CN110878343A - Cpf1 kit for quickly detecting genetic deafness pathogenic gene SLC26A4 mutation and detection method thereof - Google Patents
Cpf1 kit for quickly detecting genetic deafness pathogenic gene SLC26A4 mutation and detection method thereof Download PDFInfo
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Abstract
The invention discloses a Cpf1 kit for quickly detecting genetic deafness pathogenic gene SLC26A4 mutation and a detection method thereof, wherein the Cpf1 kit comprises a Cpf1 detection system; the Cpf1 detection system includes: a crRNA, Cpf1 protein and single stranded DNA reporter system specific for SLC26a 4; the specific crRNA is any one or more designed and synthesized aiming at mutation sites; the single-stranded DNA report system comprises a ssDNA FQ reporter used for fluorescence detection of a microplate reader and/or a ssDNA DB reporter used for detection of an immune colloidal gold test strip. The invention adopts Cpf1 to detect the SLC26A4 gene mutation site for the first time, and has the advantages of high sensitivity, strong specificity, short time consumption, no dependence on large-scale experimental equipment and the like.
Description
Technical Field
The invention relates to the field of genetic deafness gene detection, in particular to a rapid detection method and a detection kit for multiple mutation sites of a SLC26A4 gene applied to clinical diagnosis of vestibular aqueduct enlargement/Pendred syndrome, and belongs to the field of biotechnology.
Background
Deafness is a common disease with defective hearing nervous system in clinic, which seriously affects the life quality of human beings, and causes deafness due to various reasons, genetic factors, environmental factors, medicines, wounds, infection and the like. The incidence of global presbycusis is about 1/1000, of which more than 50% are caused by genetic factors. In hereditary hearing loss, about 80% of the hereditary hearing loss is autosomal recessive inheritance, although hearing loss genes have higher gene and locus heterogeneity, most of the hereditary hearing loss is caused by a few hot-spot gene mutations, including GJB2 gene, SLC26A4 gene, mitochondrial 12S rRNA gene and the like, so that the screening of the hereditary hearing loss becomes possible.
The large vestibular aqueduct syndrome is a non-syndrome type related autosomal recessive genetic disease, and has the same pathogenic gene as pendred syndrome. In 1997 Everett first reported that the SLC26A4 gene is the causative gene of vestibular aqueduct enlargement/pendred syndrome. The SLC26A4 gene is located in the autosomal 7q31 region, contains 21 exons and has an open reading frame of 2343bp, and the gene encodes a multi-transmembrane protein Pendrin containing 780 amino acid residues, mainly consists of hydrophobic amino acids, belongs to one member of an anion transfer family SLC26A and mainly mediates Cl < - >, HCO3Transport of anions such as OH-. In the inner ear, the Pendrin protein may be able to maintain the ionic balance of lymph fluid by regulating Cl-transport, and it has also been found that the Pendrin protein is involved in coordinating the development of the inner ear. At present, hundreds of mutation sites of SLC26A4 gene are related to deafness, and the mutation spectrum and the mutation frequency of different sites are greatly different among different people, and in China, the most common mutation site of SLC26A4 gene is IVS7-2A>G and c.2168A>G, and the like. The clinical manifestations of the deafness related to SLC26A4 gene mutation are congenital sensorineural or mixed deafness, sometimes fluctuating or progressive, and its fluctuation is often related to cold or head trauma, or accompanied byVertigo occurs.
Currently, no treatment method exists for deafness, and three-level preventive intervention measures for hereditary deafness can be established only by screening carriers of hot spot mutation of deafness genes before pregnancy, noninvasive and invasive prenatal diagnosis during pregnancy and postpartum gene detection. At present, the deafness detection technologies mainly comprise allele specific PCR, microarray gene chips, nucleic acid mass spectrometry, sequencing technologies and the like, and the technologies have high detection flux or high accuracy, but have the defects that the main source of a detection sample is adult venous blood or neonatal heel blood, invasive sampling is adopted, the sample preparation process is complex in operation, the detection time is long, the sequencing cost is high, the data analysis difficulty is high, the sensitivity developed based on the technologies is high, the specificity is high, and rapid and convenient noninvasive detection is few.
CRISPR-Cas (Clustered regulated short palindromic repeats, CRISPRs) is an adaptive immune system in bacteria, and Cas proteins target degradation of foreign nucleic acids through RNA-guided nucleases. Among them, the CRISPR-Cas9 protein family has been widely applied to numerous fields such as gene editing, antiviral agents, biological imaging, and the like. CRISPR-Cas12a (Cpf1) belongs to Cas enzyme second family and is used to guide RNA to guide double stranded DNA cleavage of a single RuvC catalytic domain. The Cpf1 enzyme recognizes the Thymine (Thymine, T) nucleotide rich spacer adjacent motif (PAM), catalyzes their own directed CRISPR RNA (crRNA) maturation, and produces PAM distal dsDNA breaks with staggered 5 'and 3' end incompliance. When the CRISPR/Cpf1 protein cleaves double-stranded dna (dsdna) in a sequence-specific manner, a powerful non-specific single-stranded dna (ssdna) trans-cleavage activity is induced. Based on the above properties of Cpf1, we developed a rapid and accurate detection method for detecting the mutation sites of hereditary hearing loss in clinical specimens. Genomic dsDNA is extracted from clinical samples to be tested and Recombinase Polymerase Amplification (RPA) is performed under isothermal conditions. The Cpf1-crRNA complex binds to and cleaves the target SLC26A4dsDNA, which activates the trans-cleavage of ssDNA. Fluorescent reporter molecules coupled to ssDNA generate a fluorescent signal upon cleavage. The novel method called DNA endonuclease targeting CRISPR trans-reporter gene provides a powerful platform for quickly and accurately detecting genetic mutation of genetic diseases.
Colloidal gold immunoassay is an efficient technical scheme for clinical rapid detection. When the colloidal gold particle-labeled antibody binds to the corresponding antigen, the colored immunoreactive reagent can be visually detected. The colloidal gold has the characteristics of short detection action time, long-term stable storage and relatively low cost, and the characteristics ensure that the colloidal gold is widely applicable to high specificity, high sensitivity, convenience and quickness in detection aiming at the SLC26A4 mutation site of the deafness gene clinically.
Disclosure of Invention
The invention aims to provide a high-sensitivity, strong-specificity and rapid visual deafness detection Cpf1 kit and a detection method thereof aiming at rapid detection of clinical hereditary deafness vestibular aqueduct enlargement/pendred syndrome pathogenic gene SLC26A4 mutation sites IVS7-2A > G, c.2168A > G, c.1174A > T and c.1229C > T.
In order to achieve the purpose, the invention provides a Cpf1 kit for rapidly detecting multiple sites of a pathogenic gene SLC26A4 of the hereditary deafness vestibular aqueduct/pendred syndrome, which is characterized by comprising a Cpf1 detection system suitable for mutation of multiple pathogenic sites of the vestibular aqueduct/pendred syndrome.
The Cpf1 detection system includes: specific crRNA and control crRNA for the SLC26a4 gene partial mutation site, Cpf1 protein, and single stranded dna (ssdna) reporter system.
The specific crRNA is any one or more of crRNAs designed aiming at SLC26A4 gene mutation sites IVS7-2A > G, c.2168A > G, c.1174A > T and c.1229C > T, and the sequences of the crRNAs are shown in tables 2, 4, 5, 6 and 7 (or SEQ NO.9 to SEQ NO. 38).
The single-stranded DNA (ssDNA) report system comprises a ssDNA FQ reporter used for fluorescence detection of a microplate reader and/or a ssDNA DB reporter used for detection of an immune colloidal gold test strip; wherein the ssDNA FQ reporter is ssDNA labeled by 6-carboxyfluorescein (6-FAM) and a fluorescence quencher (BHQ1), and the labeling products are as follows: 56 FAM/TTTATTT/3BHQ1/, designated ssDNA FQ reporter/56 FAM/TTTATTT/3BHQ 1/; the ssDNA DBreporter is ssDNA labeled by Digoxin (Digoxin) and Biotin (Biotin), and the labeling products are as follows: the gene is named ssDNA DB reporter/5Dig/TTTATTT/3 Bio/.
Preferably, the Cpf1 kit for rapidly detecting the SLC26A4 gene mutation site of hereditary hearing loss further comprises an immune colloidal gold test strip; the immune colloidal gold test strip comprises a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a PVC back lining; the sample pad, the combination pad, the nitrocellulose membrane and the absorbent pad are sequentially adhered to the PVC backing; the conjugate of the mouse anti-digoxin antibody marked by colloidal gold is coated on the combination pad; the cellulose nitrate membrane is respectively coated with a quality control line formed by streptavidin and a detection line formed by rabbit anti-mouse IgG antibody.
Preferably, the preparation method of the specific crRNA comprises: aiming at multiple mutation sites of SLC26A4 gene, namely IVS7-2A > G, c.1174A > T, c.1229> T and c.2168A > G, a targeting sequence containing cpf1 recognition sequence (PAM) TTTN is searched, crRNA with the length of 23nt is designed and named as IVS7-2-G-23, c.1174-T-23, c.1229-T-23 and c.2168-G-23 respectively, after the design is finished, DNA oligo is synthesized and constructed into a vector pGL3-T7-crRNA, and the target crRNA is obtained through in vitro transcription.
The invention also optimizes the Cpf1 detection system to better distinguish normal sites and mutant sites: aiming at the mutation sites c.1229C > T and c.1174A > T of the SLC26A4 gene, searching a targeting sequence containing a cpf1 recognition sequence TTTN, and respectively designing crRNAs with the lengths of 17nt, 19nt, 21nt and 23nt so as to detect the influence of the lengths of the crRNAs on distinguishing normal sites and mutation sites; aiming at SLC26A4 gene mutation sites IVS7-2A > G and c.2168A > G sites, a targeting sequence containing a cpf1 recognition sequence TTTN is searched, single point mutation is respectively introduced at different sites of crRNA to detect the influence of additionally introduced single base mutation on distinguishing normal sites and mutation sites, after the design is completed, oligo is synthesized to construct a vector LbCpf1-pGL3-T7-crRNA, the target crRNA is obtained through in vitro transcription, and the optimal crRNA for better distinguishing the normal sites and the mutation sites is found through design optimization and detection of the crRNA so as to be used for the detection of a subsequent kit.
Preferably, the preparation method of the control crRNA comprises: according to the first scheme (FIG. 1), aiming at the mutation site of the SLC26A4 gene, introducing a positive control crRNA near each site, named IVS7-2-G-ctrl, c.1174-T-ctrl, c.1229T-ctrl and c.2168-G-ctrl, respectively, and comparing the ratio of the mutation site to the positive control to determine the pure sum and heterozygous type of the detection sample; according to the second scheme (FIG. 1), two crRNAs are designed for the mutation sites, one On crRNA is completely matched with WT, one Off crRNA is completely matched with the mutation sequence and is named IVS7-2-A-21, IVS7-2-G-21, c.1174-A-21, c.1174-T-21, c.1229-C-21, c.1229-T-21, c.2168-A-21 and c.2168-G-21 respectively, and the type of the detected sample is obtained according to the detection signals of the two crRNAs. After the crRNA design is completed, oligo is synthesized and constructed into the vector pGL3-T7-crRNA, and the objective crRNA is obtained through in vitro transcription.
Preferably, the preparation method of the Cpf1 protein comprises: prokaryotic codon optimization is carried out on a Cpf1 protein nucleic acid sequence to obtain a sequence, a pET28a expression vector is constructed, low-temperature induction soluble protein expression is carried out, and a target protein is obtained through affinity purification and molecular sieve purification.
The Cpf1 kit for rapidly detecting multiple mutation sites of the deafness gene SLC26A4 gene provided by the invention can be detected by a fluorescence detection of an enzyme-labeling instrument and can also be detected by an immune colloidal gold test strip. The dna (ssDNA) reporter system in the Cpf1 detection system was ssDNA FQ reporter when fluorescence was detected using a microplate reader, and ssDNA (ssDNA) reporter when detected using an immune colloidal gold strip.
When fluorescence detection is carried out by using a microplate reader, when a mutant SLC26A4 gene site exists in a Cpf1 detection system, the endonuclease activity of the Cpf1 protein is specifically activated under the mediation of SLC26A4 specific crRNA. The activated Cpf1 protein cleaves ssDNA FQ reporter labeled with a fluorophore and a quencher, thereby releasing the activated fluorophore, and a fluorescence reading can be detected using a plate reader. Correspondingly, when the normal SLC26A4 gene sequence exists in the sample to be detected, the fluorescence reading book is shown as a base value.
When an immune colloidal gold test strip is used for detection, after a sample to be detected is added into the colloidal gold test strip after Cpf1 is cut, a mouse anti-digoxin antibody marked by colloidal gold is combined with a digoxin-labeled ssDNA (single-stranded deoxyribonucleic acid) report system, and a compound moves from a quality control line to a detection line along the direction of liquid flow; the quality control line streptavidin saturation captures a ssDNA report system marked with a biotin label, thereby displaying a strip; when the Cpf1 detects the SLC26a4 mutation site, the ssDNA reporter system labeled with digoxin and biotin is cut off, so that the color development captured by the detection line will be realized, and when the Cpf1 detects the normal sequence of the SLC26a4 gene, the ssDNA reporter system labeled with digoxin and biotin cannot be cut off, so that the color development captured by the detection line will not be realized.
The invention also provides a nucleic acid rapid detection method for multiple mutation sites of the deafness gene SLC26A4, which is characterized in that the kit for rapidly detecting the Cpf1 by using the nucleic acid for partial mutation sites of the deafness related gene SLC26A4 is adopted.
Preferably, the rapid detection method for the multiple mutation site nucleic acids of the deafness gene SLC26A4 comprises the following steps:
step a: releasing nucleic acid in a sample to be detected by using a nucleic acid quick release reagent;
step b: amplifying nucleic acid in a sample to be detected by using an isothermal amplification primer: b, adding specific primers SEQ ID NO. 40-SEQ ID NO.45 of different mutation sites of the product obtained in the step a, namely SLC26A4, into an RPA isothermal amplification system, and reacting at 37 ℃ for 20min for amplification to obtain a specific product;
step c: cleavage of SLC26a4 nucleic acid using Cpf1 detection system: adding the product obtained in the step b into a Cpf1 detection system, and reacting for 30min at 37 ℃;
step d: and detecting the pathogenic site of the SLC26A4 gene in the sample by using an enzyme-labeling instrument or an immune colloidal gold test strip.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention realizes high sensitivity, high specificity and rapid visual detection of a plurality of pathogenic mutations of a pathogenic gene SLC26A4 of the hereditary deafness vestibular aqueduct enlargement/pendred syndrome by utilizing the Cpf1 specific recognition nucleic acid and combining the immunochromatography technology. According to research findings, when detecting the mutation site of the SLC26A4 gene, the detection signals of normal people, deaf patients and carriers are different, and in order to better judge the positive result, two schemes are designed, firstly, a control sequence which does not have the mutation site and SNP and contains cpf1 recognition sequence (PAM) TTTN is selected near each specific crRNA to be detected, the fluorescence value of the crRNA designed aiming at the mutation site or the gray value of the positive strip of an immune colloidal gold test strip is compared with the result of the control sequence, and the obtained positive result is judged to be pure patients or mutation carriers according to the ratio; and secondly, designing two crRNAs for the mutation sites respectively, wherein one On crRNA is completely matched with WT, one Off crRNA is completely matched with the mutation sequence, and the type of the detection sample is a pure patient or a mutation carrier is obtained according to detection signals obtained by the two crRNAs.
(2) The invention relates to a rapid detection tool based on a pathogenic gene SLC26A4 of Cpf 1-deafness vestibular aqueduct enlargement/pendred syndrome, which comprises immunochromatography strip detection and can realize convenient and rapid result interpretation.
(3) The invention realizes the rapid, high specificity, high sensitivity and visual detection of the SLC26A4 nucleic acid by utilizing the Cpf1 to cut a specific sequence and adopting an immunoassay chromatography technology. Meanwhile, based on designing different crRNA lengths or introducing mutation sites into specific crRNA sequences, the optimal detection signal of the crRNA capable of distinguishing the normal sites from the mutation sites is explored, and the purity and the heterozygosity of SLC26A4 gene mutation are obviously distinguished. The rapid detection method of the SLC26A4 nucleic acid mutation site, which is established by the invention, provides an accurate, rapid and simple detection method for clinical diagnosis and laboratory research.
(4) The invention discloses a series of Cpf1 reaction systems for SLC26A4 nucleic acid detection, a crRNA combination and an RPA amplification primer, wherein the sequences of the Cpf1 reaction systems and the crRNA combination are sequentially shown as SEQ ID NO.9 to NO. 45. The Cpf1, crRNA and RPA amplification primer combination can be used for detecting SLC26A4 nucleic acid deafness pathogenic mutation sites IVS7-2A > G, c.1174A > T, c.1229> T and c.2168A > G. The invention adopts Cpf1 to detect hereditary hearing loss for the first time, and has the advantages of high sensitivity, strong specificity, short time consumption, high flux, no dependence on large-scale experimental equipment and the like. These advantages make the Cpf 1-based colloidal gold test strip detection method developed by the invention convenient for rapid detection and diagnosis of hereditary hearing loss in laboratories and clinical medicine.
Drawings
FIG. 1 is a schematic diagram of a method for rapidly detecting genetic deafness SLC26A4 nucleic acid pathogenic mutation based on Cpf 1;
FIG. 2 is a specific crRNA design for rapid detection of multiple mutation sites of SLC26A4 nucleic acid based on Cpf 1;
FIG. 3 shows fluorescence detection results of different crRNAs of Cpf1 for detecting SLC26A4 mutation sites;
FIG. 4 influence of crRNA length on fluorescence signal in Cpf1 detection system;
FIG. 5 is a graph showing the influence of the introduction of mutation site positions into crRNA in the Cpf1 detection system on fluorescence signals;
FIG. 6Cpf1 typing a pair of SLC26A4 IVS7-2A > G sites based on the Cpf1 rapid detection protocol;
FIG. 7Cpf1 typing two SLC26A4 IVS7-2A > G sites based on the Cpf1 rapid detection protocol.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
In the invention: RPA amplification kit Twist
Basic kit was purchased from twist amp, inc; the crRNA in vitro Transcription cassette MEGAshortscript T7 Transcription Kit and the purification cassette MEGA clear Kit were purchased from Ambion; conventional reagents such as Tris-Base, NaCl, Tris-HCl, MgCl2BSA and glycerol, etc. were purchased from ThermoFisher; nucleic acid and ssDNA probe synthesis was done by Nanjing Kinsley; the invention uses the product of NovowedThe rapid nucleic acid releasing agent of (a) to obtain a pretreated nucleic acid.
The general technical schematic diagram of the invention is shown in the attached figure 1, and comprises the following 3 parts: preparing a nucleic acid sample to be detected, designing and preparing a Cpf1 detection component, constructing a system, designing a fluorescent and colloidal gold test strip and reading results.
Example 1: rapid and sensitive detection of SLC26A4 gene mutation site fragment
1.1 nucleic acid preparation
In this case, the SLC26a2 gene fragment is obtained by referring to the SLC26a4 gene sequence in the NCBI database, designing an amplification primer according to each mutation site, using a humanized genomic DNA as a template, obtaining a target fragment through PCR amplification, and constructing the target fragment on a pUC57 vector by a homologous recombination method, wherein the target fragment is named as pUC57-IVS7-2-a (SEQ No.1), pUC57-IVS7-2-G (SEQ No.2), pUC57-1174-a (SEQ No.3), pUC57-1174-T (SEQ No.4), pUC57-1229-C (SEQ No.5), pUC57-1229-T (SEQ No.6), pUC57-2168-a (SEQ No.7), and pUC57-2168- > G (SEQ No. 8). The amplification system is shown in Table 1.
TABLE 1 SLC26A4 gene mutation site fragment amplification system
The reaction system is as follows:
and (4) obtaining a sample and then carrying out the next nucleic acid detection.
1.2 design preparation of SLC26A4 Multi-site specific crRNA
As shown in FIG. 2, the preparation of crRNA is performed according to the following scheme, and a targeting sequence containing cpf1 recognition sequence (PAM) TTTN is searched for aiming at a selected mutation site in SLC26A4 gene, and crRNA with the length of 23nt is designed and named IVS7-2-G-23, c.1174-T-23, c.1229-T-23 and c.2168-G-23 respectively. After the design is finished, the DNA oligo is synthesized by the Botanhua biotechnology company, and is constructed on a carrier LbCpf1-pGL3-T7, and the target crRNA is obtained through in vitro transcription.
The SLC26A4 crRNA provided by the invention comprises SEQ ID NO.9 to SEQ ID NO.12, and the specific information is shown in Table 2.
TABLE 2SLC26A4 Gene-specific crRNA
The detection adopts a 20 mu L system as shown in the table 3, but is not limited to the system, and comprises the following steps of adjusting the proportion of corresponding components:
TABLE 3 SLC26A4 multiple mutation site cpf1 detection system
Wherein the ssDNA reporter is ssDNA FQ reporter or ssDNA DB reporter.
1.3 full-wavelength ELIASA fluorescence detection
In the fluorescence detection of the microplate reader, Cpf1 is added with various components in turn to a target gene detection system. The components are mixed evenly and then react for 30min at 37 ℃. Wherein, the concentration of RNase Inhibitors in the reaction system is 40U/. mu.L, the concentration of Cpf1 is 200 ng/. mu.L, the concentration of ssDNA FQ reporter is 25 pmol/. mu.L, and the concentration of crRNA is 1 pmol/. mu.L.
The Cpf1 detection system was assessed for activity using fluorescence detection. The full-wavelength microplate reader is used for measuring fluorescence of detection reaction and monitoring fluorescence dynamics, wherein the excitation wavelength is 485nm, the emission wavelength is 520nm, detection is carried out once every 5 minutes, and detection lasts for 2 hours. Taking the fluorescence value detected for 30min as a reaction value. The detection of the cleavage of each site in the SLC26A4 is shown in figure 3, and the invention can realize the ultra-high sensitivity detection of multiple mutation sites of the SLC26A4 by utilizing a fluorescence method result judgment scheme.
1.4 colloidal gold test strip detection
In the colloidal gold test strip detection, various components are sequentially added into a target gene detection system by Cpf 1. The components are mixed evenly and then react for 30min at 37 ℃. Wherein, the concentration of RNase Inhibitors in the reaction system is 40U/. mu.L, the concentration of Cpf1 is 200 ng/. mu.L, the concentration of ssDNA DB reporter is 25 pnol/. mu.L, and the concentration of crRNA is 1 pmol/. mu.L.
The detection steps of the immune colloidal gold test strip are as follows: mu.L of Cpf1 cleavage product was mixed with 40. mu.L of colloidal gold dipstick buffer (4XSSC, 2% BSA and 0.05% Tween-20, pH 7.0). The test strip was immersed in the mixture and after 3 minutes of reaction, the results were visually determined and recorded by photography.
Example 2: rapid detection condition optimization of SLC26A4 gene fragment
The rapid detection of the SLC26A4 gene mutation site is helpful for definitely judging the mutation in clinical samples, and has important significance for detecting early deafness. In this embodiment, the IVS7-2A > G, c.2168A > G, c.1229C > T and c.1174A > T sites of SLC26A4 gene are taken as examples to demonstrate that the Cpf1 detection kit of the present invention is used for optimizing the detection conditions of SLC26A4 pathogenic sites.
Based on the property that Cpf1 is a highly specific recognition cleavage of a target sequence under crRNA guidance, the hypothesis was proposed: the reduction in the length of the crRNA that precisely pairs with the SLC26a4 gene may result in inefficient recognition of the Cpf1 cleavage system, which is manifested by a somewhat reduced fluorescence value but an increased difference in signal between wild-type and mutant. Based on this principle, we prepared crrnas of different lengths as shown in table 4, comparing the effect of crRNA length matching SLC26a4 gene on the detection signal. In the experiments for detecting the influence of crRNA length on the fluorescence signal value of the C.1229C > T site and the c.1174A > T site of the SLC26A4 gene, the comparison of the results is shown in FIG. 4, and the optimal crRNA length is selected according to the signal difference between the wild type and the mutant type.
TABLE 4 SLC26A4 Gene-specific Length gradient crRNA
Based on the property that Cpf1 is highly specific for recognition and then cleavage of a target sequence under crRNA guidance, the hypothesis was proposed: if a point mutation is added to the crRNA which is accurately matched with the SLC26A4 gene, the Cpf1 cleavage system cannot be efficiently identified, and the detection shows that although the fluorescence value is reduced to a certain extent, the signal difference between the normal gene and the mutant gene is improved. Based on this principle, we introduced the crRNA at the mismatch site as shown in table 5, comparing the effect of the crRNA length matched to the SLC26a4 gene on the detection signal. In the experiment for detecting the influence of the introduction of the crRNA mismatch site on the fluorescence signal value, the result alignment is shown in FIG. 5, wherein the c.2168A > G site of the SLC26A4 gene is introduced.
TABLE 5 introduction of point mutant crRNA at C.2168 locus
In the fluorescence detection, the components were added sequentially in the Cpf1 detection system. The components are mixed evenly and reacted for 30min at 37 ℃. In the present detection system, crRNA refers to the different crRNA designed as mentioned above. Wherein, the concentration of RNase Inhibitors in the reaction system is 40U/. mu.L, the concentration of Cpf1 is 200 ng/. mu.L, the concentration of ssDNA DB reporter is 25 pmol/. mu.L, and the concentration of crRNA is 1 pmol/. mu.L.
In this example, the detection activity of the Cpf1 detection system was determined using fluorescence detection. The full-wavelength microplate reader is used for measuring fluorescence of detection reaction and monitoring fluorescence dynamics, wherein the excitation wavelength is 485nm, the emission wavelength is 520nm, detection is carried out once every 5 minutes, and detection lasts for 2 hours. Taking the fluorescence value detected for 30min as a reaction value.
In this example, the assumption of the difference between the wild-type and mutant signals is verified by detecting the crRNA signal with a length gradient that is precisely matched to the SLC26a4 gene. The results are shown in fig. 4, the fluorescence signals obtained by crrnas of different SLC26a4 recognition nucleic acid segment lengths are different, and the signal difference of c.1174a > T sites detected for wild-type and mutant sequences is most significant. Based on this, the hypothesis that the influence of crRNAs with different matching lengths on the detection signal can be detected through Cpf1 is verified, and in our detection results, 21nt crRNAs have better fluorescence signal difference, so that the subsequent design and detection of the crRNAs are carried out by using 21 nt. Next, the effect of crRNA that precisely pairs with SLC26a4 gene on fluorescence or dipstick signals was examined by introducing point mutations at different positions of crRNA, verifying the hypothesis that mutations at different positions of crRNA differ in wild-type and mutant signals. As shown in FIG. 5, the fluorescence signals obtained by introducing point mutations at different sites of the crRNA of the SLC26A4 recognition nucleic acid segment are different, and the introduced point mutations have better results in the differentiation of the fluorescence signals at one and two positions (c.2168-G-mut + 1 and c.2168-G-mut +2) after the crRNA itself is mutated. Based on this, the hypothesis that introducing different point mutations in crRNA by Cpf1 could more clearly distinguish between wild-type and mutant was verified.
In the colloidal gold test strip detection, various components are sequentially added into a Cpf1 detection system. The components are mixed evenly and reacted for 30min at 37 ℃. In the present detection system, crRNA refers to the different crRNA designed as mentioned above. mu.L of Cpf1 cleavage product was mixed with 40. mu.L of colloidal gold dipstick buffer (4XSSC, 2% BSA and 0.05% Tween-20, pH 7.0). The test strip was immersed in the mixture and after 3 minutes of reaction, the results were visually determined and recorded by photography.
Example 3: rapid and sensitive typing of a pair of SLC26A4 gene mutation sites by using a scheme
The vestibular aqueduct enlargement/Pendred syndrome is an autosomal recessive genetic disease, and the typing detection of the SLC26A4 gene mutation site as a pathogenic gene is beneficial to definitely judging that a clinical sample is a normal population, a mutation carrier or a deafness patient, and has important significance for detecting early deafness. In this embodiment, the site IVS7-2A > G of SLC26A4 gene is used as an example to demonstrate that the Cpf1 detection kit of the present invention can be used to classify the pathogenic site of deafness.
Based on the Cpf1 effect on cleavage efficiency based on how well crRNA matches the target sequence, the hypothesis was presented: selecting a proper control sequence near the IVS7-2A > G site, transcribing the sequence into IVS7-2-G-ctrl in vitro, and determining whether the sample is normal, a carrier or a deaf patient by comparing the detection signal of IVS7-2-G with the detection signal of IVS 7-2-G-ctrl. Based on this principle, we prepared the control crRNA shown in Table 6, and the results of the detection are shown in FIG. 6.
TABLE 6 SLC26A4 Gene-specific control crRNA
In the Cpf1 detection system, the various components were added sequentially. The components are mixed evenly and reacted for 30min at 37 ℃. The Cpf1 detection system was assessed for activity using fluorescence detection. The full-wavelength microplate reader is used for measuring fluorescence of detection reaction and monitoring fluorescence dynamics, wherein the excitation wavelength is 485nm, the emission wavelength is 520nm, detection is carried out once every 5 minutes, and detection lasts for 2 hours. Taking the fluorescence value detected for 30min as a reaction value. The detection of IVS7-2A > G cleavage in SLC26A4 is shown in figure 4, and the invention can realize ultra-high sensitivity detection and typing of SLC26A4 mutation sites by using a fluorescence method result judgment scheme.
In the colloidal gold test strip detection, various components are sequentially added into a Cpf1 detection system. The components are mixed evenly and reacted for 30min at 37 ℃. mu.L of Cpf1 cleavage product was mixed with 40. mu.L of colloidal gold dipstick buffer (4XSSC, 2% BSA and 0.05% Tween-20, pH 7.0). The test strips were immersed in the mixture and after 3 minutes of reaction, photographic recordings and grey value analyses were carried out. The results of the grey value analysis are shown in FIG. 6.
Example 4: rapid and sensitive typing of two pairs of SLC26A4 gene mutation sites by using scheme
In this embodiment, a double crRNA system is adopted, and the site IVS7-2A > G of SLC26A4 gene is taken as an example to demonstrate that the Cpf1 detection kit of the present invention is applied to typing of deafness-causing sites.
Based on the fact that Cpf1 produces significant cleavage signals based on the extent to which crRNA perfectly matches the target sequence, the hypothesis was: aiming at the fact that IVS7-2A > G sites are matched with wild types and mutant types respectively, two crRNAs are designed, namely IVS7-2-A-crRNA and IVS7-2-G-crRNA, the type of a detected sample is judged through cutting signals generated by the two crRNAs, a sample with the cutting signal only in the IVS7-2-A-crRNA is a normal human sample, the cutting signal in both the two crRNAs is a carrier, a sample with the cutting signal only in the IVS7-2-G-crRNA is a deafness patient, and the detection result is shown in figure 7. Based on this principle, we prepared crRNA as shown in table 7.
TABLE 7 SLC26A4 Gene-specific control crRNA
In the Cpf1 detection system, the various components were added sequentially. The components are mixed evenly and reacted for 30min at 37 ℃. The Cpf1 detection system was assessed for activity using fluorescence detection. The full-wavelength microplate reader is used for measuring fluorescence of detection reaction and monitoring fluorescence dynamics, wherein the excitation wavelength is 485nm, the emission wavelength is 520nm, detection is carried out once every 5 minutes, and detection lasts for 2 hours. Taking the fluorescence value detected for 30min as a reaction value. The invention utilizes a fluorescence method result judgment scheme to realize the ultra-high sensitivity detection and typing of the SLC26A4 mutation site.
In the colloidal gold test strip detection, various components are sequentially added into a Cpf1 detection system. The components are mixed evenly and reacted for 30min at 37 ℃. mu.L of Cpf1 cleavage product was mixed with 40. mu.L of colloidal gold dipstick buffer (4XSSC, 2% BSA and 0.05% Tween-20, pH 7.0). The test strips were immersed in the mixture and after 3 minutes of reaction, photographic recordings and grey value analyses were carried out.
Example 5: rapid detection of SLC26A4 gene mutation site for nucleic acid of clinical blood sample and tissue sample
This example provides a rapid nucleic acid detection of clinical blood or tissue samples, all samples and manipulations being performed in the laboratory, in this case Cpf1 detection of DNA obtained from blood extraction. This example uses a rapid nucleic acid release agent from Novomedium to obtain pre-treated nucleic acids. The method comprises the following steps: and adding 20 mu L of nucleic acid lysate into 2 mu L of sample to be detected, standing for 3 minutes at normal temperature, adding 20 mu L of neutralizing solution, mixing uniformly, and carrying out next detection.
By using the RPA amplification primers SEQ NO.40 to SEQ NO.45, 2 mul of each sample to be detected is subjected to RPA pre-amplification by referring to the RPA isothermal amplification operation steps to obtain the sample to be detected. The specific operation is as follows:
25 mu L of 2 × Buffer, 2 mu L of RPA-F, 2 mu L of RPA-R, 2.5 mu L of magnesium acetate and 2 mu L of DNA sample are added with water to be supplemented to 50 mu L, the mixture is uniformly mixed and reacted for 20min at 37 ℃, the obtained sample is subjected to next nucleic acid detection, and 2 mu L of Buffer, 1 mu L of RNase Inhibitors, 1 mu L of Cpf1, 1 mu L of ssDNA DB reporter, 5 mu L of RPAsample, 1 mu L of crRNA and 9 mu L H are sequentially added into a Cpf1 detection system2And O. The components are mixed evenly and reacted for 30min at 37 ℃.
In this example, using scheme two, the Cpf1 cleavage product was subjected to colloidal gold test paper result determination. Cpf1 cleavage product was diluted 1:2 into colloidal gold assay buffer, followed by insertion of the colloidal gold test strip of the invention, the strip and incubation at room temperature for 3 minutes. And judging the test result of the test strip, photographing and storing the test result, and performing gray level analysis on the test result of the colloidal gold test strip. The result shows that the Cpf1 colloidal gold test strip can realize sensitive, rapid and accurate visual detection of multiple deafness-related mutation sites of the SLC26A4 gene in clinical blood samples.
Sequence listing
<110> institute of science and technology of the national institute of health and wellness
<120> Cpf1 kit for quickly detecting genetic deafness pathogenic gene SLC26A4 mutation and detection method thereof
<160>45
<170>SIPOSequenceListing 1.0
<210>1
<211>523
<212>DNA
<213>Artificial Sequence
<400>1
gcgtgtagca gcaggaagta tataaaatta ttttcttttt atagacgctg gttgagattt 60
ttcaaaatat tggtgatacc aatcttgctg atttcactgc tggattgctc accattgtcg 120
tctgtatggc agttaaggaa ttaaatgatc ggtttagaca caaaatccca gtccctattc 180
ctatagaagt aattgtggta agtagaatat gtagttagaa agttcagcat tatttggttg 240
acaaacaagg aattattaaa accaatggag tttttaacat cttttgtttt atttcagacg 300
ataattgcta ctgccatttc atatggagcc aacctggaaa aaaattacaa tgctggcatt 360
gttaaatcca tcccaagggg gtgagtgtgg tgttcctctt agtactaata cattaagtca 420
gtaagtcagt cttttttatt taaataaaac cttttattac aagcttcatt tcactgatac 480
tccttcaata gtcctatttg tgtgtgatct ggaagaaaca acc 523
<210>2
<211>523
<212>DNA
<213>Artificial Sequence
<400>2
gcgtgtagca gcaggaagta tataaaatta ttttcttttt atagacgctg gttgagattt 60
ttcaaaatat tggtgatacc aatcttgctg atttcactgc tggattgctc accattgtcg 120
tctgtatggc agttaaggaa ttaaatgatc ggtttagaca caaaatccca gtccctattc 180
ctatagaagt aattgtggta agtagaatat gtagttagaa agttcagcat tatttggttg 240
acaaacaagg aattattaaa accaatggag tttttaacat cttttgtttt atttcggacg 300
ataattgcta ctgccatttc atatggagcc aacctggaaa aaaattacaa tgctggcatt 360
gttaaatcca tcccaagggg gtgagtgtgg tgttcctctt agtactaata cattaagtca 420
gtaagtcagt cttttttatt taaataaaac cttttattac aagcttcatt tcactgatac 480
tccttcaata gtcctatttg tgtgtgatct ggaagaaaca acc 523
<210>3
<211>560
<212>DNA
<213>Artificial Sequence
<400>3
tatggcgtcc aaactcctga tgtcgtacaa ggaccccaag tacctatcac ggtaaaaatt 60
aaattggacc accacgcaga gtaggcatgg gagttttcat tcttaatgta cttcctgaaa 120
tactcagcga aggtcttgca aagattcaat ttgtaggatc gttgtcatcc agtctcttcc 180
ttaggaattc attgcctttg ggatcagcaa catcttctca ggattcttct cttgttttgt 240
ggccaccact gctctttccc gcacggccgt ccaggagagc actggaggaa agacacaggt 300
aggaacaaca gccttatgat atccatctca gagaacaagt cgaggaatgg caacagagga 360
aggctcgcac cgagcttagc aggacaattt gcctttcaga cttgtacttc ctaatctgat 420
tcacctcagg cctattcctc ttgttccact ccctcacctg aaatctctta aaaaacaaca 480
tgtatggttt tctgatacag tgattctcaa atctatttgt cagtgcttac ctgtcatgta 540
gctacactta cctgctgtgg 560
<210>4
<211>560
<212>DNA
<213>Artificial Sequence
<400>4
tatggcgtcc aaactcctga tgtcgtacaa ggaccccaag tacctatcac ggtaaaaatt 60
aaattggacc accacgcaga gtaggcatgg gagttttcat tcttaatgta cttcctgaaa 120
tactcagcga aggtcttgca aagattcaat ttgtaggatc gttgtcatcc agtctcttcc 180
ttaggaattc attgcctttg ggatcagcta catcttctca ggattcttct cttgttttgt 240
ggccaccact gctctttccc gcacggccgt ccaggagagc actggaggaa agacacaggt 300
aggaacaaca gccttatgat atccatctca gagaacaagt cgaggaatgg caacagagga 360
aggctcgcac cgagcttagc aggacaattt gcctttcaga cttgtacttc ctaatctgat 420
tcacctcagg cctattcctc ttgttccact ccctcacctg aaatctctta aaaaacaaca 480
tgtatggttt tctgatacag tgattctcaa atctatttgt cagtgcttac ctgtcatgta 540
gctacactta cctgctgtgg 560
<210>5
<211>560
<212>DNA
<213>Artificial Sequence
<400>5
tatggcgtcc aaactcctga tgtcgtacaa ggaccccaag tacctatcac ggtaaaaatt 60
aaattggacc accacgcaga gtaggcatgg gagttttcat tcttaatgta cttcctgaaa 120
tactcagcga aggtcttgca aagattcaat ttgtaggatc gttgtcatcc agtctcttcc 180
ttaggaattc attgcctttg ggatcagcaa catcttctca ggattcttct cttgttttgt 240
ggccaccact gctctttccc gcacggccgt ccaggagagc actggaggaa agacacaggt 300
aggaacaaca gccttatgat atccatctca gagaacaagt cgaggaatgg caacagagga 360
aggctcgcac cgagcttagc aggacaattt gcctttcaga cttgtacttc ctaatctgat 420
tcacctcagg cctattcctc ttgttccact ccctcacctg aaatctctta aaaaacaaca 480
tgtatggttt tctgatacag tgattctcaa atctatttgt cagtgcttac ctgtcatgta 540
gctacactta cctgctgtgg 560
<210>6
<211>560
<212>DNA
<213>Artificial Sequence
<400>6
tatggcgtcc aaactcctga tgtcgtacaa ggaccccaag tacctatcac ggtaaaaatt 60
aaattggacc accacgcaga gtaggcatgg gagttttcat tcttaatgta cttcctgaaa 120
tactcagcga aggtcttgca aagattcaat ttgtaggatc gttgtcatcc agtctcttcc 180
ttaggaattc attgcctttg ggatcagcaa catcttctca ggattcttct cttgttttgt 240
ggccaccact gctctttccc gcatggccgt ccaggagagc actggaggaa agacacaggt 300
aggaacaaca gccttatgat atccatctca gagaacaagt cgaggaatgg caacagagga 360
aggctcgcac cgagcttagc aggacaattt gcctttcaga cttgtacttc ctaatctgat 420
tcacctcagg cctattcctc ttgttccact ccctcacctg aaatctctta aaaaacaaca 480
tgtatggttt tctgatacag tgattctcaa atctatttgt cagtgcttac ctgtcatgta 540
gctacactta cctgctgtgg 560
<210>7
<211>516
<212>DNA
<213>Artificial Sequence
<400>7
ctgtagtcct agctaattgg gagggtgagg tggggggatc acttgaactt gggacgcgga 60
ggttgcagtg agcaatgatg ccactgcact ccagcctggg caatagaatg agactctgtc 120
tcaaaaacaa acaaaaattt cttttcctag gaactaacaa aacattgtgt ctttcttttg 180
aagattatgt gatagaaaag ctggagcaat gcgggttctt tgacgacaac attagaaagg 240
acacattctt tttgacggtc catgatgcta tactctatct acagaaccaa gtgaaatctc 300
aagagggtca aggttccatt ttagaaacgg taaatattca acctttctac agatgtatct 360
tttctaaact atcatgattt ctataaatgg caaacattac acaagtctag tctagctgtt 420
gaattttaag ctacctatat aacttcatgg agcctcagtt ttttcatcag taaaatggaa 480
gtaaaaacat taaccttgct aggtagatat gaagac 516
<210>8
<211>516
<212>DNA
<213>Artificial Sequence
<400>8
ctgtagtcct agctaattgg gagggtgagg tggggggatc acttgaactt gggacgcgga 60
ggttgcagtg agcaatgatg ccactgcact ccagcctggg caatagaatg agactctgtc 120
tcaaaaacaa acaaaaattt cttttcctag gaactaacaa aacattgtgt ctttcttttg 180
aagattatgt gatagaaaag ctggagcaat gcgggttctt tgacgacaac attagaaagg 240
acacattctt tttgacggtc cgtgatgcta tactctatct acagaaccaa gtgaaatctc 300
aagagggtca aggttccatt ttagaaacgg taaatattca acctttctac agatgtatct 360
tttctaaact atcatgattt ctataaatgg caaacattac acaagtctag tctagctgtt 420
gaattttaag ctacctatat aacttcatgg agcctcagtt ttttcatcag taaaatggaa 480
gtaaaaacat taaccttgct aggtagatat gaagac 516
<210>9
<211>23
<212>DNA
<213>Artificial Sequence
<400>9
ggacgataat tgctactgcc att 23
<210>10
<211>23
<212>DNA
<213>Artificial Sequence
<400>10
ggatcagcta catcttctca gga 23
<210>11
<211>23
<212>DNA
<213>Artificial Sequence
<400>11
ccgcatggcc gtccaggaga gca 23
<210>12
<211>23
<212>DNA
<213>Artificial Sequence
<400>12
acggtccgtg atgctatact cta 23
<210>13
<211>23
<212>DNA
<213>Artificial Sequence
<400>13
ccgcatggcc gtccaggaga gca 23
<210>14
<211>21
<212>DNA
<213>Artificial Sequence
<400>14
ccgcatggcc gtccaggaga g 21
<210>15
<211>19
<212>DNA
<213>Artificial Sequence
<400>15
<210>16
<211>17
<212>DNA
<213>Artificial Sequence
<400>16
<210>17
<211>23
<212>DNA
<213>Artificial Sequence
<400>17
ggatcagcta catcttctca gga 23
<210>18
<211>21
<212>DNA
<213>Artificial Sequence
<400>18
ggatcagcta catcttctca g 21
<210>19
<211>19
<212>DNA
<213>Artificial Sequence
<400>19
ggatcagcta catcttctc 19
<210>20
<211>17
<212>DNA
<213>Artificial Sequence
<400>20
ggatcagcta catcttc 17
<210>21
<211>21
<212>DNA
<213>Artificial Sequence
<400>21
acggtgcgtg atgctatact c 21
<210>22
<211>21
<212>DNA
<213>Artificial Sequence
<400>22
acggtcggtg atgctatact c 21
<210>23
<211>21
<212>DNA
<213>Artificial Sequence
<400>23
acggtccgag atgctatact c 21
<210>24
<211>21
<212>DNA
<213>Artificial Sequence
<400>24
acggtccgtc atgctatact c 21
<210>25
<211>21
<212>DNA
<213>Artificial Sequence
<400>25
ggtcgataat tgctactgcc a 21
<210>26
<211>21
<212>DNA
<213>Artificial Sequence
<400>26
ggtcgataat tgctactgcc a 21
<210>27
<211>21
<212>DNA
<213>Artificial Sequence
<400>27
ggaggataat tgctactgcc a 21
<210>28
<211>21
<212>DNA
<213>Artificial Sequence
<400>28
ggaccataat tgctactgcc a 21
<210>29
<211>21
<212>DNA
<213>Artificial Sequence
<400>29
atatggagcc aacctggaaa a 21
<210>30
<211>21
<212>DNA
<213>Artificial Sequence
<400>30
taggatcgtt gtcatccagt c 21
<210>31
<211>21
<212>DNA
<213>Artificial Sequence
<400>31
cctttcagac ttgtacttcc t 21
<210>32
<211>21
<212>DNA
<213>Artificial Sequence
<400>32
taaaatggaa ccttgaccct c 21
<210>33
<211>21
<212>DNA
<213>Artificial Sequence
<400>33
agacgataat tgctactgcc a 21
<210>34
<211>21
<212>DNA
<213>Artificial Sequence
<400>34
ggacgataat tgctactgcc a 21
<210>35
<211>21
<212>DNA
<213>Artificial Sequence
<400>35
ggatcagcaa catcttctca g 21
<210>36
<211>21
<212>DNA
<213>Artificial Sequence
<400>36
ccgcacggcc gtccaggaga g 21
<210>37
<211>21
<212>DNA
<213>Artificial Sequence
<400>37
acggtccatg atgctatact c 21
<210>38
<211>21
<212>DNA
<213>Artificial Sequence
<400>38
acggtccgtg atgctatact c 21
<210>39
<211>3744
<212>DNA
<213>Artificial Sequence
<400>39
agcaagctgg aaaaatttac caactgctac agcctgagca agaccctgcg tttcaaagcg 60
atcccggttg gcaagaccca ggaaaacatt gacaacaaac gtctgctggt tgaggacgaa 120
aagcgtgcgg aggattataa aggtgtgaag aaactgctgg atcgttacta tctgagcttt 180
atcaacgacg tgctgcacag cattaagctg aaaaacctga acaactacat cagcctgttc 240
cgtaagaaaa cccgtaccga gaaggaaaac aaagagctgg aaaacctgga aatcaacctg 300
cgtaaggaga ttgcgaaggc gttcaagggt aacgagggct acaagagcct gttcaagaaa 360
gatatcatcg aaaccatcct gccggagttc ctggacgata aggacgaaat tgcgctggtt 420
aacagcttca acggttttac caccgcgttc accggcttct ttgataaccg tgagaacatg 480
tttagcgagg aagcgaaaag caccagcatc gcgttccgtt gcattaacga aaacctgacc 540
cgttacatca gcaacatgga cattttcgag aaggttgacg cgatctttga taaacacgag 600
gtgcaggaaa tcaaggagaa aattctgaac agcgactatg atgttgaaga tttctttgag 660
ggtgaattct ttaactttgt tctgacccaa gagggcatcg acgtgtacaa cgcgatcatt 720
ggtggcttcg tgaccgaaag cggcgagaag atcaaaggcc tgaacgagta cattaacctg 780
tataaccaga agaccaaaca aaagctgccg aaatttaagc cgctgtataa gcaggtgctg 840
agcgatcgtg aaagcctgag cttctacggc gagggctata ccagcgacga ggaagttctg 900
gaagtgtttc gtaacaccct gaacaaaaac agcgagatct tcagcagcat taagaaactg 960
gaaaagctgt tcaaaaactt tgacgagtac agcagcgcgg gtatctttgt taagaacggc 1020
ccggcgatca gcaccattag caaagatatc ttcggtgaat ggaacgtgat tcgtgacaag 1080
tggaacgcgg agtatgacga tatccacctg aagaaaaagg cggtggttac cgaaaagtac 1140
gaggacgatc gtcgtaaaag cttcaaaaag attggcagct ttagcctgga acagctgcaa 1200
gagtacgcgg acgcggatct gagcgtggtt gaaaaactga aggagatcat tatccagaag 1260
gttgatgaaa tctacaaagt gtatggtagc agcgagaagc tgttcgacgc ggattttgtt 1320
ctggagaaga gcctgaaaaa gaacgacgcg gtggttgcga tcatgaagga cctgctggat 1380
agcgtgaaaa gcttcgaaaa ctacattaag gcgttctttg gtgaaggcaa agagaccaac 1440
cgtgacgaga gcttctatgg cgattttgtt ctggcgtacg acatcctgct gaaggtggac 1500
cacatctacg atgcgattcg taactatgtt acccaaaaac cgtacagcaa ggataagttc 1560
aagctgtact tccagaaccc gcaattcatg ggtggctggg acaaggataa agagaccgac 1620
tatcgtgcga ccatcctgcg ttacggtagc aagtactatc tggcgattat ggataaaaag 1680
tacgcgaaat gcctgcagaa gatcgacaaa gacgatgtta acggtaacta cgaaaagatc 1740
aactacaagc tgctgccggg cccgaacaag atgctgccga aagtgttctt tagcaaaaag 1800
tggatggcgt actataaccc gagcgaggac atccaaaaga tctacaagaa cggtaccttc 1860
aaaaagggcg atatgtttaa cctgaacgac tgccacaagc tgatcgactt ctttaaagat 1920
agcattagcc gttatccgaa gtggagcaac gcgtacgatt tcaactttag cgagaccgaa 1980
aagtataaag acatcgcggg tttttaccgt gaggttgagg aacagggcta taaagtgagc 2040
ttcgaaagcg cgagcaagaa agaggtggat aaactggtgg aggaaggtaa actgtacatg 2100
ttccaaatct acaacaagga cttcagcgat aagagccacg gcaccccgaa cctgcacacc 2160
atgtacttca agctgctgtt tgacgaaaac aaccatggtc agatccgtct gagcggtggc 2220
gcggagctgt tcatgcgtcg tgcgagcctg aagaaagagg agctggttgt gcacccggcg 2280
aacagcccga ttgcgaacaa aaacccggat aacccgaaaa agaccaccac cctgagctac 2340
gacgtgtata aggataaacg ttttagcgaa gaccaatacg agctgcacat tccgatcgcg 2400
attaacaagt gcccgaaaaa catcttcaag attaacaccg aagttcgtgt gctgctgaaa 2460
cacgacgata acccgtatgt tatcggtatt gaccgtggcg agcgtaacct gctgtacatc 2520
gtggttgtgg acggtaaagg caacattgtg gaacagtata gcctgaacga gattatcaac 2580
aactttaacg gtatccgtat taagaccgat taccacagcc tgctggacaa aaaggagaag 2640
gaacgtttcg aggcgcgtca gaactggacc agcatcgaaa acattaagga gctgaaagcg 2700
ggctatatca gccaagttgt gcacaagatt tgcgaactgg ttgagaaata cgatgcggtg 2760
atcgcgctgg aggacctgaa cagcggtttt aagaacagcc gtgttaaggt ggaaaagcag 2820
gtttaccaaa agttcgagaa gatgctgatc gataagctga actacatggt ggacaaaaag 2880
agcaacccgt gcgcgaccgg tggcgcgctg aaaggttatc agattaccaa caagttcgaa 2940
agctttaaaa gcatgagcac ccaaaacggc ttcatctttt acattccggc gtggctgacc 3000
agcaaaatcg atccgagcac cggttttgtt aacctgctga agaccaaata taccagcatt 3060
gcggatagca aaaagttcat cagcagcttt gaccgtatta tgtacgtgcc ggaggaagac 3120
ctgttcgagt ttgcgctgga ctataagaac ttcagccgta ccgacgcgga ctacatcaaa 3180
aagtggaaac tgtacagcta tggtaaccgt atccgtattt tccgtaaccc gaaaaagaac 3240
aacgtttttg actgggagga agtgtgcctg accagcgcgt ataaggaact gttcaacaaa 3300
tacggtatca actatcagca aggcgatatt cgtgcgctgc tgtgcgagca gagcgacaag 3360
gcgttctaca gcagctttat ggcgctgatg agcctgatgc tgcaaatgcg taacagcatc 3420
accggtcgta ccgatgttga ttttctgatc agcccggtga aaaacagcga cggcattttc 3480
tacgatagcc gtaactatga agcgcaggag aacgcgattc tgccgaagaa cgcggacgcg 3540
aacggtgcgt ataacatcgc gcgtaaagtt ctgtgggcga ttggccagtt caaaaaggcg 3600
gaggacgaaa agctggataa ggtgaaaatc gcgattagca acaaagaatg gctggagtac 3660
gcgcaaacca gcgttaagca cgagaacctg tacttccaat cccaccacca ccaccaccac 3720
caccaccacc accaccacca ctga 3744
<210>40
<211>20
<212>DNA
<213>Artificial Sequence
<400>40
tggattgctc accattgtcg 20
<210>41
<211>20
<212>DNA
<213>Artificial Sequence
<400>41
ggttgtttct tccagatcac 20
<210>42
<211>15
<212>DNA
<213>Artificial Sequence
<400>42
gatggtatgg cgtcc 15
<210>43
<211>20
<212>DNA
<213>Artificial Sequence
<400>43
ttcaggtgag ggagtggaac 20
<210>44
<211>20
<212>DNA
<213>Artificial Sequence
<400>44
gctaattggg agggtgaggt 20
<210>45
<211>20
<212>DNA
<213>Artificial Sequence
<400>45
ggaaccttga ccctcttgag 20
Claims (7)
1. A Cpf1 kit for rapidly detecting genetic deafness SLC26A4 gene mutation sites is characterized by comprising a Cpf1 detection system suitable for SLC26A4 gene mutation sites IVS7-2A > G, c.2168A > G, c.1174A > T and c.1229C > T;
the Cpf1 detection system includes: specific crRNA and control crRNA, Cpf1 protein and ssDNA reporter system for multiple sites of SLC26a4 gene;
the specific crRNA is any one or more of crRNAs designed aiming at SLC26A4 gene mutation sites IVS7-2A > G, c.2168A > G, c.1174A > T and c.1229C > T, and the sequences of the crRNAs are SEQ NO.9 to SEQ NO. 38;
the ssDNA report system comprises a ssDNA FQ reporter used for fluorescence detection of a microplate reader and/or a ssDNA DB reporter used for detection of an immune colloidal gold test strip; wherein the ssDNA FQ reporter is ssDNA labeled by 6-carboxyfluorescein and a fluorescence quencher, and the labeling products are as follows: 56 FAM/TTTATTT/3BHQ1/, designated ssDNA FQreporter/56 FAM/TTTATTT/3BHQ 1/; the ssDNA DB reporter is ssDNA labeled by digoxin and biotin, and the labeling products are as follows: the gene is named ssDNA DB reporter/5Dig/TTTATTT/3 Bio/.
2. The Cpf1 kit for rapid detection of SLC26A4 gene mutation site of hereditary hearing loss of claim 1, further comprising an immune colloidal gold test strip;
the immune colloidal gold test strip comprises a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a PVC back lining; the sample pad, the combination pad, the nitrocellulose membrane and the absorbent pad are sequentially adhered to the PVC backing; the conjugate of the mouse anti-digoxin antibody marked by colloidal gold is coated on the combination pad; the cellulose nitrate membrane is respectively coated with a quality control line formed by streptavidin and a detection line formed by rabbit anti-mouse IgG antibody.
3. The Cpf1 kit of claim 1 for rapid detection of the SLC26A4 gene mutation site of hereditary hearing loss, wherein the Cpf1 detection system is optimized to better distinguish the normal site from the mutation site: aiming at the mutation sites c.1229C > T and c.1174A > T of the SLC26A4 gene, searching a targeting sequence containing a cpf1 recognition sequence TTTN, and respectively designing crRNAs with the lengths of 17nt, 19nt, 21nt and 23nt so as to detect the influence of the lengths of the crRNAs on distinguishing normal sites and mutation sites; aiming at SLC26A4 gene mutation sites IVS7-2A > G and c.2168A > G sites, a targeting sequence containing a cpf1 recognition sequence TTTN is searched, single point mutation is respectively introduced at different sites of crRNA to detect the influence of additionally introduced single base mutation on distinguishing normal sites and mutation sites, after the design is completed, oligo is synthesized to construct a vector LbCpf1-pGL3-T7-crRNA, the target crRNA is obtained through in vitro transcription, and the optimal crRNA for better distinguishing the normal sites and the mutation sites is found through design optimization and detection of the crRNA so as to be used for the detection of a subsequent kit.
4. The Cpf1 kit for rapid detection of SLC26A4 gene mutation site of hereditary hearing loss as claimed in claim 1, wherein the preparation method of the specific crRNA comprises: aiming at the mutation site of the SLC26A4 gene and respective control sequences, a targeting sequence containing a cpf1 recognition sequence TTTN is searched, crRNA with the length of 21nt is designed, after the design is finished, oligo is synthesized and constructed to a vector LbCpf1-pGL3-T7-crRNA, and the target crRNA is obtained through in vitro transcription.
5. The Cpf1 kit for rapidly detecting the mutation site of the SLC26A4 gene of hereditary hearing loss of claim 1, wherein the preparation method of the Cpf1 protein comprises the following steps: prokaryotic codon optimization is carried out on cpf1 protein nucleic acid sequence to obtain sequence SEQ NO.39, pET28a expression vector is constructed, low-temperature induction soluble protein expression is carried out, and target protein is obtained through affinity purification and molecular sieve purification.
6. A method for rapidly detecting genetic deafness SLC26A4 gene mutation sites, which is characterized in that a Cpf1 kit for rapidly detecting SLC26A4 gene mutation sites IVS7-2A > G, c.2168A > G, c.1174A > T and c.1229C > T is adopted according to any one of claims 1 to 5.
7. The method for rapidly detecting the SLC26A4 gene mutation site of hereditary hearing loss according to claim 6, wherein the method for rapidly detecting the SLC26A4 gene mutation site comprises the following steps:
step a: releasing nucleic acid in a sample to be detected by using a nucleic acid quick release reagent;
step b: amplifying nucleic acid in a sample to be detected by using an isothermal amplification primer: b, adding the product obtained in the step a into an RPA isothermal amplification system by using specific primers SEQ NO. 40-SEQ NO.45 of different sites, and reacting at 37 ℃ for 20min for amplification to obtain a specific product;
step c: the Cpf1 detection system is used for detecting signals of the SLC26A4 gene mutation site and the control site: adding the product obtained in the step b into a Cpf1 detection system, and reacting for 30min at 37 ℃;
step d: detecting detection signals and control sequence signals of SLC26A4 gene IVS7-2A > G, c.2168A > G, c.1174A > T and c.1229C > T sites in a sample by using an immune colloidal gold test strip, and performing typing after a ratio is made.
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