阅读说明：本技术 叶酸代谢相关分子标志物基因突变的检测试剂盒与检测方法 (Detection kit and detection method for gene mutation of folate metabolism related molecular marker ) 是由 姚杰 赵洪友 程诚 于 2020-12-11 设计创作，主要内容包括：本发明公开了一种检测叶酸代谢相关分子标志物基因突变的crRNA、试剂盒及检测方法,其中crRNA包括：MTHFR-667突变位点的crRNA：序列如SEQ ID NO.11-12任一所示；MTHFR-1298突变位点的crRNA：序列如SEQ ID NO.13-14任一所示；MTRR-66基因突变位点的crRNA：序列如SEQ ID NO.15-16任一所示。通过对crRNA进行筛选并结合CRISPR-Cpf1系统,可在短时间内检测待测样本中是否存在叶酸代谢相关分子标志物基因突变,所述试剂盒及检测方法不仅操作简单、检测速度快、成本低、可多次重复检测,同时检测灵敏度和检测特异性也显著提高。(The invention discloses a crRNA for detecting folate metabolism related molecular marker gene mutation, a kit and a detection method, wherein the crRNA comprises: crRNA at the MTHFR-667 mutation site: the sequence is shown in any one of SEQ ID NO. 11-12; crRNA at the MTHFR-1298 mutation site: the sequence is shown in any one of SEQ ID NO. 13-14; crRNA of MTRR-66 gene mutation site: the sequence is shown in any one of SEQ ID NO. 15-16. By screening crRNA and combining with a CRISPR-Cpf1 system, whether the gene mutation of the molecular marker related to folate metabolism exists in a sample to be detected can be detected in a short time.)

1. A crDNA for detecting a mutation in a folate metabolism-related molecular marker gene, said crDNA comprising:

crDNA of MTHFR-667 mutation site: the sequence is shown in any one of SEQ ID NO. 5-6;

crDNA of MTHFR-1298 mutation site: the sequence is shown in any one of SEQ ID NO. 7-8;

crDNA of MTRR-66 gene mutation site: the sequence of the polypeptide is shown in any one of SEQ ID NO. 9-10.

2. A crRNA for detecting a folate metabolism-related molecular marker gene mutation, wherein the crRNA comprises:

crRNA at the MTHFR-667 mutation site: the sequence is shown in any one of SEQ ID NO. 11-12;

crRNA at the MTHFR-1298 mutation site: the sequence is shown in any one of SEQ ID NO. 13-14;

crRNA of MTRR-66 gene mutation site: the sequence of the polypeptide is shown in any one of SEQ ID NO. 15-16.

3. A kit for detecting a gene mutation of a folate metabolism-related molecular marker, wherein the kit comprises the crDNA of claim 1 or the crRNA of claim 2.

4. The assay kit of claim 3, further comprising a Cpf1 protein and a fluorescent probe.

5. The detection kit of claim 4, wherein the Cpf1 protein is FnCpf1 protein.

6. The detection kit of claim 4, wherein the fluorescent probe sequence is labeled with a fluorescent group at the 5 'end and a quenching group at the 3' end.

7. The detection kit according to claim 6, wherein the fluorescent group is selected from any one of FAM, VIC, HEX, TRT, Cy3, Cy5, ROX, JOE and Texas Red, and the quenching group is selected from any one of TAMRA, DABCYL, MGB, BHQ-1, BHQ-2 and BHQ-3.

8. Use of the crDNA according to claim 1, the crRNA according to claim 2, or the detection kit according to any one of claims 3 to 7 for the purpose of detecting the mutation of the folate metabolism-related molecular marker gene for non-disease diagnosis and treatment.

9. A method for detecting a mutation in a folate metabolism-related molecular marker gene for non-disease diagnostic and therapeutic purposes, said method comprising the steps of:

s1, amplifying the nucleic acid of the sample to be detected to obtain an amplification product;

s2, and detecting the amplification product, the crRNA, the Cpf1 protein, the fluorescent probe and enzyme-free water which form a detection system.

10. The assay of claim 9 wherein the assay is incubated and the change in fluorescence is detected or measured using a colloidal gold strip.

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a detection kit and a detection method for gene mutation of a folate metabolism related molecular marker.

Background

Folic acid is also called pteroylglutamic acid, belongs to B vitamins, and is an important nutrient required by human body. Folic acid acts in the form of tetrahydrofolic acid in vivo and acts as a donor for one-carbon units, is involved in DNA oxidative damage repair, cell proliferation and tissue growth, aids in protein metabolism, and, together with vitamin B12, promotes the production and maturation of erythrocytes. A plurality of enzymes in a folic acid metabolism pathway are jointly involved in folic acid metabolism and transportation, methylenetetrahydrofolate Reductase (MTHFR) and methionine synthase Reductase (MTRR) are two most critical enzymes, and a large number of researches show that polymorphism exists in the enzyme genes, so that enzyme activity is changed, folic acid metabolism is abnormal, the active folic acid level is reduced, the homocysteine (Hcy) level is increased, and the plasma Hcy level is increased and is closely related to occurrence of various birth defects such as neural tube deformity, congenital heart disease, Down's syndrome and the like, and pregnancy diseases such as pregnancy hypertension, recurrent abortion and the like.

MTHFR and MTRR are key enzymes of folate metabolism. The mutation of 667 th site and 1298 th site of MTHFR gene will cause the enzyme activity to change, wherein the enzyme activity is reduced to the minimum when the two sites are double mutated. The 66 th site of MTRR gene is the most studied gene site at present, and the A → G conversion of the MTRR gene can cause the activity of the enzyme encoded by MTRR to be reduced, so that the folate metabolism level is analyzed by the mutation detection result of the sites clinically.

At present, in clinical practice, the gene mutation detection of folate metabolism related sites of patients is generally performed by means of Quantitative real-time polymerase chain reaction (Q-PCR), nonradioactive in situ hybridization (Fish), multiplex PCR (multiplex PCR), and the like, however, these detection techniques all have the problems that only one gene can be detected at a time or the detection cost is too high, and the like, so that the detection techniques cannot be applied to large-scale clinical sample research. Therefore, it is highly desirable to provide a specific fragment detection method with simple temperature control and high sensitivity and specificity.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a detection kit and a detection method for folate metabolism related molecular marker gene mutation, wherein crRNA used in the kit is screened and combined with a CRISPR-Cpf1 system to detect whether the folate metabolism related molecular marker gene mutation exists in a sample to be detected, and the kit and the detection method are simple in operation, high in detection speed, low in cost and capable of obviously improving detection sensitivity and detection specificity.

In order to achieve the purpose, the invention adopts the technical scheme that:

a crDNA for detecting a folate metabolism-related molecular marker gene mutation, the crDNA comprising:

crDNA of MTHFR-667 mutation site: the sequence is shown in any one of SEQ ID NO. 5-6;

crDNA of MTHFR-1298 mutation site: the sequence is shown in any one of SEQ ID NO. 7-8;

crDNA of MTRR-66 gene mutation site: the sequence of the polypeptide is shown in any one of SEQ ID NO. 9-10.

The invention also provides a crRNA for detecting the gene mutation of the folate metabolism related molecular marker, which comprises:

crRNA at the MTHFR-667 mutation site: the sequence is shown in any one of SEQ ID NO. 11-12;

crRNA at the MTHFR-1298 mutation site: the sequence is shown in any one of SEQ ID NO. 13-14;

crRNA of MTRR-66 gene mutation site: the sequence of the polypeptide is shown in any one of SEQ ID NO. 15-16.

The invention also provides a detection kit for the gene mutation of the folate metabolism related molecular marker, and the detection kit comprises the crDNA or the crRNA.

Further, the detection kit further comprises a Cpf1 protein and a fluorescent probe.

Further, the Cpf1 protein is FnCpf1 protein.

Furthermore, the 5 'end of the fluorescent probe sequence is marked with a fluorescent group, and the 3' end is marked with a quenching group.

Further, the fluorescent group is selected from any one of FAM, VIC, HEX, TRT, Cy3, Cy5, ROX, JOE and Texas Red, and the quenching group is selected from any one of TAMRA, DABCYL, MGB, BHQ-1, BHQ-2 and BHQ-3.

Further, the detection kit also comprises a DNA enzyme inhibitor.

The invention also provides the use of the crDNA or the crRNA or the detection kit in the purposes of non-disease diagnosis and treatment for detecting the gene mutation of the folate metabolism related molecular marker.

The invention also provides a detection method for the gene mutation of the folate metabolism related molecular marker for non-disease diagnosis and treatment purposes, which comprises the following steps:

s1, amplifying the nucleic acid of the sample to be detected to obtain an amplification product;

s2, detecting the detection system consisting of the amplification product, the crRNA, the Cpf1 protein, the fluorescent probe and enzyme-free water.

Further, after the detection system is incubated, a colloidal gold test paper is used for detecting or determining the change of the fluorescence value. When visual detection is carried out through the colloidal gold test paper, the detection system after incubation is added to the detection area of the colloidal gold test paper, and the detection result is determined through the existence of the detection line, so that the operation is simple, convenient and quick, and the application range is wide. Or detecting the fluorescence change before and after the reaction by using a fluorescence detector in the incubated detection system so as to judge whether the gene of the folate metabolism related molecular marker gene mutation is contained in the sample to be detected.

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

the invention provides a crDNA and a crRNA for detecting folate metabolism related molecular marker gene mutation, a detection kit containing the crDNA or the crRNA and a detection method. According to the invention, the corresponding crRNA is designed based on the gene mutation site of the folate metabolism related molecule marker, the screening and verification are carried out on the crRNA, and meanwhile, the CRISPR-Cpf1 system is combined, so that whether the corresponding folate metabolism related molecule marker gene mutation exists in a sample to be detected can be detected through the change of a fluorescent signal or the existence of a detection line on a colloidal gold test paper in a short time. The kit and the detection method have the advantages of simple operation, high detection speed, low cost and repeated detection, and the detection sensitivity and the detection specificity are also obviously improved, wherein the lowest detection limit can reach 10 copies/. mu.L:

wherein in pairIn the detection of MTHFR-667 mutation site, when the reaction is carried out for 20-30 minutes, the fluorescence signal of the mutant gene is obviously higher than that of the wild-type gene, and the method can distinguish 10 copies of mutant from 10 copies of mutant⁴The sensitivity of the copied wild strain is higher than that of the common PCR;

in the detection of MTHFR-1298 mutant site, when the reaction is carried out for 20-30 minutes, the fluorescence signal of the mutant gene is obviously higher than that of the wild-type gene, and the method can distinguish 10 copies of the mutant from 10 copies of the wild-type gene⁴The sensitivity of the copied wild strain is higher than that of the common PCR;

in the detection of MTRR-66 mutation site, when the reaction is carried out for 20-30 minutes, the fluorescence signal of the mutant gene is obviously higher than that of the wild-type gene, and the method can distinguish 10 copies of mutant strains from 10 copies of mutant strains⁴The sensitivity of the copied wild strain is higher than that of the common PCR.

Drawings

FIG. 1 shows the results of screening test of 6 crRNAs in example 3 of the present invention;

FIG. 2 shows the result of specific detection of MTHFR-667 in example 3 of the present invention;

FIG. 3 shows the result of specific detection of MTHFR-1298 in example 3 of the present invention;

FIG. 4 shows the result of specific detection of MTRR-66 in example 3 of the present invention;

FIG. 5 shows the results of the sensitivity assay for MTHFR-667 in example 4 of the present invention;

FIG. 6 shows the results of the sensitivity assay for MTHFR-1298 in example 4 of the present invention;

FIG. 7 shows the results of the sensitivity assay for MTRR-66 of example 4 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 design and acquisition of crRNA targeting Gene mutation site

1. Discovery of folate metabolism detection site based on CRISPR-Cpf1 system

Common mutation site sequences of folate-generation related mutant genes are determined according to gene sequences and a large amount of clinical detection data, crRNA is designed aiming at different regions, and a CRISPR-Cpf1 system is constructed for research. The result shows that the sequence of the region shown in SEQ ID NO.1-3 of the table 1 is used as the detection site of the folic acid metabolic mutation based on the CRISPR-Cpf1 system, and the detection effect is good.

TABLE 1 detection sites for folate metabolism mutations

Wherein, MTHFR-667 mutation site, namely, the C/T polymorphism exists at the 677 th site of the MTHFR gene, and the C → T mutation exists at the 25bp position corresponding to SEQ ID NO. 1; MTHFR-1298 mutant site, namely, the A/C polymorphism exists at the 1298 th site of MTHFR gene, and the A → C mutation exists at the position corresponding to 24bp in SEQ ID NO. 2; the MTRR-66 mutation site, namely the 66 th site of MTHFR gene has A/G polymorphism, and the 22bp position corresponding to SEQ ID NO.3 has A → G mutation.

2. Design of crRNA targeting gene mutation site

(1) Design principle of targeted gene mutation site crRNA

Since the CRISPR-Cpf1 system is a novel targeted DNA gene editing system, where Cpf1 binds to the crRNA to form a monitoring complex, the guide region of the crRNA recognizes the target DNA with complementary sequences, and Cpf1 degrades the target DNA strand. Wherein the crRNA design requires: the crRNA includes a protein anchor sequence and a guide sequence in the sequence format: 5 '-anchor sequence binding to Cpf1 protein-crRNA guide-sequence-3', protein anchor sequence needs to be determined from Cpf1 protein to enable it to match and bind to the selected Cpf1 protein; the guide sequence is matched to a fragment in the targeting DNA. The crRNA guide sequence cannot be too close to the initiation codon (ATG); the length is 22-24 nucleotides.

(2) Selection of crDNA sequences

The Cpf1 protein selected by the invention is FnCpf1, so that the anchor sequence selected to be combined with the Cpf1 protein is UAAUUUCUACUCUUGUAGAU (shown as SEQ ID NO. 4); the crRNA guide sequence is designed according to the mutation site region in the sequence shown by SEQ ID NO.1-3, each mutation site is designed with two corresponding crDNAs, and the sequence is specifically as follows:

crDNA of MTHFR-667 mutation site: the sequence is shown in any one of SEQ ID NO. 5-6;

crDNA of MTHFR-1298 mutation site: the sequence is shown in any one of SEQ ID NO. 7-8;

crDNA of MTRR-66 gene mutation site: the sequence of the polypeptide is shown in any one of SEQ ID NO. 9-10.

(3) Obtaining crRNA

The obtained crDNA fragments at the 3 mutation sites are respectively subjected to the action of T7 RNA polymerase (the transcription reaction system is shown in Table 2) to generate RNA, and the RNA is recovered and purified to obtain the crRNA.

TABLE 2 transcription reaction System

The crRNA obtained was:

crRNA at the MTHFR-667 mutation site: the sequence is shown in any one of SEQ ID NO. 11-12;

crRNA at the MTHFR-1298 mutation site: the sequence is shown in any one of SEQ ID NO. 13-14;

crRNA of MTRR-66 gene mutation site: the sequence of the polypeptide is shown in any one of SEQ ID NO. 15-16.

Example 2 detection kit and detection method for folate metabolism-related molecular marker gene mutation

1. Composition of the kit

The kit comprises 6 crRNAs (the crRNAs corresponding to 3 mutation sites are obtained as shown in example 1) for folic acid metabolism detection or crDNAs (when the crDNAs are contained in the kit, an operator needs to firstly generate RNAs from crDNA fragments under the action of T7 RNA polymerase, and the CRRNAs are obtained by recovery and purification, specifically shown in example 1), a specific fluorescent probe (shown in Table 3), Cpf1 protein (FnCpf 1 is adopted in the example), enzyme-free water and a DNase inhibitor, wherein the CRRNAs are obtained from 6 mutation sites;

TABLE 3 fluorescent Probe sequences

Fluorescent probe	Sequence (5 '-3')
		Probe 1	FAM-TTTTTTTT-BHQ1
Probe 2	FAM-TTTTTTTTTT-BHQ1
		Probe 3	FAM-TTTTTTTTTTTT-BHQ1

Further, the kit may further include an amplification system, the amplification system includes isothermal amplification primer pairs, and the primer pair of each mutation site specifically includes:

the sequence of the isothermal amplification primer pair of the MTHFR-667 mutation site is shown in SEQ ID NO. 17-18;

the sequence of isothermal amplification primer pair of MTHFR-1298 mutation site is shown in SEQ ID NO. 19-20;

the sequence of the isothermal amplification primer pair of the MTRR-66 mutation site is shown as SEQ ID NO. 21-22.

2. Detection method for gene mutation of folate metabolism related molecular marker

(1) Taking 50-100ng of sample DNA to be detected, adding the sample DNA into an isothermal amplification system (RPA amplification system), wherein the RPA amplification system is shown in table 4, and the primers are corresponding isothermal amplification primer pairs shown above.

TABLE 4 RPA amplification System

(2) Mixing the obtained amplification product with the detection reagent: the corresponding crRNA, Cpf1 protein, fluorescent probe and enzyme-free water were mixed as shown in Table 5 to obtain a detection system.

TABLE 5 test System

100-250nM purified Cpf1, 50-100nM crRNA, 1-5. mu.L of synthetic fluorescent probe, 2. mu.L of DNase inhibitor, 5-10. mu.L of amplified product of the target DNA were incubated in detection buffer (NEBuffer 3) at 37 ℃ for 1-3 hours. A negative control group (the amplification product was replaced with enzyme-free water) was also set up. Incubating the detection system, adding the incubated detection system into a colloidal gold test strip sample detection area for detection, and observing whether the detection line exists or not; or after the detection system is incubated, a fluorescence detector is used for measuring fluorescence values, and the change conditions of the fluorescence values before and after reaction are statistically analyzed so as to judge whether the corresponding folate metabolism related molecular marker gene mutation exists in the DNA of the sample to be detected.

Example 3 specific detection of wild type and mutant sequences by crRNA

Synthesizing target sequences of a wild strain (WT) and a mutant strain (MUT), constructing detection systems by using 6 crRNAs designed in example 1 respectively, and carrying out detection screening, wherein the detection results are shown in FIG. 1, wherein 667-crRNA1 is a sequence shown in SEQ ID NO.11, and 667-crRNA2 is a sequence shown in SEQ ID NO. 12; 1298-crRNA1 is a sequence shown by SEQ ID NO.13, and 1298-crRNA2 is a sequence shown by SEQ ID NO. 14; 66-crRNA1 is shown in SEQ ID NO.15, and 66-crRNA2 is shown in SEQ ID NO. 16. According to the detection result, the crRNAs with more obvious change of fluorescence value in the mutant strain, namely higher sensitivity and more excellent effect compared with the wild strain are selected, and are 667-crRNA2 shown in SEQ ID NO.12, 1298-crRNA1 shown in SEQ ID NO.13 and 66-crRNA1 shown in SEQ ID NO.15 respectively.

The crRNA with relatively good effect is obtained through the screening, is used for constructing a CRISPR-Cpf1 system, and is subjected to in vitro cutting validity verification, and specifically comprises the following steps:

100-250nM purified Cpf1, 250-500nM crRNA, 1-5. mu.L of synthetic fluorescent probe, 2. mu.L of DNase inhibitor, and target DNA at different dilution concentrations were incubated in detection buffer (NEBuffer 3) at 37 ℃ for 1-3 hours. Several sets of reaction mixtures were reacted simultaneously in a portable detector (temperature set at 37 ℃ C., kinetic measurements were performed every 10min for 1 hour). And detecting the change of the fluorescence signal of the system by using a fluorescence quantitative PCR instrument.

1. Specific detection of MTHFR-667

The detection effect of MTHFR-667crRNA shown in SEQ ID NO.12 on mutant sequences of wild MTHFR-667 and MTHFR-667 was detected, and the detection results are shown in FIG. 2. FIG. 2 shows the results of systematic fluorescence values of MTHFR-667 mutation sites. The results show that the fluorescence signal is increased after wild MTHFR-667 and mutant MTHFR-667 templates with different concentrations are added, wherein the signal of the MTHFR-667 mutant template completely matched with MTHFR-667crRNA is more than 2 to 3 times of that of a wild type control group along with the time extension and is generally higher than that of the wild type, and the method can distinguish 10 copies of the mutant from 10 copies of the mutant⁴The sensitivity of the copied wild strain is higher than that of the common PCR.

2. Specific detection of MTHFR-1298

The detection effect of MTHFR-1298crRNA shown in SEQ ID NO.13 on wild MTHFR-1298 and MTHFR-1298 mutant sequences is detected, and the detection result is shown in FIG. 3. FIG. 3 shows the results of the systematic fluorescence of MTHFR-1298 mutation site. The results show that the fluorescence signal is increased after the wild MTHFR-1298 and the mutant MTHFR-1298 template with different concentrations are added, wherein the signal of the MTHFR-1298 mutant template completely matched with the MTHFR-1298crRNA is increased along with the timeThe elongation is more than 2 to 3 times of the signal of a wild type control group, and is generally higher than the wild type, and the method can be used for distinguishing 10 copies of mutant strains from 10 copies of mutant strains⁴The sensitivity of the copied wild strain is higher than that of the common PCR.

3. Specific detection of MTRR-66

The detection effect of MTRR-66crRNA shown in SEQ ID NO.15 on wild MTRR-66 and MTRR-66 mutant sequences is detected, and the detection result is shown in FIG. 4. FIG. 4 shows the results of systematic fluorescence values of MTRR-66 mutation sites. The result shows that the fluorescence signal is increased after the wild MTRR-66 and the mutant MTRR-66 templates with different concentrations are added, wherein the signal of the MTRR-66 mutant template which is completely matched with MTRR-66crRNA is more than 2 to 3 times of that of a wild control group and is generally higher than the wild type along with the time extension, and the method can distinguish the 10-copy mutant from the 10-copy mutant⁴The sensitivity of the copied wild strain is higher than that of the common PCR.

In conclusion, the results show that the change trend of the system fluorescent value of the crRNA detection target DNA designed by the invention is significantly higher than that of the negative control, namely 3 crRNAs for detecting the gene mutation of the folate metabolism related molecular marker can respectively and specifically detect corresponding mutant sequences.

Example 4 sensitivity test of crRNA to wild-type and mutant sequences

The sensitivity of 3 crRNAs with relatively good effect obtained by screening in detection example 3 specifically comprises:

1. sensitive detection of crRNA at MTHFR-667 mutation site

MTHFR-667 mutant plasmid and wild type plasmid standard substance are subjected to gradient dilution and then used as templates for PCR amplification and construction of a detection system, and then 1.0% agarose gel electrophoresis is used for detection, and the electrophoresis result is shown in FIG. 5. Negative control (NC, i.e., 0 copies/. mu.L) no band, indicating no contamination during amplification; template concentration at 10³copies/. mu.L to 10⁴When the copies/mu L is detected, a single electrophoresis band between 100-250bp can be observed, the band is clear and is consistent with the size of a target band (about 150 bp); and when the template concentration is less than 10²No amplified band was observed at copies/. mu.L, indicating thatWhen agarose gel electrophoresis is used for detecting PCR products, the sensitivity can reach about 10²About copies/. mu.L.

2. Sensitive detection of crRNA of MTHFR-1298 mutation site

MTHFR-1298 mutant plasmid and wild type plasmid standard substance are subjected to PCR amplification as templates after being subjected to gradient dilution, a detection system is constructed, and then the detection is carried out by using 1.0% agarose gel electrophoresis, wherein the electrophoresis result is shown in figure 6. Negative control (NC, i.e., 0 copies/. mu.L) no band, indicating no contamination during amplification; template concentration at 10³copies/. mu.L to 10⁴When the copies/mu L are used, a single electrophoresis band between 250-500bp can be observed, the band is clear and is consistent with the size of a target band (about 280 bp); and when the template concentration is less than 10²No amplified band was observed at copies/. mu.L, indicating that the sensitivity of the PCR product detected by agarose gel electrophoresis was about 10²About copies/. mu.L.

3. Sensitive detection of CRRNA of MTRR-66 gene mutation site

MTRR-66 mutant plasmid and wild type plasmid standard substance are subjected to gradient dilution and then used as templates for PCR amplification and detection system construction, and then 1.0% agarose gel electrophoresis is used for detection, and the electrophoresis result is shown in FIG. 7. Negative control (NC, i.e., 0 copies/. mu.L) no band, indicating no contamination during amplification; template concentration at 10³copies/. mu.L to 10⁴When the primers are applied to mu L, a single electrophoresis band between 250 and 500bp can be observed, the band is clear and is consistent with the size of a target band (about 320 bp); and when the template concentration is less than 10²No amplified band was observed at copies/. mu.L, indicating that the sensitivity of detecting PCR product by agarose gel electrophoresis was about 10²About copies/. mu.L.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

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<213> Artificial Sequence (Artificial Sequence)

<400> 22

gtattagtgt ccttttgttt cataaatatc ac 32