CN117210558A - Primer probe group for detecting sodium valproate metabolism related genes, kit, detection method and application - Google Patents
Primer probe group for detecting sodium valproate metabolism related genes, kit, detection method and application Download PDFInfo
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Abstract
The invention discloses a primer probe group, a kit, a detection method and application for detecting genes related to sodium valproate metabolism, wherein an ARMS primer and a Taqman probe are designed, under the action of Taq enzyme with high anti-interference characteristic and a matched buffer system, the qualitative detection of CYP2C9c.1075A > C, POLG c.3708G > T and POLG c.3428A > G locus genotypes of a whole blood sample is realized by detecting the change of fluorescent signals on a real-time fluorescent quantitative PCR instrument, and the direct amplification of blood is supported by arranging a premix liquid 1 and a premix liquid 2 and a nucleic acid releasing agent for releasing genomic DNA of the sample to be detected, so that the sample with high specificity and high sensitivity is easy to operate, and the sample with 0.2ng of detectable genomic DNA content is provided for the drug risk of sodium valproate.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a primer probe group, a kit, a detection method and application for detecting genes related to sodium valproate metabolism.
Background
Epilepsy commonly called as "sheep horn wind" or "sheep-depressive wind" is the most common brain chronic disease caused by abnormal discharge of neurons caused by various causes, and is characterized by repeatability and spontaneity, and has different degrees of damage to the brain, nerves and other tissues and organs of a patient, and the physical and mental health and the quality of life of the patient are seriously affected. According to the latest epidemiological data of China, the overall prevalence of domestic epilepsy is 7.0 per mill, the annual incidence is 28.8/10 ten thousand, the prevalence of active epilepsy with attacks within 1 year is 4.6 per mill, and in China epilepsy is the second most common disease of neurology and secondary headache.
At present, sodium valproate is taken as a first-line therapeutic drug commonly used for clinically treating epilepsy, and although the sodium valproate is rapidly absorbed by oral administration and has high bioavailability, the therapeutic window is narrow, the drug response shows larger individual difference, and various researches prove that the difference of sodium valproate drug response has quite deep influence on physiological and pathological factors such as age, weight, liver and kidney functions and the like except for the difference of the drug response of the sodium valproate. The research data show that sodium valproate is mainly metabolized by liver in human body, and the number of main metabolic pathways is 3: about 10% of the cells are metabolised by oxidative metabolism of the CYP450 enzyme system (CYP 450); about 50% via glucuronidase metabolism (UGTs); about 40% of the beta oxidation metabolism in the meridian granule body, and the rest 1% -3% is discharged through urine in prototype. Wherein CYP2C9 is located on chromosome 10q23.3 and has a length of about 390kb, and the polymorphism can cause change of enzyme activity, influence sodium valproate metabolism, cause individual difference of blood concentration, and CYP2C9 x 3 (1075A > C) C allele is a susceptibility gene of epilepsy. Furthermore, the POLG encoded by the mitochondrial polymerase gamma (POLG 1) gene is the only enzyme located in mitochondria and involved in replication and repair with mitochondrial DNA, its genetic mutation affects mitochondrial function, refractory seizures are caused and rare mutations in the POLG gene can cause Alpers-Huttenlocher syndrome, it has been demonstrated that POLG mutations are directly related to sodium valproate-induced hepatotoxicity, POLG c.3708G > T mutations are associated with sodium valproate-induced liver failure, patients with POLG c.3428a > G mutant homozygotes have a shortened life span, severe patients face the risk of death, and heterozygous mutant patients have a worse prognosis for liver failure. Therefore, POLG c.3708G > T and POLG c.3428a > G genotyping identifies high risk individuals with fatal consequences as having significant clinical implications for reducing adverse drug reactions.
Currently, most technologies related to detecting polymorphic loci of epileptic disease related genes in the prior art adopt a time-of-flight mass spectrometry technology, such as Chinese patent number CN109468379A, and the detection of SNP loci of anti-epileptic drug related genes by adopting the time-of-flight mass spectrometry technology is disclosed, wherein the detection process involves amplifying a section of DNA containing the SNP loci by PCR, removing the remaining dNTPs and primers in a PCR system by SAP enzyme, adding a single-base extension primer for extension reaction, and finally carrying out mass spectrometry analysis. The whole detection process has high requirement on the purity of DNA, complicated steps, certain requirements on the operation environment and equipment, high requirement on operators, precise detection equipment and high cost; in addition, chinese patent number CN113136424B discloses that the next generation gene sequencing technology is adopted to detect the gene mutation site for the personalized medicine of the antiepileptic medicine, and the method has the advantages of high flux and multiple sites, but has the defects of complicated steps, long test period, high cost, more redundant detection items and the like, and is not beneficial to large-area clinical popularization. Most of the current researches neglect detection of POLG c.3428A > G locus, and mutation of the POLG c.3428A > G locus is related to one fatal risk of adverse reaction caused by sodium valproate medication, so that the development of detection kits which are simple to operate, low in cost and capable of simultaneously detecting CYP2C9c.1075A > C, POLG c.3708G > T and POLG c.3428A > G is necessary. The invention aims to develop a detection scheme with simple operation, strong specificity, high sensitivity, strong reliability and low cost based on ARMS PCR detection technology.
Disclosure of Invention
The invention aims at: aiming at the problems, a primer probe group, a kit, a detection method and application for detecting genes related to sodium valproate metabolism are provided, so that the defects of the prior art are overcome.
The technical scheme adopted by the invention is as follows: a primer probe set for detecting a gene related to sodium valproate metabolism, comprising an ARMS primer probe composition for detecting CYP2C9c.1075a > C, POLG c.3708G > T and POLG c.3428a > G sites, the nucleotide sequences of the ARMS primer probe composition being shown in table 1:
TABLE 1
Wherein, the ARMS primer probe composition for detecting CYP2C9c.1075A > C locus is as follows: a forward wild primer with a nucleotide sequence shown as SEQ ID NO.1, a forward mutant primer with a nucleotide sequence shown as SEQ ID NO.2, a universal primer with a nucleotide sequence shown as SEQ ID NO.3 and a fluorescent probe with a nucleotide sequence shown as SEQ ID NO. 4;
the ARMS primer probe composition for detecting POLG c.3708G > T locus comprises the following components: a forward wild primer with a nucleotide sequence shown as SEQ ID NO.5, a forward mutant primer with a nucleotide sequence shown as SEQ ID NO.6, a universal primer with a nucleotide sequence shown as SEQ ID NO.7, and a fluorescent probe with a nucleotide sequence shown as SEQ ID NO. 8;
the ARMS primer probe composition for detecting POLG c.3428A > G locus is a forward wild type primer with a nucleotide sequence shown as SEQ ID NO.9, a forward mutant primer with a nucleotide sequence shown as SEQ ID NO.10, a universal primer with a nucleotide sequence shown as SEQ ID NO.11 and a fluorescent probe with a nucleotide sequence shown as SEQ ID NO. 12.
Further, the 5 'end of the fluorescent probe is connected with a fluorescent group, the 3' end of the fluorescent probe is connected with a quenching group, the fluorescent group is selected from any one of FAM, HEX, TEXAS RED and CY5 fluorescent dye, and the quenching group is selected from BHQ1 or BHQ2.
Further, the invention also comprises a kit for detecting the sodium valproate metabolism related gene, wherein the kit comprises the primer probe set, a nucleic acid releasing agent for treating a sample to be detected, a positive control and a negative control.
Further, the kit also comprises universal primers and universal probes for detecting the internal standard genes of the human genome DNA, wherein the internal standard genes are selected from RPPH, the nucleotide sequences of the universal primers for detecting the internal standard genes of the human genome DNA are shown as SEQ ID NO.13-14 in table 1, and the nucleotide sequences of the universal probes are shown as SEQ ID NO.15 in table 1.
Further, the nucleic acid releasing agent for treating the sample to be tested comprises SDS, formamide and HCl components; the positive control is DNA recombinant plasmid of all target sequence polymorphic sites of the target gene; the negative control was physiological saline.
Further, a forward wild-type primer, a universal primer and a fluorescent probe for detecting CYP2C9c.1075A > C site, a forward mutant primer, a universal primer and a fluorescent probe for detecting POLG c.3708G > T site, a forward wild-type primer, a universal primer and a fluorescent probe for detecting POLG c.3428A > G site, and a PCR buffer are blended to form a premix liquid 1;
the forward mutant primer, the universal primer and the fluorescent probe for detecting CYP2C9c.1075A > C locus are mixed with the forward wild primer, the universal primer and the fluorescent probe for detecting POLG c.3708G > T locus, and the forward mutant primer, the universal primer and the fluorescent probe for detecting POLG c.3428A > G locus, so as to form a premix liquid 2 with a PCR buffer.
In the primer probe set, 2 different reaction systems are designed in the premix liquid 1 and the premix liquid 2, the premix liquid 1 and the premix liquid 2 both comprise the wild primer probe set and the mutant primer probe set, and the inventor finds that when the premix liquid 1 and the premix liquid 2 are single reaction systems, the resource competition easily occurs due to different amplified fragments in multiple PCR, and the number of single-hole target genes is too large, so that a certain channel amplification curve is pressed, and after 2 different reaction systems are constructed, the problem is well avoided.
Further, the PCR buffer solution comprises Tris-HCl, 5-meter glycerol buffer, 10-meter buffer, taq enzyme and MgCl 2 And dNTPs.
Further, in premix 1, the content of the forward wild-type primer and the universal primer for detecting CYP2C9c.1075A > C site was 0.03. Mu.L to 0.07. Mu.L, respectively; the content of the forward mutant primer and the universal primer for detecting POLG c.3708G > T site is 0.03 mu L-0.07 mu L respectively; the content of the forward wild-type primer and the universal primer for detecting POLG c.3428A > G locus is 0.03 mu L-0.07 mu L respectively;
in the premix liquid 2, the content of the forward mutant primer and the universal primer for detecting CYP2C9c.1075A > C locus is respectively 0.03 mu L-0.06 mu L; the content of the forward wild-type primer and the universal primer for detecting POLG c.3708G > T site is 0.03 mu L-0.07 mu L, respectively, and the content of the forward mutant primer and the universal primer for detecting POLG c.3428A > G site is 0.03 mu L-0.06 mu L, respectively.
Further, the invention also comprises a detection method of the kit, which comprises the following steps:
A. releasing DNA of the sample to be tested by adopting the nucleic acid releasing agent for treating the sample to be tested;
B. directly adding the sample to be tested processed in the step S1 into the premix liquid 1 and the premix liquid 2, and fully and uniformly mixing;
C. the fluorescent quantitative PCR amplification reaction procedure is:
pre-denaturation reaction: reacting for 3min at 95 ℃;
amplification reaction: 15s at 95 ℃ and 45s at 60 ℃ for 45 cycles;
D. and judging the genotype of the sample to be tested.
Furthermore, the invention also comprises the application of the kit in genotyping detection, wherein the application aims at non-diagnosis or non-treatment.
In summary, due to the adoption of the technical scheme, the beneficial effects of the invention are as follows:
1. the invention designs ARMS primers and Taqman probes aiming at CYP2C9c.1075A > C, POLG c.3708G > T and POLGc.3428A > G sites related to sodium valproate metabolism and is matched with a PCR buffer system, so that the genome DNA as low as 0.1 ng/mu L can be accurately detected, and the sensitivity is higher; by introducing the reference gene, the whole-process quality monitoring is carried out on the detection process, so that false positive or false negative is effectively avoided;
2. according to the invention, the amplification efficiency of each site in the reaction system is kept consistent by optimizing the addition amounts of the specific primers and the common primers of each site in the first PCR reaction system and the second PCR reaction system and exploring the combination of the types of the primers for detecting each site in the same reaction system, so that the pressing phenomenon in the amplification process of each site due to mutually competing resources is reduced;
3. the detection kit provided by the invention provides the premix liquid 1 and the premix liquid 2 in a full premix form, so that the preparation steps of a PCR reaction system are obviously simplified, the operation is more convenient, the operation error is reduced, the use is convenient for users, and the accuracy is high; the nucleic acid releasing agent provided simultaneously realizes the nucleic acid extraction-free treatment step of the whole blood sample;
4. compared with other detection methods in the market, the detection technical method provided by the invention greatly reduces the operation intensity, can realize the result about 1 hour, provides a more perfect personalized risk prediction reference for the use of sodium valproate drugs, and benefits patients.
Drawings
FIG. 1A is a graph showing the results of optimizing the amounts of wild-type primer and common primer added at CYP2C9c.1075A > C site;
FIG. 1B shows the results of optimizing the addition amounts of the mutant primer and the common primer at CYP2C9c.1075A > C site;
FIG. 2A is a graph showing the results of optimizing the amounts of addition of wild-type primers and common primers at POLG c.3708G > T sites;
FIG. 2B shows the results of optimizing the addition amounts of the mutant primers and the common primers at the POLG c.3708G > T site;
FIG. 3A shows the optimized results of the addition of wild-type primers and common primers at POLG c.3428A > G site;
FIG. 3B shows the results of optimizing the addition amounts of the mutant primers and the common primers at POLG c.3428A > G site;
FIG. 4 is an amplification result for a known genotype sample for primer types contained in premix 1 and premix 2 that detect CYP2C9c.1075A > C, POLG c.3708G > T and POLGc.3428A > G sites;
FIG. 5 is an amplification result of primer types two covering detection CYP2C9c.1075A > C, POLG c.3708G > T and POLGc.3428A > G sites in premix liquid 1 and premix liquid 2 for a known genotype sample;
FIG. 6 is a graph showing the amplification results of a whole blood sample I using the detection kit of the present invention;
FIG. 7 is a graph showing the amplification result of a whole blood sample II using the detection kit of the present invention;
FIG. 8 is a graph showing the amplification result of a whole blood sample III using the detection kit of the present invention;
FIG. 9 is a diagram showing the detection sensitivity of the detection kit of the present invention.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings.
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The following terms or definitions are provided solely to aid in the understanding of the invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.
Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the invention are intended to be identical to what is commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.
As used herein, the terms "comprising," "including," "having," "containing," or "involving," are inclusive or open-ended and do not exclude additional unrecited elements or method steps.
The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.
The indefinite or definite article "a" or "an" when used in reference to a singular noun includes a plural of that noun.
The terms "about" and "substantially" in this invention mean the range of accuracy that one skilled in the art can understand yet still guarantee the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.
The term "nucleic acid" or "nucleic acid sequence" in the present invention refers to any molecule, preferably a polymeric molecule, comprising ribonucleic acid, deoxyribonucleic acid, or analogue units thereof. The nucleic acid may be single-stranded or double-stranded. The single-stranded nucleic acid may be a nucleic acid that denatures one strand of double-stranded DNA. Alternatively, the single-stranded nucleic acid may be a single-stranded nucleic acid that is not derived from any double-stranded DNA.
The features and capabilities of the present invention are described in further detail below in connection with examples.
Example 1
POLG1 on human chromosome 15q26.1 encodes 23 exons of the mitochondrial DNA polymerase gamma (POLG) catalytic subunit, a key enzyme in the replication and repair of mitochondrial DNA (mtDNA). The POLG mutant population often has refractory seizures, and the patient may die from status epilepticus. One POLG1 variant c.3708g > T in exon 23 has been shown to be associated with VPA-induced liver failure, with a frequency of 8.3% in the european population and 14.8% -15.9% in the finnish population, with african and asian accounting for about 1%. The POLG1 c.3428a > G variant in exon 21 is considered a common polymorphism with a frequency of 4.1% in the general european population, 2.8% in the finnish population and 2.9% in the north american population, and is closely related to disease phenotypes such as Alpers-Huttenlocher syndrome (AHS), which are rare diseases with frequent staining recessive inheritance, with an annual incidence of 0.5/100000, heterozygous variants c.3708G > T and c.3428a > G being closely related to VPA-induced hepatotoxicity (VHT) when the POLG1 c.3428a > G variant is found with other pathogenic mutations in POLG 1. CYP2C9 is located on 10q23.3 chromosome, CYP2C9 x 3 (1075A > C) gene polymorphism is related to epileptic susceptibility and VPA blood concentration, wherein the occurrence rate of CYP2C9 x 3 in caucasian is 7.5% -19.0%, asian is about 3%, the polymorphism can cause change of enzyme activity, influence VPA metabolism and cause individual difference of blood concentration. Individual differences in VPA metabolism will directly affect the selection of clinical drug use and genotype data can be predictive of drug efficacy.
Therefore, the CYP2C9c.1075A > C, POLG c.3708G > T and POLG c.3428A > G are selected as SNP loci of genes related to sodium valproate metabolism for detection.
According to the principle of allele-specific PCR, each specific primer can only be combined with a DNA template of a corresponding genotype and amplified, and specific detection primers and probes are designed and used for PCR amplification and fluorescence detection; in addition, in order to monitor the quality in the detection process and effectively avoid false positive or false negative, the detection kit of the invention also introduces an internal standard gene RPPH, and the finally screened optimal primer probe group is shown in the following table 1:
TABLE 1
Specifically, the probe adopts a Taqman fluorescent probe; the 5 'end of the probe sequence is marked with a fluorescence report group, and the 3' end is marked with a fluorescence quenching group; preferably, a probe for detecting CYP2C9c.1075A > C gene locus is marked by FAM fluorescent dye, and the 3' end is marked by BHQ1 or BHQ2 fluorescent dye; the probe for detecting POLG c.3708G > T gene locus is marked by HEX fluorescent dye, and the 3' end is marked by BHQ1 or BHQ2 fluorescent dye; the probe for detecting POLG c.3428A > G gene locus is marked by TEXAS RED fluorescent dye, and the 3' end is marked by BHQ1 or BHQ2 fluorescent dye; the probe for detecting the RPPH gene locus is marked by CY5 fluorescent dye, and the 3' -end is marked by BHQ1 or BHQ2 fluorescent dye.
Example 2
In the initial stage of the establishment of the Polymerase Chain Reaction (PCR), the skilled person usually chooses to store each component required for the PCR reaction, such as a plurality of components such as enzymes and buffers, separately and prepare them on site when the PCR reaction is performed. The operation is complex, the error of adding error or adding quantity is easy to occur, the instability of the reaction system is influenced, the detection efficiency is reduced, and the probability of pollution is high. Therefore, the detection kit provided by the invention provides the fully premixed premix liquid 1 and the premix liquid 2, and all components required by the PCR reaction are mixed, so that the preparation steps of a PCR reaction system are simplified, and the use of workers is facilitated.
The premix liquid 1 comprises a forward wild type primer with a nucleotide sequence shown as SEQ ID NO.1, a universal primer with a nucleotide sequence shown as SEQ ID NO.3 and a fluorescent probe with a nucleotide sequence shown as SEQ ID NO.4, a forward mutant primer with a nucleotide sequence shown as SEQ ID NO.6, a universal primer with a nucleotide sequence shown as SEQ ID NO.7, a fluorescent probe with a nucleotide sequence shown as SEQ ID NO.8, a forward wild type primer with a nucleotide sequence shown as SEQ ID NO.9, a universal primer with a nucleotide sequence shown as SEQ ID NO.11, a fluorescent probe with a nucleotide sequence shown as SEQ ID NO.12, and a PCR buffer;
the premix liquid 2 comprises a forward mutant primer with a nucleotide sequence shown as SEQ ID NO.2, a universal primer with a nucleotide sequence shown as SEQ ID NO.3 and a fluorescent probe with a nucleotide sequence shown as SEQ ID NO.4, a forward wild primer with a nucleotide sequence shown as SEQ ID NO.5, a universal primer with a nucleotide sequence shown as SEQ ID NO.7, a fluorescent probe with a nucleotide sequence shown as SEQ ID NO.8, a forward mutant primer with a nucleotide sequence shown as SEQ ID NO.10, a universal primer with a nucleotide sequence shown as SEQ ID NO.11, a fluorescent probe with a nucleotide sequence shown as SEQ ID NO.12, and a PCR buffer;
the PCR buffer solution comprises Tris-HCl, 5-meter glycerol buffer, 10-meter buffer, taq enzyme and MgCl 2 And dNTPs; the total volume of the PCR reaction liquid of the detection kit provided by the invention is 20 mu L, wherein the premix liquid 1 and the premix liquid 2 are respectively 18 mu L, and the addition amount of a sample to be detected is 2 mu L; the premix final recipe is shown in Table 2.
TABLE 2 premix liquid formulation table
In addition, in order to simplify the nucleic acid extraction step of the sample to be detected, the detection kit is provided with the nucleic acid release agent containing SDS, formamide and HCl, wherein the SDS can generate better synergistic effect with strong alkali, so that the protein and the nucleic acid are fully separated, the whole blood sample can be directly diluted and cracked, the genome DNA in the sample to be detected is released, and an experimenter can extract the DNA of the sample to be detected by processing the sample to be detected in one step, so that the detection efficiency is further improved.
The internal composition of the detection kit of the present invention is shown in table 3 below:
TABLE 3 internal composition of kit
In addition, the premix solutions 1 to 2 in the detection kit of the present embodiment may be prepared in 5. Mu.L, and the addition amount of the sample to be measured is set to 15. Mu.L.
In order to facilitate the interpretation of genotype results, the detection kit provided by the invention designs two reaction systems I, namely a reaction system I corresponding to the premix liquid 1 and a reaction system II corresponding to the premix liquid 2.
The storage conditions of the kit are as follows: and storing at-20+/-5 ℃ in a dark place.
The kit is suitable for the instrument: full automatic PCR analysis System Fascan48E, independently developed by the company Siamion technologies Co.
Multiplex PCR requires specific amplification of multiple sites in the same reaction system, is studied intensively because of its high efficiency, high throughput, low cost, and is widely used in fields of scientific research, disease diagnosis, and the like. However, the amplification effect and the number of actually amplified fragments are often unsatisfactory due to incompatibility of amplification conditions between a plurality of targets, and nonspecific amplification, primer dimer and the like are liable to be formed, and for this purpose, the inventors examined the amounts of primers for detecting target genes in the premix liquid 1 and the premix liquid 2, respectively, in order to coordinate the amplification efficiencies of the target genes.
1. Optimization of addition amount of specific primer and common primer of CYP2C9c.1075A > C site
The experimental method comprises the following steps: 20 mu L of PCR reaction system is added with 0.03 mu L, 0.05 mu L and 0.07 mu L of CYP2C9-WF and CYP2C9-R respectively, and then a synthetic plasmid with the DNA content of 0.5ng and containing CYP2C9c.1075A > C wild type and a synthetic plasmid with the DNA content of 0.5ng and containing CYP2C9c.1075A > C mutant type are sequentially used as amplification templates, the amplification results are shown in figure 1A, and the amplification efficiency of the CYP2C9-WF and CYP2C9-R primers of 0.05 mu L is high by combining the fluorescence height value and Ct value of the amplification curve, and meanwhile, the PCR kit has good specificity.
And (2) adding 0.03 mu L, 0.04 mu L, 0.05 mu L and 0.06 mu L of CYP2C9-MF and CYP2C9-R into a 20 mu L PCR reaction system respectively, sequentially taking a synthetic plasmid with the DNA content of 0.5ng and containing CYP2C9c.1075A > C mutant genotype and a synthetic plasmid with the DNA content of 0.5ng and containing CYP2C9c.1075A > C wild genotype as an amplification template, wherein the amplification result is shown in FIG. 1B, the amplification efficiency of combining the fluorescence height value and Ct value of the amplification curve is high, and the amplification efficiency of the CYP2C9-MF and the CYP2C9-R primer with the concentration of 0.04 mu L is high, and meanwhile, the primer has good specificity.
2. Optimization of addition amounts of specific primers and common primers for POLG c.3708G > T sites
The 20. Mu.L PCR reaction system is added with 0.03. Mu.L, 0.05. Mu.L and 0.07. Mu.L POLG c.3708-WF and POLG c.3708-R respectively, and then the synthetic plasmid with the DNA content of 0.5ng and containing the POLG c.3708G > T wild type and the synthetic plasmid with the DNA content of 0.5ng and containing the POLG c.3708G > T mutant type are sequentially used as amplification templates, the amplification results are shown in figure 2A, and the amplification efficiency of the primers POLG c.3708-WF and POLGc.3708-R of 0.05. Mu.L is high by combining the fluorescence height value and Ct value of the amplification curve, and meanwhile, the primer has good specificity.
And (2) adding 0.03 mu L, 0.05 mu L and 0.07 mu L of POLG c.3708-MF and POLG c.3708-R into a 20 mu L PCR reaction system respectively, sequentially taking a synthetic plasmid containing 0.5ng of DNA and 0.5ng of POLG c.3708G > T mutant genotype and a synthetic plasmid containing 0.5ng of POLG c.3708G > T wild genotype as an amplification template, wherein the amplification results are shown in FIG. 2B, and the amplification efficiency of the primers POLG c.3708-MF and POLG c.3708-R of 0.07 mu L is high by combining the fluorescence height value and Ct value of the amplification curve, and meanwhile, the primer has good specificity.
3. Optimization of addition amounts of specific primers and common primers for POLG c.3428A > G sites
The 20 mu L PCR reaction system is added with 0.03 mu L, 0.05 mu L and 0.07 mu L POLG c.3428-WF and POLG c.3428-R respectively, and then the synthetic plasmid with the DNA content of 0.5ng and containing the POLG c.3428A > G wild type and the synthetic plasmid with the DNA content of 0.5ng and containing the POLG c.3428A > G mutant type are sequentially used as amplification templates, the amplification results are shown in figure 3A, and the amplification efficiency of the primers of the POLG c.3428-WF and the POLG c.3428-R of 0.05 mu L is high by combining the fluorescence height value and Ct value of the amplification curve, and the amplification result has good specificity.
And (3) adding 0.03 mu L, 0.04 mu L, 0.05 mu L and 0.06 mu L of POLG c.3428-MF and POLG c.3428-R into a 20 mu L PCR reaction system respectively, sequentially taking a synthetic plasmid with the DNA content of 0.5ng and containing the POLG c.3428A > G mutant genotype and a synthetic plasmid with the DNA content of 0.5ng and containing the POLG c.3428A > G wild genotype as an amplification template, wherein the amplification result is shown in FIG. 3B, and the amplification efficiency of the primers POLG c.3708-MF and POLG c.3708-R of 0.04 mu L is higher by combining the fluorescence height value and Ct value of the amplification curve, and meanwhile, the primer has good specificity.
In addition, in order to reduce the risk of missed detection due to the fact that the high abundance templates are easy to detect and the low abundance templates have poor amplification effects caused by the competition of different amplified fragments in multiplex PCR, the inventors adjusted the primer types in premix liquid 1 and premix liquid 2 for detecting CYP2C9c.1075A > C, POLG c.3708G > T and POLG c.3428A > G sites for this purpose.
Specifically, whole blood samples with the genotype of CYP2C9-AA, POLG c.3708-GG and POLG c.3428-AA which are subjected to first-generation sequencing are taken as amplification templates for amplification detection, and a PCR amplification program is as follows: pre-denaturation reaction: reacting for 3min at 95 ℃; amplification reaction: 15s at 95℃and 45s at 60℃for 45 cycles.
Experimental example: the premix liquid 1 contains a wild type primer of CYP2C9c.1075A > C, POLG c.3428A > G site and a mutant primer of POLG c.3708G > T site; the premix liquid 2 contains a mutant primer of CYP2C9c.1075A > C and POLGc.3428A > G site and a wild primer of POLGc.3708G > T site, and the detection result is shown in figure 4;
comparative example: wild-type primers encompassing the CYP2C9c.1075A > C, POLG c.3428A > G, POLG c.3708G > T locus in premix 1; the premix liquid 2 contains a mutant primer of CYP2C9c.1075A > C, POLG c.3428A > G and POLGc.3708G > T locus; the detection results are shown in FIG. 5.
As can be seen from fig. 5, when the premix 1 simultaneously contains a wild-type primer mixture for detecting the CYP2C9c.1075a > C, POLG c.3428a > G and polgc.3708G > T sites, the POLG c.3708G > T (HEX channel) is significantly suppressed due to the competitive amplification relationship between the sites, and the amplification curve of the CYP2C9c.1075a > C, POLG c.3428a > G site has no significant "S" type amplification, and is in an oblique straight line. After the wild type primer of POLG c.3708G > T site in the premix liquid 1 and the mutant type primer of POLG c.3708G > T site in the premix liquid 2 are interchanged, the detection result is shown in figure 4 aiming at the same sample, and according to the amplification curve of figure 4, the amplification effect of the target gene is obviously improved, and a good S curve is formed. The amplification effects are similar by similarly replacing the primer types in premix liquid 1 and premix liquid 2 for detecting other sites.
Example 3
The embodiment provides the method for using the kit provided in the embodiment 2, which specifically includes the following steps:
s1, releasing DNA of a sample to be tested, taking 10 mu L of uniformly mixed whole blood samples, respectively adding the 10 mu L of uniformly mixed whole blood samples into the nucleic acid releasing agent, numbering, and uniformly vortex mixing for later use;
s2, preparing reagents, calculating the reaction test number [ the number of samples to be tested+positive control (1) +negative control (2) ], taking out the detection kit from the refrigerator, melting and shaking the components at room temperature, mixing uniformly, and centrifuging at 2000rpm for 10 seconds;
s3, adding samples, respectively taking 2 mu L of treated samples to be tested, negative control and positive control, adding the samples to be tested, the negative control and the positive control into a reaction tube which is packaged, covering a tube cover, uniformly mixing by vortex 3S, and transferring the reaction tube to a detection area after centrifuging at 6000rpm for 5S;
s4, PCR amplification and fluorescence detection, placing the reaction tubes on a fluorescence PCR amplification instrument according to a certain sequence, setting the reaction system to be 20 mu L, and carrying out PCR amplification according to the following procedures:
pre-denaturation reaction: reacting for 3min at 95 ℃;
amplification reaction: 15s at 95 ℃ and 45s at 60 ℃ for 45 cycles;
detection fluorescence selection: the genes were detected (FAM channel, TEXAS RED channel, HEX channel), and the internal standard gene (CY 5 channel).
S5, data analysis
1) Kit validity determination
Positive control: FAM, TEXAS RED and HEX channels Ct value is less than or equal to 36.5, and the amplification curve has obvious exponential growth phase;
negative control: each channel has no amplification, and Ct is more than 36.5 or has no Ct value;
if the conditions are not met, the experiment is regarded as invalid, and instruments, reagents, amplification conditions, experimental operation and the like should be checked;
2) Sample validity determination
Reference gene: in all sample detection, the Ct value of a CY5 channel is less than or equal to 36.5, and an amplification curve has obvious exponential growth phase. If this condition is not met, the sample is considered an invalid sample.
Under the conditions of the PCR reaction system and the PCR amplification program, on the premise that the internal reference gene signals form a normal amplification S-shaped curve, observing whether a target detection fluorescent signal in a specific PCR reaction system forms an amplification S-shaped curve, if so, judging that the DNA sample to be detected is positive for the polymorphism type represented by the specific reaction system, and judging the genotype of the sample to be detected according to the difference (delta Ct) between the Ct value of the mutant reaction system and the Ct value of the wild reaction system, wherein the judging mode of the specific genotype detection result is shown in the table 4.
TABLE 4 determination method of genotype test results
FIG. 6 is a diagram showing the amplification result of the whole blood sample I by the detection kit of the present invention, wherein the hole A represents the amplification result of the PCR reaction system I, and the hole B represents the amplification result of the PCR reaction system II. As can be seen, the fluorescent signals of the internal standard genes in the first and second reaction systems were both qualified, and the genotype of CYP2C9c.1075, POLG c.3708, and POLG c.3428 of the samples was determined to be AA according to the genotype determination method shown in Table 4.
Similarly, FIG. 7 shows a graph of the amplification result of a whole blood sample II using the detection kit of the present invention, and can determine that the genotype of CYP2C9c.1075 of the whole blood sample is AC, that of POLG c.3708 is GG, and that of POLG c.3428 is AA.
Similarly, FIG. 8 shows a graph of the amplification result of a whole blood sample III using the detection kit of the present invention, and can determine that the genotype of CYP2C9c.1075 of the whole blood sample is AC, that of POLG c.3708 is GG, and that of POLG c.3428 is AA.
Example 4
The sensitivity of the detection kit provided by the invention was verified by using the detection kit provided by example 2 and the detection method provided by example 3. Specifically, after the DNA concentration of a whole blood sample with a known genotype (CYP 2C9c.1075 genotype is AC, POLG c.3708 genotype is GG, POLG c.3428 genotype is AA) is measured by using a nucleic acid concentration measuring instrument, the whole blood sample is diluted to 0.1 ng/mu L, the sample to be measured is respectively added into a first PCR reaction system and a second PCR reaction system for 3 times of repeated amplification detection, the amplification results are shown as the figure 9, and the detection results of the detection kit can be both normally typed and accord with the detection standard, so that the kit can accurately detect the genotype of the sample with the genomic DNA concentration of 0.1 ng/mu L, and the sensitivity is quite high.
Example 5
In this example, a whole blood sample was used as an amplified sample, and the kit provided in example 2 and the detection method provided in example 3 were used to test, wherein genotypes of test persons were confirmed by Sanger gene sequencing, and the total number of each sample was 30, and the detection results are shown in table 5:
TABLE 5 results of accuracy testing
Example 6
The clinical significance of genotypes associated with sodium valproate metabolism is shown in table 6 below:
TABLE 6 output of sodium valproate related Gene locus results and clinical significance
In summary, according to the detection result of the kit, the risk of using the drug can be analyzed from the angle of interaction between pharmacogenomics and the drug, and a reference basis is provided for clinical drug selection. It should be noted that the detection result is only for reference of the clinician and cannot be used as the only basis for making the clinical diagnosis and treatment scheme.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. A primer probe set for detecting a gene related to sodium valproate metabolism, comprising an ARMS primer probe composition for detecting the CYP2C9c.1075a > C, POLG c.3708G > T and POLG c.3428a > G sites, the nucleotide sequence of the ARMS primer probe composition being as shown in table 1:
TABLE 1
Wherein, the ARMS primer probe composition for detecting CYP2C9c.1075A > C locus is as follows: a forward wild primer with a nucleotide sequence shown as SEQ ID NO.1, a forward mutant primer with a nucleotide sequence shown as SEQ ID NO.2, a universal primer with a nucleotide sequence shown as SEQ ID NO.3 and a fluorescent probe with a nucleotide sequence shown as SEQ ID NO. 4;
the ARMS primer probe composition for detecting POLG c.3708G > T locus comprises the following components: a forward wild primer with a nucleotide sequence shown as SEQ ID NO.5, a forward mutant primer with a nucleotide sequence shown as SEQ ID NO.6, a universal primer with a nucleotide sequence shown as SEQ ID NO.7, and a fluorescent probe with a nucleotide sequence shown as SEQ ID NO. 8;
the ARMS primer probe composition for detecting POLG c.3428A > G locus is a forward wild type primer with a nucleotide sequence shown as SEQ ID NO.9, a forward mutant primer with a nucleotide sequence shown as SEQ ID NO.10, a universal primer with a nucleotide sequence shown as SEQ ID NO.11 and a fluorescent probe with a nucleotide sequence shown as SEQ ID NO. 12.
2. The primer probe set of claim 1, wherein the fluorescent probe has a fluorescent group attached to the 5 'end and a quenching group attached to the 3' end, wherein the fluorescent group is selected from any one of FAM, HEX, TEXAS RED and CY5, and the quenching group is selected from BHQ1 or BHQ2.
3. A kit for detecting a gene related to sodium valproate metabolism, characterized in that the kit comprises the primer probe set of claim 1 or 2, a nucleic acid releasing agent for treating a sample to be tested, a positive control, and a negative control.
4. The kit according to claim 3, wherein the kit further comprises universal primers and universal probes for detecting human genomic DNA internal standard genes, wherein the internal standard genes are selected from RPPH, the nucleotide sequences of the universal primers for detecting human genomic DNA internal standard genes are shown as SEQ ID NO.13-14 in Table 1, and the nucleotide sequences of the universal probes are shown as SEQ ID NO.15 in Table 1.
5. The kit of claim 3, wherein the nucleic acid releasing agent for treating the sample to be tested comprises SDS, formamide, HCl component; the positive control is DNA recombinant plasmid of all target sequence polymorphic sites of the target gene; the negative control was physiological saline.
6. The kit of claim 3, wherein the forward wild-type primer, universal primer and fluorescent probe for detecting the CYP2C9c.1075a > C site are blended with the forward mutant primer, universal primer and fluorescent probe for detecting the POLG c.3708G > T site and the forward wild-type primer, universal primer and fluorescent probe for detecting the POLG c.3428a > G site to form premix 1 with the PCR buffer;
the forward mutant primer, the universal primer and the fluorescent probe for detecting CYP2C9c.1075A > C locus are mixed with the forward wild primer, the universal primer and the fluorescent probe for detecting POLG c.3708G > T locus, and the forward mutant primer, the universal primer and the fluorescent probe for detecting POLG c.3428A > G locus, so as to form a premix liquid 2 with a PCR buffer.
7. The kit of claim 6, wherein the PCR buffer comprises Tris-HCl, 5-glycerol-containing buffer, 10-glycerol-containing buffer, taq enzyme, mgCl 2 And dNTPs.
8. The kit according to claim 6, wherein the content of the forward wild-type primer and the universal primer for detecting CYP2C9c.1075A > C site in the premix solution 1 is 0.03. Mu.L to 0.07. Mu.L, respectively; the content of the forward mutant primer and the universal primer for detecting POLG c.3708G > T site is 0.03 mu L-0.07 mu L respectively; the content of the forward wild-type primer and the universal primer for detecting POLG c.3428A > G locus is 0.03 mu L-0.07 mu L respectively;
in the premix liquid 2, the content of the forward mutant primer and the universal primer for detecting CYP2C9c.1075A > C locus is respectively 0.03 mu L-0.06 mu L; the content of the forward wild-type primer and the universal primer for detecting POLG c.3708G > T site is 0.03 mu L-0.07 mu L, respectively, and the content of the forward mutant primer and the universal primer for detecting POLG c.3428A > G site is 0.03 mu L-0.06 mu L, respectively.
9. A method of detecting a kit according to any one of claims 3 to 8, comprising the steps of:
A. releasing DNA of the sample to be tested by adopting the nucleic acid releasing agent for treating the sample to be tested;
B. directly adding the sample to be tested processed in the step S1 into the premix liquid 1 and the premix liquid 2, and fully and uniformly mixing;
C. the fluorescent quantitative PCR amplification reaction procedure is:
pre-denaturation reaction: reacting for 3min at 95 ℃;
amplification reaction: 15s at 95 ℃ and 45s at 60 ℃ for 45 cycles;
D. and judging the genotype of the sample to be tested.
10. Use of a kit according to any one of claims 3-8 for genotyping detection, wherein the use is for non-diagnostic or non-therapeutic purposes.
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