CN114317710A - Primer probe combination for detecting SNP typing of polymorphic site of GLP1R gene, kit and application thereof - Google Patents
Primer probe combination for detecting SNP typing of polymorphic site of GLP1R gene, kit and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of genetic engineering, in particular to a primer probe combination for detecting SNP typing of polymorphic sites of GLP1R gene, a kit and application thereof, wherein the primer probe combination comprises 1 pair of primers, a probe PA and a probe PG; the kit comprises the primer probe combination and is used for detecting the rs6923761 polymorphic site of the GLP1R gene. When the kit disclosed by the invention is used for detecting the gene mutation related to the drug administration of sitagliptin and vildagliptin, the detection specificity is strong, the sensitivity is high, the safety is high, the operation is simple, the time consumption is short, the result is easy to read, and the kit has a good clinical application value.
Description
Technical Field
The invention relates to a primer probe combination and application thereof, in particular to a primer probe combination for detecting GLP1R gene polymorphic site SNP typing, a kit and application thereof.
Background
Diabetes is one of the important chronic metabolic diseases threatening the health of people at present, and the incidence rate of the diabetes is on the rising trend year by year. The prevalence rate of diabetes in China in 2017 is 11.2%, the number of patients is about 1.56 hundred million, the first in the world is, and the type of diabetes is mainly type 2 diabetes. The incretin-like peptide1 is secreted by intestinal L cells, is combined with a GLP-1 receptor (GLP 1R) on the surface of an islet B cell, and regulates the secretion of insulin through a G protein coupling way. In recent years, researches show that although the GLP-1 level of a type 2 diabetic patient is not changed after meal, the insulin secretion function is obviously damaged; in addition, some type 2 diabetic patients were insensitive to exogenous GLP-1 stimulation, suggesting a possible correlation with dysfunction of the GLP1R gene. The GLP1R gene polymorphic site can obviously influence the response of islet B cells after healthy people infuse GLP-1, and the GLP1R rs6923761 gene polymorphic site is related to the reduction of GLP-1 stimulated insulin secretion, so that the detection of the GLP1R gene polymorphic site in type 2 diabetes patients can be helpful to clarify the reason of individual difference and guide disease treatment.
Vildagliptin tablets belong to oral hypoglycemic drugs, belong to dipeptidyl peptidase 4 inhibitors, also known as DPP-4 inhibitors, can enhance insulin sensitivity, and enhance glucose-dependent insulin secretion mainly through inhibition effect on dipeptidyl peptidase 4. Vildagliptin has certain blood sugar reducing effect on patients with fasting blood sugar, postprandial blood sugar and hyperglycemia. The polymorphic locus of the GLP1R rs6923761 gene is related to the curative effect of sitagliptin or vildagliptin. Patients with AA genotype and type 2 diabetes may have reduced response to sitagliptin or vildagliptin compared to patients with GG genotype. Patients with GG genotype and type 2 diabetes may have increased response to sitagliptin or vildagliptin compared to patients with AA genotype. Therefore, before clinical medication, the kit has great guiding significance for carrying out GLP1R gene polymorphism detection, changing the drug of a patient carrying AA or AG genotype or increasing the dosage of sitagliptin or vildagliptin, and is also beneficial to monitoring the blood sugar level.
Disclosure of Invention
Aiming at the problems, the invention provides a primer probe combination for detecting SNP typing of polymorphic sites of GLP1R gene, a kit and application thereof.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a primer probe combination for detecting SNP typing of polymorphic sites of a GLP1R gene is disclosed, wherein a forward primer of the primer probe combination for detecting SNP typing of the polymorphic sites of rs6923761 of the GLP1R gene is shown as SEQ ID NO: 1, and the reverse primer is shown as SEQ ID NO: 2, and the probe PA is shown as SEQ ID NO: 3, and the probe PG is shown as SEQ ID NO: 4, respectively.
A kit comprising the primer probe combination for detecting the SNP typing of the polymorphic site of the GLP1R gene.
Further, the kit contains a primer probe combination solution;
the concentration ratio of the forward primer, the reverse primer, the probe PA and the probe PG in the primer probe combination solution is 1: 1: 1: 0.5.
furthermore, the reagent in the kit also comprises a reaction solution.
Further, the reaction solution was composed of 0.2 parts by volume of Taq DNA polymerase, 10 parts by volume of buffer Mix for SNP # 3, 3 parts by volume of Mega buffer Mix for SNP, and 3.8 parts by volume of ddH2And O is prepared.
An application of the kit in detection of the rs6923761 polymorphic site of the GLP1R gene.
Further, the specific steps of the application are that the nucleic acid of the sample to be detected is taken and respectively mixed with the reagent in the kit, the mixture is oscillated, mixed evenly and centrifuged to prepare a reaction system to be detected, then PCR amplification reaction is carried out, each reaction obtains 2 amplification curve graphs and CT values, and then the genotype of the nucleic acid of the sample to be detected is judged according to the obtained amplification curve graphs and CT values.
Further, the reaction system to be detected is 17 muL of reaction liquid, 1 muL of primer probe combination liquid and 2 muL of sample nucleic acid to be detected;
the concentration ratio of the forward primer, the reverse primer, the probe PA and the probe PG in the primer probe combination solution is 1: 1: 1: 0.5;
the reaction solution was composed of 0.2 volume parts of Taq DNA polymerase, 10 volume parts of buffer Mix for SNP 3#, 3 volume parts of Mega buffer Mix for SNP and 3.8 volume parts of ddH2And O is prepared.
Further, the amplification curve algorithm is set as an absolute fluorescence value method, the manual threshold value is adjusted to 100 for analysis, and the standard for judging the genotype of the nucleic acid of the sample to be detected is as follows:
when the CT value of FAM is less than or equal to 38 and the CT value of HEX is more than 38/no CT value, the genotype is judged as GG type;
when the CT value of HEX is less than or equal to 38 and the CT value of FAM is more than 38/no CT value, the genotype is judged as AA;
when the CT value of FAM is less than or equal to 38 and the CT value of HEX is less than or equal to 38, determining the genotype as AG type;
when the CT value of FAM is > 38/no CT value and the CT value of HEX is > 38/no CT value, then retesting is required.
Further, the PCR amplification reaction conditions were 95 ℃ pre-denaturation for 2min, followed by 95 ℃ denaturation for 5s and 58 ℃ annealing for 15s for 40 cycles.
The primer probe combination, the kit and the application thereof for detecting SNP typing of polymorphic sites of GLP1R gene have the beneficial effects that:
the primer probe combination and the kit for detecting SNP typing of polymorphic sites of GLP1R gene can detect human GLP1R (rs6923761) gene by adopting fluorescent quantitative PCR, have high accuracy, and have 100% of accuracy compared with first-generation sequencing; the detection limit is lower than that of the existing products on the market and is 0.1 ng/mu L; the precision CV value is less than or equal to 4.28 percent; the anti-interference performance is strong, wherein heme, triglyceride, bilirubin and the like have no influence on detection results, and the detection results also have no cross reaction with Escherichia coli nucleic acid, staphylococcus aureus nucleic acid, Enterobacter cloacae nucleic acid and the like;
when the kit is used for detecting the gene mutation related to the drug administration of sitagliptin and vildagliptin, the detection specificity is strong, the sensitivity is high, the safety is high, the operation is simple, the time consumption is short, and the result is easy to read;
by optimizing the kit, the application process of the whole kit is simple and rapid, the PCR program only needs 50min, the cost is lower, and the detection speed is obviously higher than that of the first-generation sequencing detection speed;
the kit disclosed by the invention has low demand for each sample, and can be used for detection only by 10-50 ng of DNA;
the kit can assist in judging the medication of the sitagliptin and the vildagliptin so as to accurately judge the medication condition, provides sufficient basis for disease diagnosis, provides a solid foundation for a treatment scheme, and has good clinical application value.
Drawings
FIG. 1 is a diagram showing the results of a screening experiment using a first set of primer probe combinations in example 1 of the present invention;
FIG. 2 is a diagram showing the results of a screening experiment using a second set of primer probe combinations in example 1 of the present invention;
FIG. 3 is a diagram showing the results of a screening experiment using a third set of primer probe combinations in example 1 of the present invention;
FIG. 4 is a graph showing the results of a concentration screening experiment of the primer probe combination A in example 1 of the present invention;
FIG. 5 is a graph showing the results of concentration screening experiments for the primer probe composition B in example 1 of the present invention;
FIG. 6 is a graph showing the results of concentration screening experiments for primer probe combination C in example 1 of the present invention;
FIG. 7 is a graph showing the results of concentration screening experiments for the primer probe combination solution D in example 1 of the present invention;
FIG. 8 is a graph showing the results of a concentration screening experiment of the primer probe combination E in example 1 of the present invention;
FIG. 9 is a graph showing the results of the screening test for the detection limit of the reagent kit in example 3 of the present invention;
FIG. 10 is a graph showing the results of the precision test of the reagent kit in example 4 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Reagent: AK Taq DNA polymerase, AK buffer Mix for SNP 3#, Mega buffer Mix for SNP were purchased from Fipeng BioSciagnosis, Inc.
Example 1 design and screening of primer Probe combinations
One) design of primer-probe combination
1) Primer probe combination design
The polymorphic site information of the GLP1R gene rs6923761 is inquired on the NCBI network, and the specific gene sequence is as follows:
wherein, the mutation direction is as follows: g is greater than the ratio of A to G,
gene frequency: GG-0.971, AG-0.029, AA-close to 0.
Introducing into biological software such as Beacon Designer, oligo, Primer 5 and the like according to the inquired polymorphic site information of the GLP1R gene rs6923761, and designing a Primer pair by combining a Primer probe design principle and a personal probe design experience; and designing two probes (PG and PA) corresponding to the primer pair according to the mutation direction and the designed primer pair. In the process of relating to the probe, in order to distinguish genotypes by using the probe in the application process, when the probe is designed, FAM fluorescence is modified on 5 'of a PG probe, and HEX fluorescence is modified on 5' of a PA probe. After the design is finished, the sequence of the designed primer probe combination is introduced into NCBI for specific comparison, the specificity is 100 percent and is not consistent with other gene sequences, and the screening design result is shown in Table 1. It should be noted that, in the design process of the primer probe combination, due to the contingency of the effect of the application of the primer probe combination, we have carried out multiple designs, and only three of them are listed here.
Primer probe combination for detecting SNP typing of rs6923761 polymorphic site of GLP1R gene designed in Table 1
| Name (R) | Sequence 5 'to 3' | 5' modification | 3' modification |
| F1 | TGGGCTACGCACTCTCCTT | ||
| R1 | TGGGCTGCTTCATTCCTCTATC | ||
| PA1 | TCCTCAGCTTCAGG | HEX | MGB |
| PG1 | TCCTCCTCGGCTTC | FAM | MGB |
| F2 | GTGGGCTACGCACTCTCCTT | ||
| R2 | TTGGGCTGCTTCATTCCTCTA | ||
| PA2 | ATCCTCCTCAGCTTC | HEX | MGB |
| PG2 | CTCCTCGGCTTCAGGT | FAM | MGB |
| F3 | GTGGGCTACGCACTCTCCTT | ||
| R3 | GTCCGGGCCACCTTACCT | ||
| PA3 | CGATCCTCCTCAGCT | HEX | MGB |
| PG3 | CGATCCTCCTCGGC | FAM | MGB |
The designed primer probe combinations were each synthesized by general biology companies.
2) Primer probe combination screening
The PCR amplification experiments were performed for the primer probe combinations designed in Table 1, respectively, and the specific procedures were as follows:
the primer probe combination liquid in the table 1 is prepared by taking the primer probe combination respectively, and the preparation method comprises the following steps: mu.L of 100. mu.M forward primer, 1. mu.L of 100. mu.M reverse primer, 1. mu.L of 100. mu.M probe PA and 1. mu.L of 100. mu.M probe PG are mixed, and 10. mu.L of DEPC water is added to supplement the mixture to obtain the corresponding primer-probe combination solution (namely, 1. mu.L of 100. mu.M F1, 1. mu.L of 100. mu.M R1, 1. mu.L of 100. mu.M PA1, 1. mu.L of 100. mu.M PG1 and 6. mu.L of DEPC water are used to prepare the primer-probe combination solution 1, and the like).
Taking 0.2. mu.L of AK Taq DNA polymerase, 10. mu.L of AK buffer Mix for SNP 3#, 3. mu.L of Mega buffer Mix for SNP and 3.8. mu.L of ddH2Mixing O, and centrifuging to obtain reaction solution (the amount of the reaction solution)The required amount for 1 reaction well, when the experimental amount is expanded, only the corresponding times are expanded).
General biologies were entrusted with the synthesis of two genotypic plasmids in table 2;
TABLE 2 GLP1R Gene rs6923761 polymorphic site genotype plasmids
Two types of genotype plasmids in the table 2 were diluted to a concentration of 0.0005 ng/. mu.L with 0.1TE buffer solution, respectively, to obtain an AA type plasmid diluent (obtained by diluting GLP1R-A plasmid) and a GG type plasmid diluent (obtained by diluting GLP1R-G plasmid).
And mixing the AA type plasmid diluent and the GG type plasmid diluent in equal volume to obtain the AG type plasmid diluent.
The AA type plasmid diluent, the GG type plasmid diluent and the AG type plasmid diluent are collectively called plasmid diluent.
And respectively and sequentially adding 17 mu L of reaction liquid, 1 mu L of primer probe combination liquid and 2 mu L of plasmid diluent into each reaction hole in the eight-row reaction system, uniformly mixing, and centrifuging to obtain a corresponding reaction system.
Wherein, each kind of primer probe combination liquid adds to 6 reaction holes, two reaction holes in these 6 reaction holes add AA type plasmid diluent, two reaction holes add GG type plasmid diluent, two last reaction holes add AG type plasmid diluent (that is, there are 2 reaction holes that add primer probe combination liquid 1 and AA type plasmid diluent, there are 2 reaction holes that add primer probe combination liquid 1 and GG type plasmid diluent, there are 2 reaction holes that add primer probe combination liquid 1 and AG type plasmid diluent, analogize with this).
And (3) respectively taking the reaction system and the control reaction system, moving the reaction system and the control reaction system to a fluorescence quantitative PCR instrument, and carrying out PCR amplification reaction under the conditions of pre-denaturation at 95 ℃ for 2min, denaturation at 95 ℃ for 5s and annealing at 58 ℃ for 15s for 40 cycles, wherein the detection results are shown in table 3 and figures 1-3 (the manual threshold value is adjusted to 100).
TABLE 3 summary of the results of the primer probe combination screening experiments
And (3) screening the quality of the probe according to the CT value and the fluorescence intensity, ideally, the reaction hole only added with the AA type plasmid diluent only has HEX fluorescence (green) line, the reaction hole only added with the GG type plasmid diluent only has FAM fluorescence (blue) line, and the reaction hole added with the AG type plasmid diluent has both fluorescence lines. As can be seen from fig. 1, FAM fluorescence (blue) lines and HEX fluorescence (green) lines in the reaction systems corresponding to the three plasmid dilutions are all high and low, indicating that the primer probe combination cannot distinguish the three plasmid dilutions; FIG. 2 shows that there is almost no line in the reaction system corresponding to AA type plasmid diluent, HEX fluorescence (green) is too high and FAM fluorescence (blue) is also line in the reaction system corresponding to GG type plasmid diluent, FAM fluorescence (blue) is not line and HEX fluorescence (green) is slightly line in the reaction system corresponding to AG type plasmid diluent, which indicates that the primer probe combination cannot distinguish three types of plasmid diluents, so this group is discarded; as can be seen from fig. 3, only HEX fluorescence (green) in the reaction system corresponding to the AA-type plasmid diluent has an appearance, only FAM fluorescence (blue) in the reaction system corresponding to the GG-type plasmid diluent has an appearance, and both HEX fluorescence (green) and FAM fluorescence (blue) in the reaction system corresponding to the AG-type plasmid diluent have appearances, which indicates that 3 plasmid diluents can be clearly distinguished, and the experimental result is in line with expectations.
Meanwhile, the true AA type nucleic acid sample is used for replacing the AA type plasmid diluent, the true GG type nucleic acid sample is used for replacing the GG type plasmid diluent, and the AG type nucleic acid sample is used for replacing the AG type plasmid diluent.
Therefore, the forward primer of the primer probe combination for detecting the SNP typing of the polymorphic site rs6923761 of the GLP1R gene is shown as SEQ ID NO: 1, and the reverse primer is shown as SEQ ID NO: 2, and the probe PA is shown as SEQ ID NO: 3, and the probe PG is shown as SEQ ID NO: 4, respectively.
However, in the reaction system corresponding to the AG-type plasmid dilution, the FAM fluorescence (blue) line is higher than the HEX fluorescence (green), and the two fluorescences need to be adjusted to be consistent as much as possible, so the ratio of the combined amounts of the primer and the probe needs to be adjusted to reduce the amount of the probe PG.
3) Proportional adjustment of the amount of primer probe combination
Preparing primer probe combination solution with different proportions according to a preparation method in the primer probe combination screening in the step 2), wherein the difference from the step 2) is only that:
only 0.6 mu L of probe PG is added into the primer probe combination liquid A;
only 0.7 mu L of probe PG is added into the primer probe combination liquid B;
only 0.8 mu L of probe PG is added into the primer probe combination solution C;
only 0.5 microliter of the probe PG is added into the primer probe combination solution D;
only 0.4 microliter of the probe PG is added into the primer probe combination solution E;
and then, according to the PCR amplification experiment in the primer probe combination screening in the step 2), the primer probe combination solutions A to E are measured, and the measurement results are shown in tables 4 to 5 and figures 4 to 8.
Table 4 dosage ratio screening test results summary table (one)
TABLE 5 dosage ratio screening Experimental results List (II)
As can be seen from tables 4 to 5 and FIGS. 4 to 8, when the primer probe composition solution D was used, the FAM fluorescence (blue) line was substantially the same as the HEX fluorescence (green) line in the reaction system corresponding to the AG-type plasmid dilution, and 3 types of genotyping were confirmed. Therefore, the final ratio of the primer to probe combinations is 1 μ L of forward primer, 1 μ L of reverse primer, 1 μ L of probe PA, 0.5 μ L of probe PG and 6.5 μ L of DEPC water (this is a 10-person formulation, and if the amount is large, the ratio is enlarged proportionally).
4) Preparation and application of kit
Preparing a kit by using the primer probe combination determined in the step 2) of primer probe combination screening and the dosage determined in the step 3) of primer probe combination dosage proportion adjustment, wherein the primer probe combination solution is prepared by mixing the following components in a volume ratio of 1: 1: 1: 0.5: 6.5 forward primer at a concentration of 100. mu.M, reverse primer at a concentration of 100. mu.M, probe PA at a concentration of 100. mu.M, probe PG at a concentration of 100. mu.M and DEPC water (the concentration ratio of forward primer, reverse primer, probe PA and probe PG is 1: 1: 1: 0.5); the reaction solution was composed of 0.2. mu.L of AK Taq DNA polymerase, 10. mu.L of AK buffer Mix for SNP 3#, 3. mu.L of Mega buffer Mix for SNP and 3.8. mu.L of ddH2Mixing O uniformly, and centrifuging to obtain the product (the amount of the reaction solution is the required amount of 1 reaction hole, and only needs to be enlarged by a corresponding multiple when the experimental amount is enlarged).
During detection, 17 mu L of reaction liquid, 1 mu L of primer probe combination liquid and 2 mu L of sample nucleic acid to be detected are respectively and sequentially added into each reaction hole in the eight-linked rows, and after uniform mixing, the reaction system to be detected is obtained by centrifugation.
And moving the eight rows into a fluorescence quantitative PCR instrument for PCR amplification reaction, wherein the reaction conditions comprise pre-denaturation at 95 ℃ for 2min, denaturation at 95 ℃ for 5s and annealing at 58 ℃ for 15s for 40 cycles, each reaction hole obtains an amplification curve graph and a CT value of the two fluorescence of FAM and HEX, and the detection results are respectively analyzed according to the two amplification curve graphs and the CT value obtained from each reaction hole to judge the genotype of the nucleic acid of the sample to be detected. Setting the amplification curve algorithm as an absolute fluorescence value method, adjusting the manual threshold value to 100 for analysis, and judging the genotype of the nucleic acid of the sample to be detected according to the following standard:
when the CT value of FAM is less than or equal to 38 and the CT value of HEX is more than 38/no CT value, the genotype is judged as GG type;
when the CT value of HEX is less than or equal to 38 and the CT value of FAM is more than 38/no CT value, the genotype is judged as AA;
when the CT value of FAM is less than or equal to 38 and the CT value of HEX is less than or equal to 38, determining the genotype as AG type;
when the CT value of FAM is > 38/no CT value and the CT value of HEX is > 38/no CT value, then retesting is required.
Example 2 test for validation of kit
Extracting whole blood nucleic acid samples of 50 persons, determining the concentration, entrusting the Okinsoniaceae biological company to carry out first-generation sequencing on polymorphic sites of rs6923761 of GLP1R gene, and taking a first-generation sequencing result as a gold standard;
the preparation and application of the kit in example 1 were used to detect the above 50 whole blood nucleic acid samples, and the detected gene results were compared with the first-generation sequencing results to verify the accuracy of the kit and the application of the kit, and the specific results are shown in the following table:
TABLE 6 summary of the results of the correctness verification tests
As can be seen from the above table, the kit and the application thereof have good correctness.
Example 3 detection Limit test of kit
Taking any of the two genotypes of whole blood nucleic acid samples in Table 6 (whole blood samples No. 1 and No. 3 selected in this example), preparing dilutions of GG type nucleic acid sample with different concentrations and dilutions of AG type nucleic acid sample with different concentrations at 0.01, 0.1, 0.5, 1, 10, and 100 ng/. mu.L, respectively;
the preparation and application of the kit in example 1 were used to measure dilutions of the GG-type whole blood nucleic acid sample and dilutions of the AG-type whole blood nucleic acid sample at different concentrations, respectively, and the specific results are shown in Table 7 and FIG. 9.
TABLE 7 summary of the results of the different concentrations of plasmid dilutions
As can be seen from Table 7 and FIG. 9, the kit of the present invention detected three genotypes of the whole blood nucleic acid sample at a concentration of 0.1 ng/. mu.L or more.
The stability of the dilutions of the GG-type whole blood nucleic acid sample at a concentration of 0.1 ng/. mu.L and the dilutions of the AG-type whole blood nucleic acid sample was measured again, and the results are shown in Table 8.
TABLE 8 Total blood nucleic acid sample dilution test results List with concentration of 0.1 ng/. mu.L
As can be seen from Table 8, the primary positive detection rate of the GG type whole blood nucleic acid sample diluent with the concentration of 0.1 ng/. mu.L detected by the kit disclosed by the invention is 95%, and the primary positive detection rate of the AG type whole blood nucleic acid sample diluent with the concentration of 0.1 ng/. mu.L detected by the kit disclosed by the invention is 95%, so that the industrial requirements (the common detection limit of 20 times and the primary positive detection rate of more than or equal to 90%) are met, and therefore, the lowest detection Limit (LOD) of three genotypes of the kit disclosed by the invention is 0.1 ng/. mu.L.
Example 4 precision testing of the kit
Using the preparation and application of the kit in example 1, 10 replicates (in the same time, place, person and instrument, and in a short time) were performed on whole blood nucleic acid samples of 3 genotypes, respectively, and CV values were calculated, and the specific results are shown in table 9 and fig. 10.
TABLE 9 summary of the results of the precision test
As can be seen from Table 9 and FIG. 10, the CV values of the primer probe combinations of the present invention are all less than or equal to 4.28%, which is lower than the industry standard (CV value is less than or equal to 5%), and the reagents have good precision.
Example 5 interference assay of the kit
The preparation and application of the kit in example 1 were used to extract nucleic acids from whole blood added with three genotypes having heme concentration less than or equal to 200g/L, triglyceride concentration less than or equal to 1000mg/dL, or bilirubin concentration less than or equal to 2mg/dL, and the three extracted nucleic acid samples were tested, with the specific results shown in Table 10.
TABLE 10 summary of interference test results
As can be seen from Table 10, the detection performance of the kit of the present invention is not affected by heme, triglyceride and bilirubin, and the anti-interference ability is strong.
The nucleic acid samples to which the E.coli nucleic acid, S.aureus nucleic acid or E.cloacae nucleic acid was added at a concentration of 0.01 ng/. mu.L were each tested by using the preparation and application of the kit of example 1, and the specific results are shown in Table 11.
TABLE 11 summary of interference test results
As can be seen from Table 11, the kit of the present invention has no cross-reactivity with Escherichia coli nucleic acid, Staphylococcus aureus nucleic acid, Enterobacter cloacae nucleic acid, etc., which are potential non-human genome nucleic acid interfering substances.
EXAMPLE 6 actual clinical sample testing of the kit
In the embodiment, the clinical sample is a whole blood sample collected from a hospital in the corridor city area (principle that a collector voluntarily) and the nucleic acid of the sample to be detected is extracted from the whole blood sample, the detection is carried out by using the kit and the detection method, and the genotype of the nucleic acid sample is detected and judged by comparing the nucleic acid sample with an internal reference substance and a positive reference substance for 50 min.
And (3) entrusting the organism company of department of Onychosanthes to perform first-generation sequencing on the rs6923761 polymorphic locus of the GLP1R gene for the nucleic acid of the sample to be detected extracted from the whole blood sample and the nucleic acid of the sample to be detected extracted from the whole blood sample, wherein the sequencing time is 2 d.
The actual clinical sample detection rate of the kit is higher than that of the existing kit.
The specific test results of the actual clinical samples are shown in the following table:
TABLE 12 summary of test results of actual clinical specimens
| Nucleic acid of sample to be tested | Number of samples detected by the kit of the invention | Number of samples detected by existing |
| AA | ||
| 1 | 1 | |
| |
2 | 2 |
| GG | 141 | 141 |
It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all 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.
Sequence listing
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Primer probe combination and kit for detecting SNP typing of polymorphic sites of GLP1R gene and application of primer probe combination and kit
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Claims (10)
1. A primer probe combination for detecting SNP typing of a polymorphic site of a GLP1R gene is characterized in that a forward primer of the primer probe combination for detecting SNP typing of the polymorphic site of the GLP1R gene is shown as SEQ ID NO: 1, and the reverse primer is shown as SEQ ID NO: 2, and the probe PA is shown as SEQ ID NO: 3, and the probe PG is shown as SEQ ID NO: 4, respectively.
2. A kit comprising the primer probe combination for detecting SNP typing of polymorphic sites of GLP1R gene according to claim 1.
3. The kit according to claim 2, wherein the kit contains a primer probe combination solution;
the concentration ratio of the forward primer, the reverse primer, the probe PA and the probe PG in the primer probe combination solution is 1: 0.5.
4. The kit according to claim 2 or 3, wherein the reagent in the kit further comprises a reaction solution.
5. The kit according to claim 4, wherein the reaction solution comprises 0.2 parts by volume of Taq DNA polymerase, 10 parts by volume of buffer Mix for SNP # 3, 3 parts by volume of Mega buffer Mix for SNP, and 3.8 parts by volume of ddH2And O is prepared.
6. Use of the kit of any one of claims 2-5 for detecting rs6923761 polymorphic site of a GLP1R gene.
7. The application of claim 6, wherein the application comprises the specific steps of taking the nucleic acid of the sample to be detected, respectively mixing with the reagents in the kit, oscillating, mixing uniformly, centrifuging to prepare a reaction system to be detected, carrying out PCR amplification reaction, obtaining 2 amplification curve graphs and CT values for each reaction, and judging the genotype of the nucleic acid of the sample to be detected according to the obtained amplification curve graphs and CT values.
8. The kit according to claim 7, wherein the reaction system to be tested is 17 μ L of reaction solution, 1 μ L of primer probe combination solution and 2 μ L of sample nucleic acid to be tested;
the concentration ratio of the forward primer, the reverse primer, the probe PA and the probe PG in the primer probe combination solution is 1: 0.5;
the reaction solution was composed of 0.2 volume parts of Taq DNA polymerase, 10 volume parts of buffer Mix for SNP 3#, 3 volume parts of Mega buffer Mix for SNP and 3.8 volume parts of ddH2And O is prepared.
9. The use according to claim 7 or 8, wherein the criterion for determining the genotype of the nucleic acid in the test sample is:
when the CT value of FAM is less than or equal to 38 and the CT value of HEX is more than 38/no CT value, the genotype is judged as GG type;
when the CT value of HEX is less than or equal to 38 and the CT value of FAM is more than 38/no CT value, the genotype is judged as AA;
when the CT value of FAM is less than or equal to 38 and the CT value of HEX is less than or equal to 38, determining the genotype as AG type;
when the CT value of FAM is > 38/no CT value and the CT value of HEX is > 38/no CT value, then retesting is required.
10. The use of claim 7 or 8, wherein the PCR amplification reaction is performed under conditions of 5 ℃ pre-denaturation for 2min, 95 ℃ denaturation for 5s, and 58 ℃ annealing for 15s for 40 cycles.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111607704.1A CN114317710A (en) | 2021-12-24 | 2021-12-24 | Primer probe combination for detecting SNP typing of polymorphic site of GLP1R gene, kit and application thereof |
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