WO2021042884A1 - 用于检测叶酸基因多态性的探针、基因芯片及试剂盒 - Google Patents

用于检测叶酸基因多态性的探针、基因芯片及试剂盒 Download PDF

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WO2021042884A1
WO2021042884A1 PCT/CN2020/102826 CN2020102826W WO2021042884A1 WO 2021042884 A1 WO2021042884 A1 WO 2021042884A1 CN 2020102826 W CN2020102826 W CN 2020102826W WO 2021042884 A1 WO2021042884 A1 WO 2021042884A1
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probe
gene
gene chip
folic acid
kit
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肖杰
王丽玲
余义文
陈建奇
宋家武
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珠海赛乐奇生物技术股份有限公司
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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention belongs to the technical field of biological detection, and particularly relates to a probe, a gene chip and a kit for detecting the polymorphism of folate gene.
  • Folic acid is a water-soluble B vitamin. As the most important carrier of one carbon unit in the body, its main physiological function is to participate in many cell metabolism processes in the body. Folic acid is indispensable in the metabolism of methionine. It acts in the form of 5-methyltetrahydrofolate. The lack of folic acid will cause adverse effects on the human body, especially the development of pregnant women and fetuses. Folic acid can also have a certain effect on the gene expression of cancer cells, inhibit their growth, and have certain anti-cancer effects.
  • Folic acid metabolism refers to the ability of folic acid to be absorbed and utilized by the human body, and mainly refers to the mutual conversion process between homocysteine and methionine.
  • other diseases are also related to folic acid metabolism abnormalities, such as cleft lip and palate, Complications in late pregnancy, different types of neurological and mental diseases, osteoporosis and colorectal cancer and other cancers.
  • senile dementia and other related brain diseases are directly or indirectly related to the lack of folic acid.
  • Deficiency may also cause a variety of diseases such as pregnancy toxemia, scurvy, parasitic diseases and rheumatoid arthritis, as well as other symptoms such as glossitis, diarrhea, poor growth, mental atrophy and mental deterioration.
  • the more mature gene sites for folic acid metabolism are: the C677T and A1298C sites of MTHFR (methylenetetrahydrofolate reductase), and the A66G site on MTRR (methionine synthesizing reductase).
  • MTHFR catalyzes 5,10-methylenetetrahydrofolate to produce 5-methyltetrahydrofolate, which is a one-carbon unit donor for HCY (homocysteine) metabolism to produce methionine.
  • HCY homocysteine
  • Gene polymorphism at C677T site will cause alanine to be replaced by valine, which will lead to a decrease in enzyme activity, produce moderate hyperhomocysteinemia (HHCY), and also reduce the content of folic acid in the blood .
  • HHCY moderate hyperhomocysteinemia
  • the Whole Genome Consortium has confirmed that the C677 locus genotype is related to the HCY level in the blood of healthy people. Further studies have shown that double mutations at the two sites A1298C and C677T will cause the enzyme activity to decrease more severely.
  • MTRR is another important enzyme in HCY metabolism. It catalyzes the formation of methionine from HCY with the participation of vitamin B12.
  • the most common mutation position of this gene is the mutation of A to G at 66, which leads to a decrease in enzyme activity and an increase in the risk of HHCY.
  • SNP typing methods mainly include: PCR-RFLP (restriction fragment length polymorphism polymerase chain reaction), PCR-chip hybridization method, first-generation DNA sequencing method and TaqMan -MGB (dual-labeled real-time fluorescent probe) genotyping method.
  • PCR-RFLP mainly relies on the restriction enzyme cleavage site of the target, which increases the difficulty of the experiment.
  • subsequent electrophoresis analysis easily causes laboratory pollution and cannot be carried out on a large scale.
  • the TaqMan-MGB probe technology can not only perform gene quantitative analysis, but also analyze gene mutations.
  • First-generation sequencing is the gold standard for genetic testing, but its operation process is too long, the cost is too high, and the equipment is expensive and it is difficult to be suitable for large-scale development in hospitals.
  • the present invention provides a probe, gene chip and kit for detecting polymorphism of folate gene.
  • the invention has the advantages of high specificity, good accuracy, no nucleic acid extraction and rapid detection of multiple sites.
  • a probe for detecting polymorphism of folate gene the sequence of the probe is:
  • MTHFR677-C probe AAAAAAAAAACGTGATGATGAAATCGGCTC;
  • MTHFR677-T probe AAAAAAAAAACGTGATGATGAAATCGACTC;
  • MTHFR1298-A probe AAAAAAAAAACAAAGACACTTTCTTCACTGGTC;
  • MTHFR1298-C probe AAAAAAAAAAAAGACACTTGCTTCACTGGT;
  • MTRR66-A probe AAAAAAAAAACAGCTTGCTCACATATTTCTTC;
  • MTRR66-G probe AAAAAAAAAACACAGCTTGCTTGCTCACACA.
  • a gene chip for detecting folic acid gene polymorphism which contains the above-mentioned probe for detecting folic acid gene polymorphism.
  • a method for preparing a gene chip for detecting folic acid gene polymorphism includes the following steps:
  • the substrate in step (1) is a silicon wafer (Si), the thickness of the silicon wafer is 2.5 mm and the diameter is 20 cm.
  • the coating in step (1) is to plate silicon nitride with a thickness of 47.5 nm and a TSPS (T structure polyamino dimethyl silane hydrocarbon) film with a thickness of 47.5 nm and 13.5 nm on the silicon wafer.
  • TSPS T structure polyamino dimethyl silane hydrocarbon
  • the coating in step (2) is a polyphenylalanine-lysine coating, and the thickness of the polyphenylalanine-lysine coating is 15nm; 6-hydrazinonicotinic acid succinate
  • concentration of imide ester hydrochloride is 1-10 ⁇ mol/L, and the treatment time of 6-hydrazinonicotinic acid succinimidyl ester hydrochloride is 20 min.
  • an aldehyde group or amino group is used to modify one end of the probe.
  • a kit for detecting folic acid gene polymorphism including the gene chip, PCR reaction system, hybridization buffer, washing solution, BW reaction solution (horseradish peroxidase + sodium citrate buffer) and TMB (3,3',5,5'-Tetramethylbenzidine) color developing solution.
  • the PCR reaction system includes the following components: 5 ⁇ Buffer, dUTP mix, F-primer, R-primer, Hot taq, UNG enzyme (uracil DNA glycosylase) and dd H 2 O (double steaming water).
  • UNG enzyme to the PCR reaction system can remove the contamination of the amplified product and reduce false positive results.
  • sequence of the F-primer is:
  • MTHFR677-F 5'-TTGAGGCTGACCTGAAGCACTTGAAGGAG-3';
  • MTHFR1298-F 5'-CTTTGGGGAGCTGAAGGACTACTA-3';
  • MTRR66-FP 5'-CCTTGAAGTGATGAGGAGG-3'.
  • sequence of the R-primer is:
  • MTHFR677-R 5'-CCTGGATGGGAAAGATCCCG-3';
  • MTHFR1298-R 5'-CACTTTGTGACCATTCCGGTTT-3';
  • MTRR66-RP 5'-CCACTGTAACGGCTCTAACC-3'.
  • the hybridization buffer includes 0.3 mol/L trisodium citrate, 3 mol/L sodium chloride, 0.3 mol/L sodium lauryl sulfate, and Triton X-100 0.1% aqueous solution.
  • the washing liquid contains washing liquid A and washing liquid B
  • the washing liquid A is a 0.01-0.1 mol/L NaOH solution
  • the washing liquid B is a 0.1 ⁇ SSC solution.
  • a method for detecting folic acid gene polymorphism, using the kit for detection includes the following steps:
  • TMB color developing solution for color development. Before and after adding TMB color developing solution, the gene chip is washed and air-dried with 0.1 ⁇ SSC solution, and the detection result is read by taking photos or naked eyes.
  • the test object contains DNA fragments.
  • the detection performed by the kit and the test substance is a hybridization reaction.
  • the test substance contains a target gene fragment that can specifically bind to the probe, the region of the gene chip corresponding to the probe appears blue.
  • the amplification conditions in step (1) are: 95°C for 10 minutes; 94°C denaturation for 30s, 56°C renaturation for 30s, 72°C extension for 30s, repeating 40 cycles; 72°C for 5min extension.
  • the amount of PCR amplification product added in step (2) is 10 ⁇ L, and the hybridization buffer is 100 ⁇ L; the reaction conditions are: reaction at 50-60°C for 30-60 minutes.
  • the 0.01-0.1mol/L NaOH solution is preheated before use, the preheating temperature is 50°C, and each wash is 5-10s, and the wash is 3 times; the temperature of the 0.1 ⁇ SSC solution is 50°C, The washing time is 1 min.
  • the addition amount of the BW reaction solution in step (4) is 100 ⁇ L, and the reaction time is 10 min.
  • the added amount of TMB color developing solution is 100 ⁇ L, and the reaction time is 5 minutes; the 0.1 ⁇ SSC solution is used to wash 3 times before and after adding TMB color developing solution, each time is 1 minute.
  • the gene chip method is used to realize the one-time multi-site determination of the folate gene, which reduces the detection time and cost;
  • the present invention has the advantages of good specificity, high accuracy, rapidity and convenience through the selection of specific probes and primers, combined with the method of gene chip;
  • test results can be directly read by taking pictures or naked eyes, which is simple and convenient;
  • This method uses the extraction-free PCR amplification method for gene amplification, eliminating the complicated operation of nucleic acid extraction.
  • Figure 1 is the detection results of the gene chip and kit prepared in Example 1-2 of the present invention on the mixed plasmids of MTHFR677-C plasmid, MTHFR1298-A plasmid and MTRR66-A plasmid;
  • Figure 2 is the detection result of the gene chip and kit prepared in Example 1-2 of the present invention on the mixed plasmids of MTHFR677-T plasmid, MTHFR1298-C plasmid and MTRR66-G plasmid;
  • Figure 3 shows the mixing of the gene chip and kit prepared in Example 1-2 of the present invention on MTHFR677-C plasmid, MTHFR1298-A plasmid, MTRR66-A plasmid, MTHFR677-T plasmid, MTHFR1298-C plasmid and MTRR66-G plasmid Plasmid test results.
  • a probe for detecting polymorphism of folate gene the sequence of the probe is:
  • MTHFR677-C probe AAAAAAAAAACGTGATGATGAAATCGGCTC;
  • MTHFR677-T probe AAAAAAAAAACGTGATGATGAAATCGACTC;
  • MTHFR1298-A probe AAAAAAAAAACAAAGACACTTTCTTCACTGGTC;
  • MTHFR1298-C probe AAAAAAAAAAAAGACACTTGCTTCACTGGT;
  • MTRR66-A probe AAAAAAAAAACAGCTTGCTCACATATTTCTTC;
  • MTRR66-G probe AAAAAAAAAACACAGCTTGCTTGCTCACACA.
  • a gene chip containing the above-mentioned probe for detecting polymorphism of folate gene, and its preparation process includes the following steps:
  • a silicon wafer (Si) with a thickness of about 2.5mm and a diameter of 20cm is plated with a silicon nitride film of 47.5nm and a TSPS film of 13.5nm by a rotary vacuum coater and a vacuum vapor deposition method to prepare the corresponding biosensor , And covered with 15nm polyphenylalanine-lysine coating, and finally treated with 10 ⁇ mol/L 6-hydrazinonicotinic acid succinimidyl ester hydrochloride for 20 minutes, and then rinse the chip with clean water.
  • the probe is spotted on the processed chip and reacted at room temperature to prepare a gene chip containing the probe.
  • the spotted PolyA plays a role in positioning on the gene chip; and when the test result does not show color at the PolyA spotted spot, it proves that the color developer is invalid.
  • a kit for detecting folic acid gene polymorphism including the gene chip in Example 1, PCR reaction system, hybridization buffer, washing solution, BW reaction solution and TMB color developing solution.
  • the sequence of the F-primer in the PCR reaction system is:
  • MTHFR677-F 5'-TTGAGGCTGACCTGAAGCACTTGAAGGAG-3';
  • MTHFR1298-F 5'-CTTTGGGGAGCTGAAGGACTACTA-3';
  • MTRR66-FP 5'-CCTTGAAGTGATGAGGAGG-3'.
  • the sequence of the R-primer in the PCR reaction system is:
  • MTHFR677-R 5'-CCTGGATGGGAAAGATCCCG-3';
  • MTHFR1298-R 5'-CACTTTGTGACCATTCCGGTTT-3';
  • MTRR66-RP 5'-CCACTGTAACGGCTCTAACC-3'.
  • the hybridization buffer includes 0.3mol/L trisodium citrate, 3mol/L sodium chloride, 0.3mol/L sodium lauryl sulfate and Triton X-100 0.1% aqueous solution.
  • the washing liquid contains washing liquid A and washing liquid B, washing liquid A is 0.01-0.1 mol/L NaOH solution, and washing liquid B is 0.1 ⁇ SSC solution.
  • Hot-Taq enzyme, dUTP, and UNG enzyme were purchased from Shenzhen Feipeng Biological Co., Ltd.
  • the primers and probes are self-designed and synthesized by Shanghai Shenggong Biological Engineering Co., Ltd.
  • the gene chips and kits prepared in Example 1-2 were used for detection.
  • the object to be detected was a plasmid containing the target gene fragment.
  • the above plasmids were synthesized and provided by Shanghai Shenggong Bioengineering Co., Ltd.
  • the target gene fragment includes one of the MTHFR677 (C/T) site, MTHFR1298 (A/C) site and MTRR66 (A/G) site.
  • the specific sequences of the target gene fragment are as follows:
  • the MTHFR677-C plasmid, MTHFR1298-A plasmid and MTRR66-A plasmid were mixed to obtain a mixed plasmid, and the mixed plasmid was dissolved in 1 mL TE buffer so that the concentration of the plasmid was 2 ⁇ g/mL, that is, 2000 ng/mL. Take 5 ⁇ L of the plasmid solution as the test substance and add it to the PCR reaction system so that the total reaction volume is 50 ⁇ L to prepare a PCR reaction mixture.
  • the detection results are shown in Figure 1.
  • the MTHFR677-C probe, MTHFR1298-A probe and MTRR66-A probe are all blue at the spot of the gene chip (area 1, 2, and 3 respectively), and PolyA spotted The place (area 7) also showed blue color, confirming that using the gene chip and kit prepared in Example 1-2, when the MTHFR677/MTHFR1298/MTRR66 site is not mutated, accurate results can be obtained by detecting it.
  • Example 4 is basically the same as Example 3, except that the test substance used is a mixed plasmid of MTHFR677-T plasmid, MTHFR1298-C plasmid and MTRR66-G plasmid.
  • the analyte was detected using the gene chip and kit prepared in Example 1-2.
  • the detection results are shown in Figure 2.
  • the MTHFR677-T probe, MTHFR1298-C probe and MTRR66-G probe are all blue at the spot of the gene chip (area 4, 5, 6 respectively), and PolyA spotted The place (area 7) also showed blue, confirming that the gene chip and kit prepared in Example 1-2 can accurately detect the mutations at the MTHFR677/MTHFR1298/MTRR66 site.
  • Example 5 is basically the same as Example 3, except that the test substances used are MTHFR677-C plasmid, MTHFR1298-A plasmid, MTRR66-A plasmid, MTHFR677-T plasmid, MTHFR1298-C plasmid and MTRR66-G plasmid The mixed plasmid.
  • the analyte was detected using the gene chip and kit prepared in Example 1-2.
  • MTHFR677-C probe, MTHFR1298-A probe, MTRR66-A probe, MTHFR677-T probe, MTHFR1298-C probe and MTRR66-G probe are at the spot of the gene chip (Area 1, 2, 3, 4, 5, 6 respectively) are blue, and PolyA spot (area 7) is also blue. It is confirmed that the gene chip and kit prepared in Example 1-2 are used in When the 6 types of plasmids are mixed, they also have good specificity and can also get accurate detection results.

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Abstract

本发明公开了用于检测叶酸基因多态性的探针、基因芯片及试剂盒。所述探针包括MTHFR677-C探针、MTHFR677-T探针、MTHFR1298-A探针、MTHFR1298-C探针、MTRR66-A探针和MTRR66-G探针。所述基因芯片上含有所述探针。所述试剂盒包括所述探针和基因芯片。

Description

用于检测叶酸基因多态性的探针、基因芯片及试剂盒 技术领域
本发明属于生物检测技术领域，特别涉及用于检测叶酸基因多态性的探针、基因芯片及试剂盒。
背景技术
叶酸是一种水溶性的B族维生素，作为体内最重要的一碳单位载体，其主要生理功能为参与机体许多细胞代谢过程。叶酸在甲硫氨酸代谢中必不可少，以5-甲基四氢叶酸的形式起作用，如果缺乏叶酸会对人体造成不利的影响，特别是对孕妇和胎儿的发育造成很大的危害。叶酸也可对癌细胞的基因表达有一定影响，抑制其生长，具备一定的抗癌功效。
叶酸代谢能力是指叶酸被人体吸收与利用的能力，主要指参与同型半胱氨酸与甲硫氨酸之间的相互转化过程。近年来对其代谢过程机制的研究发现，除了早产、流产先天性心脏病、神经管畸形、唐氏综合征与叶酸代谢有关外，其他的疾病也与叶酸的代谢异常相关，像是唇腭裂、妊娠晚期并发症、不同类型的神经和精神疾病、骨质疏松征及大肠癌等癌症，另外，许多文献报道，老年期痴呆症等有关脑病变疾病与叶酸的缺乏有着直接或者间接的关系，叶酸缺乏还可能引起妊娠毒血症、坏血病、寄生虫病和类风湿性关节炎等多种疾病，以及其他诸如舌炎、腹泻、生长不良、精神萎缩和智力退化等症状。
人与人之间因基因不同，对叶酸的代谢吸收能力也不同。通过基因检测，可了解自身的叶酸代谢能力，实现个体化补充叶酸，降低相关疾病的发病风险。
目前对叶酸代谢研究较为成熟的基因位点有：MTHFR(亚甲基四氢叶酸还原酶)的C677T和A1298C位点、MTRR(甲硫氨酸合成还原酶)上的A66G位点。
MTHFR催化5,10-亚甲基四氢叶酸生成5-甲基四氢叶酸，后者是HCY(同型半胱氨酸)代谢产生甲硫氨酸的一碳单位供体。C677T位点的基因多态性会导致丙氨酸被缬氨酸代替，这会导致酶活性下降，产生中度的高同型半胱氨酸血症(HHCY)，也会降低血液中叶酸的含量。全基因组联合会已经证实了C677位点基因型与健康人群血液中的HCY水平相关。进一步的研究表明，A1298C和C677T两个位点的双重突变会导致酶活性降低更加严重。
MTRR是HCY代谢中另一种重要的酶，它在维生素B12的参与下催化HCY形成甲硫氨酸。该基因最常见的突变位置为66处的A突变为G，导致酶活下降，HHCY的风险上升。
对于单基因位点SNP(单核苷酸多态性)分型方法主要有：PCR-RFLP(限制性片段长度多态性聚合酶链反应)、PCR-芯片杂交法、一代DNA测序法和TaqMan-MGB(双标记实时荧光探针)基因分型法。PCR-RFLP主要依赖于靶标的限制性酶切位点，实验难度增大， 同时后续的电泳分析容易引起实验室污染，无法大规模开展。TaqMan-MGB探针技术既可进行基因定量分析，又可分析基因突变。一代测序是基因检测的金标准，但是其操作流程过长，成本过高，同时仪器昂贵也难以适合在医院大规模开展。
但是，上述几种检测方法仍面临以下两个问题：(1)仍需要对唾液或者血样样本进行复杂的核酸提取和纯化步骤；(2)无法在一管中实现多SNP位点准确分型。
因此，提供一种更优秀的用于检测叶酸基因多态性的探针、基因芯片及试剂盒是十分有必要的。
发明内容
针对现有技术的不足，本发明提供一种用于检测叶酸基因多态性的探针、基因芯片及试剂盒。本发明具有特异性高、准确性好、免核酸提取和可快速检测多位点的优点。
一种用于检测叶酸基因多态性的探针，所述探针的序列为：
MTHFR677-C探针：AAAAAAAAAACGTGATGATGAAATCGGCTC；
MTHFR677-T探针：AAAAAAAAAACGTGATGATGAAATCGACTC；
MTHFR1298-A探针：AAAAAAAAAACAAAGACACTTTCTTCACTGGTC；
MTHFR1298-C探针：AAAAAAAAAAAAGACACTTGCTTCACTGGT；
MTRR66-A探针：AAAAAAAAAACAGCTTGCTCACATATTTCTTC；
MTRR66-G探针：AAAAAAAAAACACAGCTTGCTTGCTCACACA。
一种用于检测叶酸基因多态性的基因芯片，含有上述用于检测叶酸基因多态性的探针。
一种用于检测叶酸基因多态性的基因芯片的制备方法，包括以下步骤：
(1)在衬底上镀膜，得到生物传感器；
(2)在生物传感器上覆盖涂层，经6-肼基烟酸琥珀酰亚胺酯盐酸盐处理后，水洗；
(3)将经修饰的探针点样在生物传感器上，制得基因芯片。
优选的，步骤(1)中所述衬底为硅片(Si)，硅片的厚度为2.5mm，直径20cm。
优选的，步骤(1)中镀膜为在硅片上镀上厚度为47.5nm的氮化硅及13.5nm的TSPS(T结构聚氨二甲基硅烷烃)薄膜。
优选的，步骤(2)中所述涂层为多聚苯丙氨酸-赖氨酸涂层，多聚苯丙氨酸-赖氨酸涂层的厚度为15nm；6-肼基烟酸琥珀酰亚胺酯盐酸盐的浓度为1-10μmol/L，6-肼基烟酸琥珀酰亚胺酯盐酸盐处理的时间为20min。
优选的，步骤(3)中采用醛基或氨基对探针的一端进行修饰。
一种用于检测叶酸基因多态性的试剂盒，包括所述基因芯片、PCR反应体系、杂交缓冲 液、洗涤液、BW反应液(辣根过氧化物酶+柠檬酸钠缓冲液)和TMB(3,3',5,5'-四甲基联苯胺)显色液。
优选的，所述PCR反应体系包括以下组分：5×Buffer，dUTP mix，F-引物，R-引物，Hot taq，UNG酶(尿嘧啶DNA糖基化酶)和dd H 2O(双蒸水)。
PCR反应体系中加入UNG酶能够去除扩增产物污染，减少假阳性结果。
进一步优选的，所述F-引物的序列为：
MTHFR677-F：5'-TTGAGGCTGACCTGAAGCACTTGAAGGAG-3'；
MTHFR1298-F：5'-CTTTGGGGAGCTGAAGGACTACTA-3'；
MTRR66-FP：5'-CCTTGAAGTGATGAGGAGG-3'。
进一步优选的，所述R-引物的序列为：
MTHFR677-R：5'-CCTGGATGGGAAAGATCCCG-3'；
MTHFR1298-R：5'-CACTTTGTGACCATTCCGGTTT-3'；
MTRR66-RP：5'-CCACTGTAACGGCTCTAACC-3'。
优选的，所述杂交缓冲液包括0.3mol/L柠檬酸三钠、3mol/L氯化钠、0.3mol/L十二烷基硫酸钠和Triton X–100 0.1％水溶液。
优选的，所述洗涤液中含有洗涤液A和洗涤液B，洗涤液A为0.01-0.1mol/L的NaOH溶液，洗涤液B为0.1×SSC溶液。
一种叶酸基因多态性的检测方法，采用所述试剂盒进行检测，包括以下步骤：
(1)将待测物加入至PCR反应体系中进行扩增，得到PCR扩增产物；
(2)在基因芯片上加入PCR扩增产物和杂交缓冲液，进行反应；
(3)用0.01-0.1mol/L的NaOH溶液和0.1×SSC溶液对基因芯片进行洗涤，洗涤后风干；
(4)取BW反应液于基因芯片上，在室温条件下反应；
(5)加入TMB显色液进行显色，加入TMB显色液的前后均使用0.1×SSC溶液对基因芯片进行洗涤并风干，通过拍照或肉眼读取检测结果。
所述待测物中含有DNA片段。
用所述试剂盒与待测物进行的检测为杂交反应，当待测物中含有与探针能特异性结合的目的基因片段时，该探针所对应基因芯片区域显蓝色。
优选的，步骤(1)中扩增的条件为：95℃反应10min；94℃变性30s、56℃复性30s、72℃延伸30s，重复40个循环；72℃延伸5min。
优选的，步骤(2)中PCR扩增产物的加入量为10μL，杂交缓冲液为100μL；反应的条 件为：在50-60℃的条件下反应30-60min。
优选的，步骤(3)中0.01-0.1mol/L的NaOH溶液使用前预热，预热温度为50℃，每次洗涤5-10s，洗涤3次；0.1×SSC溶液的温度为50℃，进行洗涤的时间为1min。
优选的，步骤(4)中BW反应液的加入量为100μL，反应时间为10min。
优选的，步骤(5)中TMB显色液的加入量为100μL，反应时间为5min；用0.1×SSC溶液在加入TMB显色液的前后各洗涤3次，每次1min。
相对于现有技术，本发明的有益效果如下：
(1)采用基因芯片的方法，实现了叶酸基因的一次性多位点测定，减少了检测时间和成本；
(2)本发明通过特异性探针和引物的选择，结合基因芯片的方法，具有特异性好、准确性高和快速方便的优势；
(3)通过基因芯片的可见光光学放大，能达到较高的信号分辨率；
(4)检测结果可由拍照或肉眼直接读取，简单方便；
(5)本方法采用免提取的PCR扩增法进行基因扩增，省去核酸提取的复杂操作。
附图说明
图1为本发明实施例1-2制得的基因芯片和试剂盒对MTHFR677-C质粒、MTHFR1298-A质粒和MTRR66-A质粒的混合质粒的检测结果；
图2为本发明实施例1-2制得的基因芯片和试剂盒对MTHFR677-T质粒、MTHFR1298-C质粒和MTRR66-G质粒的混合质粒的检测结果；
图3为本发明实施例1-2制得的基因芯片和试剂盒对MTHFR677-C质粒、MTHFR1298-A质粒、MTRR66-A质粒、MTHFR677-T质粒、MTHFR1298-C质粒和MTRR66-G质粒的混合质粒的检测结果。
具体实施方式
为了让本领域技术人员更加清楚明白本发明所述技术方案，现列举以下实施例进行说明。需要指出的是，以下实施例对本发明要求的保护范围不构成限制作用。
实施例1
一种用于检测叶酸基因多态性的探针，其探针的序列为：
MTHFR677-C探针：AAAAAAAAAACGTGATGATGAAATCGGCTC；
MTHFR677-T探针：AAAAAAAAAACGTGATGATGAAATCGACTC；
MTHFR1298-A探针：AAAAAAAAAACAAAGACACTTTCTTCACTGGTC；
MTHFR1298-C探针：AAAAAAAAAAAAGACACTTGCTTCACTGGT；
MTRR66-A探针：AAAAAAAAAACAGCTTGCTCACATATTTCTTC；
MTRR66-G探针：AAAAAAAAAACACAGCTTGCTTGCTCACACA。
一种含有上述用于检测叶酸基因多态性的探针的基因芯片，其制备过程包括以下步骤：
通过旋转真空镀膜机和真空气相沉淀法在厚度约为2.5mm，直径20cm的硅片(Si)上镀上厚度为47.5nm的氮化硅及13.5nm的TSPS的薄膜，制备出相应的生物传感器，并覆盖15nm的多聚苯丙氨酸-赖氨酸涂层，最后经过10μmol/L 6-肼基烟酸琥珀酰亚胺酯盐酸盐处理20分钟，用清水洗净芯片。将上述用于检测叶酸基因多态性的探针的5'端用醛基修饰后，将探针点样至处理好的芯片上，在室温下反应，制备出包含探针的基因芯片。
每种探针在芯片上设置两个平行，故探针在芯片上的排列方法如表1所示：
表1：基因芯片探针点样排列
Figure PCTCN2020102826-appb-000001
点样PolyA在基因芯片上起定位作用；且检测结果在PolyA点样处不显色时，证明显色剂失效。
实施例2
一种用于检测叶酸基因多态性的试剂盒，包括实施例1中的基因芯片、PCR反应体系、杂交缓冲液、洗涤液、BW反应液和TMB显色液。
其中PCR反应体系的组成如表2所示：
表2：PCR反应体系组成
物料名称 浓度 加入量
5×Buffer   10μL
dUTP mix 25μmol/L 0.4μL
F-引物 200μmol/L 0.05μL
R-引物 200μmol/L 0.05μL
Hot taq 5U/μL 0.3μL
UNG酶 1U/μL 0.2μL
ddH 2O   加水补到45μL
其中PCR反应体系中F-引物的序列为：
MTHFR677-F：5'-TTGAGGCTGACCTGAAGCACTTGAAGGAG-3'；
MTHFR1298-F：5'-CTTTGGGGAGCTGAAGGACTACTA-3'；
MTRR66-FP：5'-CCTTGAAGTGATGAGGAGG-3'。
其中PCR反应体系中R-引物的序列为：
MTHFR677-R：5'-CCTGGATGGGAAAGATCCCG-3'；
MTHFR1298-R：5'-CACTTTGTGACCATTCCGGTTT-3'；
MTRR66-RP：5'-CCACTGTAACGGCTCTAACC-3'。
其中杂交缓冲液包括0.3mol/L柠檬酸三钠、3mol/L氯化钠、0.3mol/L十二烷基硫酸钠和Triton X–100 0.1％水溶液。
其中洗涤液中含有洗涤液A和洗涤液B，洗涤液A为0.01-0.1mol/L的NaOH溶液，洗涤液B为0.1×SSC溶液。
其中Hot-Taq酶、dUTP、UNG酶均购自深圳菲鹏生物股份有限公司。
其中引物、探针为自行设计，由上海生工生物工程有限公司合成。
实施例3
采用实施例1-2中制得的基因芯片和试剂盒进行检测，待检测物为含有目标基因片段的质粒，以上质粒均由上海生工生物工程有限公司合成和提供。
其中目的基因片段中包括MTHFR677(C/T)位点、MTHFR1298(A/C)位点和MTRR66(A/G)位点之一，目的基因片段的具体序列分别如下所示：
TTGAAGGAGAAGGTGTCTGCGGGAG[C/T]CGATTTCATCATCACGCAGCTTTTC；
TGGGGGGAGGAGCTGACCAGTGAAG[A/C]AAGTGTCTTTGAAGTCTTCGTTCTT；
AGGCAAAGGCCATCGCAGAAGAAAT[A/G]TGTGAGCAAGCTGTGGTACATGGAT。
将MTHFR677-C质粒、MTHFR1298-A质粒和MTRR66-A质粒混合，得到混合质粒，将混合质粒用1mL TE缓冲液溶解，使得质粒的浓度为2μg/mL即2000ng/mL。取5μL质粒溶液作为待测物加入PCR反应体系中，使得反应总体积为50μL，制得PCR反应混合液。
将上述PCR反应混合液放入PCR仪进行PCR扩增，PCR扩增的条件为：
95℃反应10min；94℃变性30s、56℃复性30s、72℃延伸30s，重复40个循环；72℃延 伸5min。
扩增完成后，将PCR扩增产物95℃加热3分钟,迅速置于冰上；
取10μL PCR扩增产物至所制得芯片上；
取100μL杂交缓冲液于芯片上，在55℃反应60分钟；
用预热温度为50℃的0.05mol/L NaOH溶液洗涤芯片3次，每次5-10s；
将芯片置于50℃的0.1×SSC中洗涤1min，对芯片表面进行风干；
取100μL BW反应液于芯片上，室温下反应10分钟；
用0.1×SSC溶液清洗芯片3次，每次1分钟，对芯片表面进行风干；
取100μL TMB显色液于芯片表面，反应5分钟；
用0.1×SSC溶液清洗芯片3次，每次1分钟，对芯片表面进行风干，通过拍照或肉眼读取检测结果。
检测结果如图1所示，MTHFR677-C探针、MTHFR1298-A探针和MTRR66-A探针在基因芯片的点样处(分别为区域1、2、3)均显蓝色，PolyA点样处(区域7)也显蓝色，证实采用实施例1-2制备的基因芯片和试剂盒，当MTHFR677/MTHFR1298/MTRR66位点未发生突变时，对其进行检测可得到准确结果。
实施例4
实施例4与实施例3基本相同，区别之处仅在于，所用待测物为MTHFR677-T质粒、MTHFR1298-C质粒和MTRR66-G质粒的混合质粒。
采用实施例1-2制得的基因芯片和试剂盒对该待测物进行检测。
检测结果如图2所示，MTHFR677-T探针、MTHFR1298-C探针和MTRR66-G探针在基因芯片的点样处(分别为区域4、5、6)均显蓝色，PolyA点样处(区域7)也显蓝色，证实采用实施例1-2制备的基因芯片和试剂盒可以准确地对MTHFR677/MTHFR1298/MTRR66位点的突变进行检测。
实施例5
实施例5与实施例3基本相同，区别之处仅在于，所用待测物为MTHFR677-C质粒、MTHFR1298-A质粒、MTRR66-A质粒、MTHFR677-T质粒、MTHFR1298-C质粒和MTRR66-G质粒的混合质粒。
采用实施例1-2制得的基因芯片和试剂盒对该待测物进行检测。
检测结果如图3所示，MTHFR677-C探针、MTHFR1298-A探针、MTRR66-A探针、MTHFR677-T探针、MTHFR1298-C探针和MTRR66-G探针在基因芯片的点样处(分别为区域1、2、3、4、5、6)均显蓝色，PolyA点样处(区域7)也显蓝色，证实采用实施例1-2 制备的基因芯片和试剂盒，在6种类别质粒混合的情况下，也具备良好的特异性，同样能得到准确的检测结果。

Claims (10)

  1. 一种用于检测叶酸基因多态性的探针，其特征在于，所述探针的序列为：
    MTHFR677-C探针：AAAAAAAAAACGTGATGATGAAATCGGCTC；
    MTHFR677-T探针：AAAAAAAAAACGTGATGATGAAATCGACTC；
    MTHFR1298-A探针：AAAAAAAAAACAAAGACACTTTCTTCACTGGTC；
    MTHFR1298-C探针：AAAAAAAAAAAAGACACTTGCTTCACTGGT；
    MTRR66-A探针：AAAAAAAAAACAGCTTGCTCACATATTTCTTC；
    MTRR66-G探针：AAAAAAAAAACACAGCTTGCTTGCTCACACA。
  2. 一种用于检测叶酸基因多态性的基因芯片，其特征在于，含有权利要求1所述的用于检测叶酸基因多态性的探针。
  3. 一种权利要求2所述用于检测叶酸基因多态性的基因芯片的制备方法，其特征在于，包括以下步骤：
    (1)在衬底上镀膜，得到生物传感器；
    (2)在生物传感器上覆盖涂层，经6-肼基烟酸琥珀酰亚胺酯盐酸盐处理后，水洗；
    (3)将经修饰的探针点样在生物传感器上，制得基因芯片。
  4. 根据权利要求3所述的制备方法，其特征在于，步骤(3)中采用醛基或氨基对探针的一端进行修饰。
  5. 一种用于检测叶酸基因多态性的试剂盒，其特征在于，包括权利要求2所述的基因芯片、PCR反应体系、杂交缓冲液、洗涤液、BW反应液和TMB显色液。
  6. 根据权利要求5所述的试剂盒，其特征在于，所述PCR反应体系包括以下组分：5×Buffer，dUTP mix，F-引物，R-引物，Hot taq，UNG酶和dd H2O。
  7. 根据权利要求6所述的试剂盒，其特征在于，所述F-引物的序列为：
    MTHFR677-F：5'-TTGAGGCTGACCTGAAGCACTTGAAGGAG-3'；
    MTHFR1298-F：5'-CTTTGGGGAGCTGAAGGACTACTA-3'；
    MTRR66-FP：5'-CCTTGAAGTGATGAGGAGG-3'。
  8. 根据权利要求6所述的试剂盒，其特征在于，所述R-引物的序列为：
    MTHFR677-R：5'-CCTGGATGGGAAAGATCCCG-3'；
    MTHFR1298-R：5'-CACTTTGTGACCATTCCGGTTT-3'；
    MTRR66-RP：5'-CCACTGTAACGGCTCTAACC-3'。
  9. 根据权利要求5所述的试剂盒，其特征在于，所述杂交缓冲液包括0.3mol/L柠檬酸三 钠、3mol/L氯化钠、0.3mol/L十二烷基硫酸钠和Triton X–100 0.1％水溶液。
  10. 一种叶酸基因多态性的检测方法，其特征在于，采用权利要求5-9中任一项所述的试剂盒进行检测，包括以下步骤：
    (1)将待测物加入至PCR反应体系中进行扩增，得到PCR扩增产物；
    (2)在基因芯片上加入PCR扩增产物和杂交缓冲液，进行反应；
    (3)用0.01-0.1mol/L的NaOH溶液和0.1×SSC溶液对基因芯片进行洗涤，洗涤后风干；
    (4)取BW反应液于基因芯片上，在室温条件下反应；
    (5)加入TMB显色液进行显色，加入TMB显色液的前后均使用0.1×SSC溶液对基因芯片进行洗涤并风干，通过拍照或肉眼读取检测结果。
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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1982471B (zh) * 2005-12-14 2011-12-14 北京华安佛医药研究中心有限公司 多态性位点基因型预测血管紧张素转换酶抑制剂类药物作用效果的用途、方法和试剂盒
CN104131087A (zh) * 2014-07-11 2014-11-05 武汉千麦医学检验所有限公司 一种针对叶酸代谢基因进行基因分型的试剂盒
CN105506122A (zh) * 2016-01-08 2016-04-20 珠海赛乐奇生物技术股份有限公司 用于检测il28b基因多态性的探针、基因芯片和试剂盒
CN107841537A (zh) * 2017-09-28 2018-03-27 苏州旷远生物分子技术有限公司 一种全预混mthfr和mtrr多重pcr基因多态性检测试剂盒和方法
CN108251510A (zh) * 2018-03-13 2018-07-06 普迈德（北京）科技有限公司 一种叶酸代谢能力基因分型的试剂盒、检测方法及其应用
CN109234374A (zh) * 2018-08-13 2019-01-18 武汉千麦医学检验所有限公司 一种针对叶酸代谢基因进行基因分型的试剂盒
CN109234375A (zh) * 2018-08-13 2019-01-18 武汉千麦医学检验所有限公司 一种针对叶酸代谢基因进行基因分型的方法
CN110616259A (zh) * 2019-09-02 2019-12-27 珠海赛乐奇生物技术股份有限公司 用于检测叶酸基因多态性的探针、基因芯片及试剂盒

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1982471B (zh) * 2005-12-14 2011-12-14 北京华安佛医药研究中心有限公司 多态性位点基因型预测血管紧张素转换酶抑制剂类药物作用效果的用途、方法和试剂盒
CN104131087A (zh) * 2014-07-11 2014-11-05 武汉千麦医学检验所有限公司 一种针对叶酸代谢基因进行基因分型的试剂盒
CN105506122A (zh) * 2016-01-08 2016-04-20 珠海赛乐奇生物技术股份有限公司 用于检测il28b基因多态性的探针、基因芯片和试剂盒
CN107841537A (zh) * 2017-09-28 2018-03-27 苏州旷远生物分子技术有限公司 一种全预混mthfr和mtrr多重pcr基因多态性检测试剂盒和方法
CN108251510A (zh) * 2018-03-13 2018-07-06 普迈德（北京）科技有限公司 一种叶酸代谢能力基因分型的试剂盒、检测方法及其应用
CN109234374A (zh) * 2018-08-13 2019-01-18 武汉千麦医学检验所有限公司 一种针对叶酸代谢基因进行基因分型的试剂盒
CN109234375A (zh) * 2018-08-13 2019-01-18 武汉千麦医学检验所有限公司 一种针对叶酸代谢基因进行基因分型的方法
CN110616259A (zh) * 2019-09-02 2019-12-27 珠海赛乐奇生物技术股份有限公司 用于检测叶酸基因多态性的探针、基因芯片及试剂盒

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