Screening 5 pairs for exon 6-261 locus ABO blood group genotyping was performed. The results are shown in FIG. 2, where ABO-6-T7-F1 and ABO-6-R1 (FIG. 2) are preferred, and the amplified product appears as a distinct, single band in the electropherogram, rather than multiple, stray bands. The pair of primers only amplify target gene fragments, and no nonspecific amplification product appears.
6. Screening 8 pairs for exon 7-796 for ABO blood typing results are shown in FIG. 3, where ABO-7-T7-F1 and ABO-7-R1 (FIG. 3) are best, and amplified products appear as distinct, single bands in the electropherogram, rather than multiple, stray bands. The pair of primers only amplify target gene fragments, and no nonspecific amplification product appears.
7. The multiplex PCR reaction system is as follows: mixing two groups of primers of exons 6-261 and exons 7-796 sites in different proportions under the same PCR reaction conditions as 3) and 4), wherein the two groups of primers are specifically as follows: primer set A for exons 6-261 is: ABO-6-T7-F1/ABO-6-R1, primer set B for exon 7-796 is: ABO-7-T7-F1/ABO-7-R1, multiplex PCR reactions were performed.
The results are shown in fig. 4 b. As can be seen from the figure, two sets of primers, in which exons 6-261 and exons 7-796 were mixed in a ratio of 2:8, perform best, the amounts of amplified products generated at the two gene loci are substantially balanced, and the brightness is comparable in the electrophoretogram.
3. CRISPR/Cas13a detection
Obtaining crRNA
CRISPR/Cas13 detection technology crRNA length is 64nt, and can be obtained through commercial synthesis or in vitro transcription. The crRNA sequence is specifically shown in the sequence table 1 6-261-G1、6-261-G2、6-261-G3、6-261-D1、6-261-D2、6-261-D3、7-796-C1、7-796-C2、7-796-C3、7-796-A1、7-796-A2、7-796-A3.
The CRISPR CRRNA reaction generated by in vitro transcription contained 100. Mu.M of the synthesized ssDNA template with the upstream T7 promoter, 100. Mu. M T7-3G IVT primer and 1X standard Taq buffer, denatured at 95℃for 5min and then gradient annealed at 4℃with a slope of 0.1℃per second in a PCR thermal cycler.
CRISPR CRRNA produced by in vitro transcription Using the commercial RNA Synthesis kit NEBT7Quick HIGH YIELD RNA SYNTHESIS KIT, transcription product overnight at 37 ℃.
The CRISPR CRRNA product produced by in vitro transcription was treated with DNase I and purified according to the instructions of RNA Clean & Concentrator TM of ZYMO, and the purified crRNA was quantified. Finally, the crRNA produced was stored at-80 ℃ for further use. Specific sequence information for the crRNA obtained is 6-261-G1、6-261-G2、6-261-G3、6-261-D1、6-261-D2、6-261-D3、7-796-C1、7-796-C2、7-796-C3、7-796-A1、7-796-A2、7-796-A3, and is shown in Table 1.
CRISPR/Cas13a reaction
CRISPR/Cas13a reaction system (20 μl): comprises 1. Mu.L of PCR amplification product (obtained in step two of this example), 2. Mu.L of 10 XCas 13a buffer, 0.8. Mu. L rNTPs (25 mM/seed), 0.2. Mu.L of ssRNA reporter probe 100. Mu.M), 0.2. Mu.L of Cas13a nuclease (10 pmol/. Mu.L), 0.8. Mu. L T7 RNA polymerase (50U/. Mu.L), 0.5. Mu.L of RNase inhibitor (40U/. Mu.L) and 0.5. Mu.L of crRNA (1. Mu.M).
Cas13a nuclease (Shanghai emeter harbor, 32117), cas13 enzyme buffer (Shanghai emeter harbor, 32117), T7 RNA polymerase (NEW ENGLAND BioLabs, M0251L), rNTPs (NEW ENGLAND BioLabs, N0450), RNase inhibitor (Beijing Soy Bao, R8061) are commercially available at a concentration of Cas13a nuclease (10 pmol/. Mu.L), cas13 buffer 10×, T7 RNA polymerase (50U/. Mu.L), rNTPs cocktail (25 mM/seed), RNase inhibitor (40U/. Mu.L). The reporter probe is ssRNA, the length is 5nt, and the 5' -end modifies the fluorescent group 6-FAM;3' -end modification of the quenching group BHQ1.
CRISPR/Cas13a reaction conditions: the fluorescence intensity of each reaction was read automatically during cycling using ABI 7500 at 37 ℃ for 20min and the optical data was analyzed using 7500 software v 2.340.
3. Screening and parting
CRISPR CRRNA 3 pairs of ABO blood group genotyping were screened for each locus. The method comprises the following steps: aiming at the 6-261 locus, the crRNA adopts 6-261-G1 (G1 for short), 6-261-G2 (G2 for short) and 6-261-G3 (G3 for short); 3 pairs of 6-261-D1 (D1), 6-261-D2 (D2) and 6-261-D3 (D3); for 7-796 sites, crRNA adopts 7-796-C1 (C1 for short), 7-796-C2 (C2 for short) and 7-796-C3 (C3 for short); 3 pairs of 7-796-A1 (A1), 7-796-A2 (A2) and 7-796-A3 (A3). The detailed sequence information of the primers and crrnas is listed in table 1.
The results are shown in FIG. 4a, with the best performing primer pairs and crRNA pairs shown in the figure.
In FIG. 4 c,6-261-G2 and 6-261-D3 can specifically detect three genotypes GG, GD, DD (D is a abbreviation for delete, referring to nucleotide G deletion) of exons 6-261. Wherein the genotype is homozygous in which GG is the nucleotide 194 of SEQ ID No.1 marked by exon 6-261 and G is homozygous in which DD is the nucleotide 194 of SEQ ID No.1 marked by exon 6-261 and the genotype is homozygous in which GD is the nucleotide 194 of SEQ ID No.1 marked by exon 6-261 and G is heterozygous containing G (inserted G) and G is deleted.
In FIG. 4 d,7-796-C1 and 7-796-A3 were able to detect the three genotypes CC, CA, AA of exons 7-796. . Wherein the genotype is homozygous for CC with C at nucleotide 362 of SEQ ID No.18 marked with exon 7-796, wherein the genotype is homozygous for A with AA at nucleotide 362 of SEQ ID No.18 marked with exon 7-796, wherein the genotype is heterozygous for C and A with CA at nucleotide 362 of SEQ ID No.18 marked with exon 7-796.
In summary, the CRISPR/Cas13a reaction system of the ABO blood typing method based on CRISPR/Cas13a is as follows: 1. Mu.L of PCR amplification product (obtained in step two of this example, using primers ABO-6-T7-F1& ABO-6-R1 and ABO-7-T7-F1& ABO-7-R1), 2. Mu.L of 10 XCas 13a buffer, 0.8. Mu. LrNTPs (25 mM/seed), 0.2. Mu.L of ssRNA reporter probe 100. Mu.M, 0.2. Mu.L of Cas13a nuclease (10 pmol/. Mu.L), 0.8. Mu. L T7 RNA polymerase (50U/. Mu.L), 0.5. Mu.L of RNase inhibitor (40U/. Mu.L) and 0.5. Mu.L of crRNA (1. Mu.M), wherein 4 crRNAs (6-261-G2, 6-261-D3, 7-796-C1 and 7-796-A3) were added to the system, respectively, i.e.four CRISPR 13a reactions were performed per PCR amplification product obtained in step two (each time corresponding to different crRNAs, otherwise identical).
The blood group genotype result judgment criteria of the above detection method are as follows:
1) if the CRISPR/Cas13a (crRNA-6-261-G2) reaction detects a FAM fluorescent signal, the CRISPR/Cas13a (crRNA-6-261-D3) reaction does not detect a FAM fluorescent signal, the CRISPR/Cas13a (crRNA-7-796-C1) reaction detects a FAM fluorescent signal, and the CRISPR/Cas13a (crRNA-7-796-A3) reaction does not detect a FAM fluorescent signal, blood typing is AA;
2) If the CRISPR/Cas13a (crRNA-6-261-G2) reaction detects a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-6-261-D3) reaction detects a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-7-796-C1) reaction detects a FAM fluorescence signal, and the CRISPR/Cas13a (crRNA-7-796-A3) reaction does not detect a FAM fluorescence signal, the blood group genotype is AO;
3) If the CRISPR/Cas13a (crRNA-6-261-G2) reaction detects a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-6-261-D3) reaction does not detect a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-7-796-C1) reaction does not detect a FAM fluorescence signal, and the CRISPR/Cas13a (crRNA-7-796-A3) reaction detects a FAM fluorescence signal, the blood group genotyping is BB;
4) If the CRISPR/Cas13a (crRNA-6-261-G2) reaction detects a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-6-261-D3) reaction detects a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-7-796-C1) reaction detects a FAM fluorescence signal, and the CRISPR/Cas13a (crRNA-7-796-A3) reaction detects a FAM fluorescence signal, the blood group genotype is BO;
5) If the CRISPR/Cas13a (crRNA-6-261-G2) reaction detects a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-6-261-D3) reaction does not detect a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-7-796-C1) reaction detects a FAM fluorescence signal, and the CRISPR/Cas13a (crRNA-7-796-A3) reaction detects a FAM fluorescence signal, the blood group genotype is AB;
6) If the CRISPR/Cas13a (crRNA-6-261-G2) reaction does not detect a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-6-261-D3) reaction detects a FAM fluorescence signal, the CRISPR/Cas13a (crRNA-7-796-C1) reaction detects a FAM fluorescence signal, and the CRISPR/Cas13a (crRNA-7-796-A3) reaction does not detect a FAM fluorescence signal, the blood group genotyping is OO.
4. Sensitivity test of detection method for ABO blood group genotyping based on CRISPR/Cas13a
Oral swab samples of AA, AO, BB, BO, OO and AB genotypes were used for further validation. Genomic DNA was extracted and quantified according to the manufacturer's instructions (beijing Tiangen biochemistry, DP 322) to evaluate the sensitivity of the assay method of the invention. DNA sample concentrations were 5000, 500, 50 and 5 pg/. Mu.L, negative control was nuclease free deionized water.
The sensitivity of 261-G, 261-D, 796-C and 796-A to detection of gradient diluted DNA samples (5000, 500, 50 and 5 pg/. Mu.L and negative control) was verified using the CRISPR/Cas13a detection method of step three, with DNA template addition volumes of 1. Mu.L for the different concentrations. Fluorescence values at the 20 minute endpoint were collected and multiple comparisons between different groups were performed by two-way anova.
As shown in FIGS. 5 a-d, the results of the detection of the different genotypes were very accurate when the detection limit of the DNA template concentration was 50 pg/. Mu.L.
5. ABO blood group genotyping detection method specificity test based on CRISPR/Cas13a
DNA samples of six healthy persons without blood relationship were extracted and qualitatively analyzed, and genotypes of the six samples were identified by sequencing (a in FIG. 6), and genotypes of each sample were AA, AO, BB, BO, OO and AB, respectively.
Four crRNAs (6-261-G2, 6-261-D3, 7-796-C1 and 7-796-A3) screened in step three were used for multiplex PCR amplification product detection of six DNA samples based on CRISPR/Cas13 a.
According to the real-time fluorescence curves shown in FIG. 6 b, 6-261-G2, 6-261-D3, 7-796-C1 and 7-796-A3 can specifically detect the corresponding alleles; from the 20 minute endpoint fluorescence heat map shown in fig. 6 c, a clear distinction is made between the six genotypes AA, AO, BB, BO, OO and AB. Thus, the method established by the present study can reliably determine ABO blood group genotypes.
Example 2 application of detection method of ABO blood group genotyping based on CRISPR/Cas13a
Sample to be measured: DNA samples of 26 healthy persons without blood relationship. Specific information on the samples is shown in Table 2.
DNA samples of 26 healthy persons without blood relationship were extracted and qualitatively analyzed, and these samples had different phenotypes (A, B, O and AB) and genotypes (AA, AO, BB, BO, OO and AB). Of the 26 samples, 8 were in ABO blood group phenotype A, 2 were in AA genotype and 6 were in AO genotype; 6 persons with the ABO blood group phenotype of B, 2 persons with the BB genotype and 4 persons with the BO genotype; 2 people with an ABO blood group phenotype of AB and 10 people with an ABO blood group phenotype of OO. The ABO phenotype of all samples was identified by serological means and their genotypes by Sanger sequencing (beijing day-hui).
TABLE 2 blood group information of samples in this embodiment
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Note that: d is the abbreviation of delete, del, referring to the nucleotide G deletion.
The sequencing results for exons 6-261 and exons 7-796 are shown in FIG. 7 a. 20 minute endpoint fluorescence calorimeter graphs for multiplex PCR amplification products based on CRISPR/Cas13a (6-261-G2, 6-261-D3, 7-796-C1 and 7-796-A3) for 26 DNA samples are shown in FIG. 7 b.
The results showed that the ABO genotyping results based on CRISPR/Cas13a were 100% identical to the serological and sequencing results. The detection method of the ABO blood group genotyping SNPs based on CRISPR/Cas13a is reliable.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.
Claims (10)
1. Composition for genotyping detection of blood groups, characterized in that it comprises a primer set a and a primer set B specific for the nucleic acid to be detected, a crRNA set 1 and a crRNA set 2 specific for the nucleic acid to be detected, a Cas13a protein and a fluorescent reporter probe; the primer group A consists of a primer ABO-6-T7-F1 and a primer ABO-6-R1; the primer group B consists of a primer ABO-7-T7-F1 and a primer ABO-7-R1; the crRNA group 1 consists of crRNA-6-261-G2 and crRNA-6-261-D3; the crRNA group 2 consists of crRNA-7-796-C1 and crRNA-7-796-A3; the fluorescent reporter probe is single-stranded RNA with one end connected with a fluorescent group and the other end connected with a quenching group;
The primer ABO-6-T7-F1 is (b 1) or (b 2) as follows:
(b1) A single-stranded DNA molecule shown in a sequence 2 of a sequence table;
(b2) A DNA molecule which has the same function as the sequence 2 and is obtained by substituting and/or deleting and/or adding nucleotides in the sequence 2;
The primer ABO-6-R1 is (b 3) or (b 4) as follows:
(b3) A single-stranded DNA molecule shown in a sequence 7 of a sequence table;
(b4) A DNA molecule having the same function as sequence 7 and obtained by subjecting sequence 7 to nucleotide substitution and/or deletion and/or addition;
The primer ABO-7-T7-F1 is as follows (b 5) or (b 6):
(b5) A single-stranded DNA molecule shown in a sequence 19 of a sequence table;
(b6) A DNA molecule having the same function as sequence 19 obtained by subjecting sequence 19 to nucleotide substitution and/or deletion and/or addition;
the primer ABO-7-R1 is (b 7) or (b 8) as follows:
(b7) A single-stranded DNA molecule represented by sequence 27 of the sequence listing;
(b8) A DNA molecule having the same function as sequence 27 and obtained by subjecting sequence 27 to nucleotide substitution and/or deletion and/or addition;
The crRNA-6-261-G2 of the nucleic acid to be detected is (c 1) or (c 2) as follows:
(c1) A single-stranded DNA molecule shown in a sequence 13 of a sequence table;
(c2) A DNA molecule having the same function as sequence 13 obtained by subjecting sequence 13 to nucleotide substitution and/or deletion and/or addition;
The crRNA-6-261-D3 of the nucleic acid to be detected is (c 3) or (c 4) as follows:
(c3) A single-stranded DNA molecule shown in a sequence 17 of a sequence table;
(c4) A DNA molecule having the same function as sequence 17 obtained by subjecting sequence 17 to nucleotide substitution and/or deletion and/or addition;
the crRNA-7-796-C1 of the nucleic acid to be detected is (C5) or (C6) as follows:
(c5) A single-stranded DNA molecule shown in a sequence 35 of a sequence table;
(c6) A DNA molecule having the same function as the sequence 35 by substitution and/or deletion and/or addition of nucleotides to the sequence 35;
the crRNA-7-796-A3 of the nucleic acid to be detected is (c 7) or (c 8) as follows:
(c7) A single-stranded DNA molecule represented by sequence 40 of the sequence listing;
(c8) A DNA molecule having the same function as sequence 40 by substitution and/or deletion and/or addition of nucleotides to sequence 40;
The nucleotide sequence of the single-stranded RNA of the fluorescent reporter probe is the sequence FAM-UUUUU-BHQ1 in the sequence table.
2. The composition of claim 1, wherein: the composition further comprises reagents for performing nucleic acid amplification.
3. A kit for genotyping of blood groups, characterized in that it comprises a composition according to claim 1 or 2.
4. Use of a composition according to claim 1 or 2 in any of the following:
a1 Preparing a product for genotyping to identify or identify blood groups;
a2 Blood typing.
5. Use of the kit of claim 3 in any of the following:
a1 Preparing a product for genotyping to identify or identify blood groups;
a2 Blood typing.
6. CrRNA set 1 and crRNA set 2 as set forth in claim 1 or 2.
7. The primer set a or the primer set B described in claim 1 or 2.
8. A method for detecting blood group genotyping, comprising the steps of:
A1 Extracting DNA of a sample to be detected;
A2 Using the cDNA as a template, and performing PCR amplification by using the primer group A and the primer group B in the claim 1 to obtain an amplification product;
A3 Using a CRISPR-Cas13a detection system to detect the amplification product; the CRISPR/Cas13a detection system comprises crRNA set 1 and crRNA set 2 in the composition of claim 1 or 2.
9. The method of claim 8, wherein the content ratio of primer set a to primer set B is 2:8.
10. The method of claim 8 or 9, wherein the CRISPR-Cas13a detection system further comprises a Cas13a protein, a Cas13a buffer, and a fluorescent reporter probe.
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