技术领域Technical field
本发明属于分子生物学技术领域，特别涉及一种用于制备产前胎儿21-三体综合征检测的PCR反应体系的特异性引物。The invention belongs to the technical field of molecular biology, and particularly relates to a specific primer for preparing a PCR reaction system for prenatal fetal trisomy 21 detection.
背景技术Background technique
21-三体综合症即唐氏综合症(DS，Down's syndrome)是当前最常见的遗传疾病。在新生儿中，21-三体综合症的发病率高达1/700，并且发病率会随着产妇年龄的增加而上升。由于该疾病无法治疗，因此预防手段主要是通过产前筛查来进行，但传统的侵入性技术，包括妊娠早期绒毛活检、妊娠中期羊膜腔穿刺、妊娠中晚期脐血穿刺等技术，存在较高的孕妇宫内感染、早产及流产风险。此外，细胞遗传学诊断需大约4周时间，存在费时、易培养失败以及不能检测较小的染色体结构畸变等缺点。唐氏筛查技术基于血清学检测进行，但其准确度相对较低，因此只能作为预筛选手段。Trisomy 21, also known as Down's syndrome (DS, Down's syndrome), is currently the most common genetic disease. Among newborns, the incidence of trisomy 21 is as high as 1/700, and the incidence increases with maternal age. Since the disease cannot be treated, prevention measures are mainly carried out through prenatal screening. However, traditional invasive techniques, including chorionic villus biopsy in the first trimester, amniocentesis in the second trimester, and umbilical cord blood puncture in the second and third trimesters, have higher risks. pregnant women’s risk of intrauterine infection, premature delivery and miscarriage. In addition, cytogenetic diagnosis takes approximately 4 weeks and has the disadvantages of being time-consuming, prone to culture failure, and unable to detect minor chromosomal structural aberrations. Down syndrome screening technology is based on serological testing, but its accuracy is relatively low, so it can only be used as a pre-screening method.
随着孕妇外周血中胎儿游离DNA的发现，研究者发现其可用于指示胎儿遗传病的风险，即胎儿游离DNA中包含了胎儿的所有遗传信息。香港中文大学卢煜明教授与霍普金斯大学团队合作，以二代测序为基础，开发出基于孕妇外周血中游离胎儿DNA(cfDNA)的产前检测方法，该方法主要基于二代测序技术进行，对比不同染色体上的基因丰度差异或突变情况，确定三体及遗传病风险。该技术检测灵敏度高且数据分析结果较为准确，因而目前已在国内外临床上广泛推广应用。但二代测序操作程序繁杂，单次运行成本偏高，此外，cfDNA片段化严重，提取得率低等问题，加大了DNA文库的制备难度。With the discovery of fetal cell-free DNA in the peripheral blood of pregnant women, researchers have found that it can be used to indicate the risk of fetal genetic diseases, that is, fetal cell-free DNA contains all the genetic information of the fetus. Professor Lu Yuming of the Chinese University of Hong Kong collaborated with a team from Hopkins University to develop a prenatal detection method based on cell-free fetal DNA (cfDNA) in the peripheral blood of pregnant women based on second-generation sequencing. This method is mainly based on second-generation sequencing technology. Compare gene abundance differences or mutations on different chromosomes to determine the risk of trisomies and genetic diseases. This technology has high detection sensitivity and relatively accurate data analysis results, so it has been widely promoted and applied clinically at home and abroad. However, the second-generation sequencing operation procedures are complicated and the cost of a single operation is high. In addition, cfDNA is seriously fragmented and the extraction yield is low, which makes the preparation of DNA libraries more difficult.
发明内容Contents of the invention
进一步的研究结果表明，孕妇和胎儿在部分位点甲基化水平上存在差异，从而提示我们可利用特异性核酸内切酶或甲基化免疫沉淀的方法来富集孕妇外周血中的胎儿游离DNA，从而在去除母体DNA的情况下，检测胎儿三体风险。但考虑到荧光定量PCR等分子检测技术本身的误差，以及胎儿/母体DNA含量的个体差异性等因素，有必要通过多组位点的组合检测，提高诊断准确率。Further research results show that there are differences in the methylation levels of some sites between pregnant women and fetuses, suggesting that we can use specific endonucleases or methylation immunoprecipitation methods to enrich fetal ionocytes in the peripheral blood of pregnant women. DNA to detect the risk of fetal trisomy without maternal DNA. However, considering the errors of molecular detection technologies such as fluorescence quantitative PCR and the individual differences in fetal/maternal DNA content, it is necessary to improve the diagnostic accuracy through combined detection of multiple sets of sites.
本发明提供一种用于产前胎儿21-三体综合征的检测方法，包括以下步骤：The invention provides a method for detecting prenatal fetal trisomy 21, which includes the following steps:
(1)待测样本采集：(1) Collection of samples to be tested:
采集待测母体外周血500μL-1mL，采用通用核酸柱式抽提试剂盒进行血液游离DNA提取，所获得DNA样品经微量紫外分光光度计检测获得浓度及纯度，DNA样品纯度为1.8～2.0，将DNA样品稀释得到待测DNA样本；Collect 500 μL-1mL of maternal peripheral blood to be tested, and use a universal nucleic acid column extraction kit to extract blood-free DNA. The obtained DNA sample is tested with a micro-UV spectrophotometer to obtain the concentration and purity. The purity of the DNA sample is 1.8 to 2.0. The DNA sample is diluted to obtain the DNA sample to be tested;
(2)甲基化DNA免疫共沉淀：(2) Co-immunoprecipitation of methylated DNA:
按照甲基化DNA免疫共沉淀试剂盒使用说明书要求，对待测DNA样本(Sample)、阳性对照(Positive control)、阴性对照(Negative control)进行甲基化DNA分离，在最终洗脱甲基化DNA环节加入ddH2O溶解洗脱甲基化DNA；According to the instruction manual of the methylated DNA co-immunoprecipitation kit, separate the methylated DNA from the DNA sample (Sample), positive control (Positive control), and negative control (Negative control), and finally elute the methylated DNA. Add ddH2 O to dissolve and elute methylated DNA;
(3)酶切：(3) Enzyme digestion:
按照HpaII酶切反应体系处理，将待测DNA样本、阳性对照、阴性对照的甲基化DNA配制酶切反应体系，配制好的酶切反应体系在37℃水浴2h进行酶切反应，反应后在80-90℃水浴20-30min灭活HpaII酶，得到cut待测DNA样本、cut阳性对照、cut阴性对照；According to the HpaII enzyme digestion reaction system, prepare the enzyme digestion reaction system from the DNA sample to be tested, the methylated DNA of the positive control, and the negative control. The prepared enzyme digestion reaction system is subjected to the enzyme digestion reaction in a 37°C water bath for 2 hours. After the reaction, Inactivate the HpaII enzyme in a water bath at 80-90°C for 20-30 minutes to obtain the cut DNA sample to be tested, cut positive control, and cut negative control;
(4)配制PCR反应体系：(4) Prepare PCR reaction system:
将获得的cut待测DNA样本、cut阳性对照、cut阴性对照分别使用9组特异性引物配制PCR反应体系，每个样品每组引物配置3-6个体系重复，9组特异性引物中包括1组常规染色体位点检测引物和8组21号染色体位点检测引物；The obtained cut DNA samples, cut positive controls, and cut negative controls were prepared using 9 sets of specific primers to prepare a PCR reaction system. Each sample was configured with 3-6 system repeats for each set of primers. The 9 sets of specific primers included 1 One set of conventional chromosome locus detection primers and 8 sets of chromosome 21 locus detection primers;
(5)PCR反应：(5)PCR reaction:
使用实时荧光定量PCR仪进行PCR反应和荧光测定，PCR过程如下：Use a real-time fluorescence quantitative PCR instrument to perform PCR reaction and fluorescence measurement. The PCR process is as follows:
第一阶段、预变性：PCR反应体系在95℃下反应5min；The first stage, pre-denaturation: the PCR reaction system reacts at 95°C for 5 minutes;
第二阶段、循环反应：PCR反应体系在95℃下反应10sec后在60℃下反应30sec；The second stage, cycle reaction: the PCR reaction system reacts at 95°C for 10 seconds and then at 60°C for 30 seconds;
第三阶段、获得溶解曲线：PCR反应体系在95℃下反应15sec后，在60℃下反应30sec；再升温至95℃反应15sec，得到溶解曲线；The third stage is to obtain the dissolution curve: after the PCR reaction system reacts at 95°C for 15 seconds, react at 60°C for 30 seconds; then increase the temperature to 95°C and react for 15 seconds to obtain the dissolution curve;
(6)分析：(6)Analysis:
使用实时荧光定量PCR仪自带的分析软件分析实验结果获得Cq值，将获得的Cq值进行分析，Cq值包括待测DNA样本Cq值：Cq样本，阳性对照Cq值：Cq阳性对照，阴性对照Cq值：Cq阴性对照；各自将21号染色体上8个位点的Cq值与常规染色体的Cq值相比，获得比值R；若4个以上位点R阳性对照≈R样本＞R阴性对照，则说明胎儿21三体概率极高，建议进行羊水穿刺做核型分析确认；若1-4个位点R阳性对照≈R样本＞R阴性对照，则建议复查；若所有位点R阳性对照＞R样本≈R阴性对照，则说明胎儿21三体概率极低，可放心生育。Use the analysis software that comes with the real-time fluorescence quantitative PCR instrument to analyze the experimental results to obtain the Cq value. Analyze the obtained Cq value. The Cq value includes the Cq value of the DNA sample to be tested: Cqsample , the positive control Cq value: Cqpositive control , and the negative control. Cq value: Cqnegative control ; compare the Cq values of 8 sites on chromosome 21 with the Cq values of conventional chromosomes to obtain the ratio R; if more than 4 sites Rpositive control ≈ Rsample > Rnegative control , It means that the probability of fetal trisomy 21 is extremely high, and it is recommended to perform amniocentesis for karyotyping confirmation; if 1-4 sites Rpositive control ≈ Rsample > Rnegative control , it is recommended to reexamine; if all sites Rpositive control > Rsample ≈ Rnegative control , which means that the probability of fetal trisomy 21 is extremely low and you can give birth with confidence.
作为优选，在(1)待测样本采集步骤中，用去离子水将待测样本DNA样品浓度稀释为50ng/μl。Preferably, in (1) the sample collection step to be tested, the DNA sample concentration of the sample to be tested is diluted to 50ng/μl with deionized water.
作为优选，在(2)甲基化DNA免疫共沉淀步骤中，所述的阳性对照为21三体综合征胎儿羊水穿刺细胞中提取的DNA与健康成年人体的外周血中获得的血细胞DNA按1:20比例混合获得的标准品，浓度为50ng/μl；所述的阴性对照为健康女性成年人体的外周血中获得的血细胞DNA，浓度为50ng/μl；甲基化DNA分离过程中每组的DNA含量不少于0.8μg。Preferably, in (2) the methylated DNA co-immunoprecipitation step, the positive control is DNA extracted from amniocentesis cells of fetuses with trisomy 21 and blood cell DNA obtained from peripheral blood of healthy adults. :20 ratio of the standard obtained by mixing, the concentration is 50ng/μl; the negative control is blood cell DNA obtained from the peripheral blood of healthy female adults, the concentration is 50ng/μl; the methylated DNA isolation process of each group The DNA content is not less than 0.8μg.
作为优选，在(3)酶切步骤中，酶切反应体系中，DNA样本、HpaII酶、10x Buffer、ddH2O的体积比为40:1:5:4，每个样本配制的酶切反应体系体积分别为50μl；酶切反应体系中DNA样本浓度为50ng/μl；HpaII酶的浓度为5U/μl。Preferably, in the enzyme digestion step (3), in the enzyme digestion reaction system, the volume ratio of DNA sample, HpaII enzyme, 10x Buffer, and ddH2 O is 40:1:5:4. The enzyme digestion reaction prepared for each sample The system volume is 50 μl respectively; the DNA sample concentration in the enzyme digestion reaction system is 50ng/μl; the concentration of HpaII enzyme is 5U/μl.
作为优选，在(4)配制PCR反应体系步骤中，PCR反应体系中，荧光定量PCR 2×预混液、上游引物、下游引物、样本/对照、ddH2O的体积比为25:1:1:1:22；每个样品每组配制的PCR反应体系体积分别为20μl。Preferably, in the step of (4) preparing the PCR reaction system, in the PCR reaction system, the volume ratio of fluorescence quantitative PCR 2× master mix, upstream primer, downstream primer, sample/control, and ddH2 O is 25:1:1: 1:22; the volume of the PCR reaction system prepared for each sample and each group is 20 μl.
作为优选，在(4)配制PCR反应体系步骤中，所述的9组特异性引物如下：Preferably, in the step of (4) preparing the PCR reaction system, the 9 sets of specific primers are as follows:
第1组用于3号常规染色体位点的检测：Group 1 is used for the detection of conventional chromosome 3 loci:
上游引物Ch3-F：AGCTGGCACCCGCTGGUpstream primer Ch3-F: AGCTGGCACCCGCTGG
下游引物Ch3-R：GTGTGGGGTTGCACGCGDownstream primer Ch3-R: GTGTGGGGTTGCACCGG
第2组用于21号染色体位点1的检测：Group 2 is used for the detection of chromosome 21 locus 1:
上游引物ERG-F：CAGGAGGCTGAGGCAGGGUpstream primer ERG-F: CAGGAGGCTGAGGCAGGG
下游引物ERG-R：GGTACAGGTTGGGAGTTTGGGDownstream primer ERG-R: GGTACAGGTTGGGAGTTTGGG
第3组用于21号染色体位点2的检测：Group 3 is used for the detection of chromosome 21 locus 2:
上游引物AIRE-F：TAGTAAGGGAGGGCCGAGAAUpstream primer AIRE-F: TAGTAAGGGAGGGCCGAGAA
下游引物AIRE-R：CAGAGCCAGAACGCACAGAGDownstream primer AIRE-R: CAGAGCCAGAACGCACAGAG
第4组用于21号染色体位点3的检测：Group 4 is used for the detection of chromosome 21 locus 3:
上游引物SIM-F：ACCGGGCCTTCTGTCTGTCUpstream primer SIM-F: ACCGGGCCTTCTGTCTGTC
下游引物SIM-R：TCTGCACGCTTCAATCCTTCDownstream primer SIM-R: TCTGCACGCTTCAATCCTTC
第5组用于21号染色体位点4的检测：Group 5 is used for the detection of chromosome 21 locus 4:
上游引物NIPK4-F：GAGCCAGGTCTGTTCTCCACGUpstream primer NIPK4-F: GAGCCAGGTCTGTTTCTCCACG
下游引物NIPK4-R：AGCCGATGCCCTTGTTGCDownstream primer NIPK4-R: AGCCGATGCCCTTGTTGC
第6组用于21号染色体位点5的检测：Group 6 is used for the detection of chromosome 21 locus 5:
上游引物YBEY-1F：GGTTCGTGCACTCGCTGAGUpstream primer YBEY-1F: GGTTCGTGCACTCGCTGAG
下游引物YBEY-1R：GCCTTCTCCTTCTGGAACATCTDownstream primer YBEY-1R: GCCTTCTCCTTCTGGAACATCT
第7组用于21号染色体位点6的检测：Group 7 is used for the detection of chromosome 21 locus 6:
上游引物JAM2-F：TTTTGGGCCACATCATTCTUpstream primer JAM2-F:TTTTGGGCCACATCATTCT
下游引物JAM2-R：GCTGGGATTACAGGCGTGAGDownstream primer JAM2-R: GCTGGGATTACAGGCGTGAG
第8组用于21号染色体位点7的检测：Group 8 is used for the detection of chromosome 21 locus 7:
上游引物DSCAM-F：CCTAAGACCTTTGCCTAGCTTCTUpstream primer DSCAM-F: CCTAAGACCTTTGCCTAGCTTCT
下游引物DSCAM-R：TCCTTTATGGGATTTCTGTTGTGDownstream primer DSCAM-R: TCCTTTATGGGATTTCTGTTGTG
第9组用于21号染色体位点8的检测：Group 9 is used for the detection of chromosome 21 locus 8:
上游引物PCNT-F：AGCAGACACGGGCTCGGTUpstream primer PCNT-F: AGCAGACACGGGCTCGGT
下游引物PCNT-R：CGGTGCTCAGCAACCACCDownstream primer PCNT-R: CGGTGCTCAGCAACCACC
每组上游引物和下游引物的浓度分别为10μM。The concentrations of each set of upstream primers and downstream primers were 10 μM respectively.
本发明提供了一组胎儿特异性甲基化位点及其对应引物序列，该位点分别位于21及3号染色体上，这些位点上母体则相应的无甲基化，因此可在特异性富集后，通过对比21号染色体及3号染色体上的基因相对丰度，确定胎儿21-三体风险。本发明涉及8个位点的检测，其结果可相互验证，从而提高诊断准确性。The present invention provides a set of fetal-specific methylation sites and their corresponding primer sequences. The sites are respectively located on chromosomes 21 and 3. The mother has no methylation at these sites, so it can be used in specific After enrichment, the risk of fetal trisomy 21 was determined by comparing the relative abundance of genes on chromosome 21 and chromosome 3. The present invention involves the detection of 8 sites, and the results can be mutually verified, thereby improving diagnostic accuracy.
本发明提供的特异性引物所涉及的3号染色体上的位点在不同孕妇外周血中含量及甲基化水平基本稳定，涉及的21号染色体上的8个位点则可保证在不同孕妇外周血中4个以上具有显著的胎儿-孕妇差异；3号常规染色体位点与21染色体的8个位点具有极强的丰度一致性，确保相对丰度检测的准确性。The content and methylation level of the sites on chromosome 3 involved in the specific primers provided by the invention are basically stable in the peripheral blood of different pregnant women. The 8 sites on chromosome 21 involved can ensure that the content and methylation level in the peripheral blood of different pregnant women are stable. More than 4 sites in the blood have significant fetal-pregnant differences; the regular chromosome 3 locus has extremely strong abundance consistency with 8 sites on chromosome 21, ensuring the accuracy of relative abundance detection.
本发明的有益效果：Beneficial effects of the present invention:
本发明可以快速进行产前胎儿三体检测，检测时间短、成本低、有望替代无创DNA检测中的二代测序环节，减少产妇家庭的检测费支出，减轻年轻家庭的经济压力，带来良好的经济效益和社会效益。The present invention can quickly perform prenatal fetal trisomy detection with short detection time and low cost. It is expected to replace the second-generation sequencing link in non-invasive DNA detection, reduce the detection expenses of maternal families, reduce the economic pressure on young families, and bring good results. Economic benefits and social benefits.
附图说明Description of the drawings
图1为本发明案例一检测结果R值对比图；Figure 1 is a comparison chart of the R value of the test results of Case 1 of the present invention;
图2为本发明案例二检测结果R值对比图；Figure 2 is a comparison chart of the R value of the test results of Case 2 of the present invention;
图3为本发明案例三检测结果R值对比图；Figure 3 is a comparison chart of the R value of the test results of case three of the present invention;
图4为本发明案例四检测结果R值对比图；Figure 4 is a comparison chart of the R value of the test results of Case 4 of the present invention;
图5为本发明案例五检测结果R值对比图；Figure 5 is a comparison chart of the R value of the test results of Case 5 of the present invention;
图6为本发明案例六检测结果R值对比图。Figure 6 is a comparison chart of the R value of the test results of Case 6 of the present invention.
具体实施方式Detailed ways
本发明提供一种用于产前胎儿21-三体综合征的检测方法，包括以下步骤：The invention provides a method for detecting prenatal fetal trisomy 21, which includes the following steps:
(1)待测样本采集：(1) Collection of samples to be tested:
采集待测母体外周血500μL-1mL，采用通用核酸柱式抽提试剂盒(南京诺唯赞生物科技股份有限公司、FastPure Blood/Cell/Tissue/Bacteria DNA Isolation Mini Kit)进行血液游离DNA提取，所获得DNA样品经微量紫外分光光度计(Thermo，NanoDrop One)检测获得浓度及纯度，DNA样品纯度为1.8～2.0，用去离子水将待测样本DNA样品浓度稀释为50ng/μl得到待测DNA样本；Collect 500 μL-1mL of maternal peripheral blood to be tested, and use a universal nucleic acid column extraction kit (Nanjing Novezan Biotechnology Co., Ltd., FastPure Blood/Cell/Tissue/Bacteria DNA Isolation Mini Kit) to extract blood free DNA. Obtain the DNA sample and detect the concentration and purity with a micro-UV spectrophotometer (Thermo, NanoDrop One). The purity of the DNA sample is 1.8~2.0. Use deionized water to dilute the DNA sample concentration of the sample to be tested to 50ng/μl to obtain the DNA sample to be tested. ;
(2)甲基化DNA免疫共沉淀：(2) Co-immunoprecipitation of methylated DNA:
按照艾美捷甲基化DNA免疫共沉淀试剂盒(MethylampTMMethylated DNA CaptureKit)使用说明书要求，对待测DNA样本(Sample)、阳性对照(Positive control)、阴性对照(Negative control)进行甲基化DNA分离，所述的阳性对照为21三体综合征胎儿羊水穿刺细胞中提取的DNA与健康成年人体的外周血中获得的血细胞DNA按1:20比例混合获得的标准品，浓度为50ng/μl；所述的阴性对照为健康女性成年人体的外周血中获得的血细胞DNA，浓度为50ng/μl；甲基化DNA分离过程中每组的DNA含量为0.8μg；在最终洗脱甲基化DNA环节加入10μlddH2O溶解洗脱甲基化DNA。According to the instruction manual of MethylampTM Methylated DNA CaptureKit, methylated DNA was measured on the DNA sample (Sample), positive control (Positive control), and negative control (Negative control). Separation, the positive control is a standard obtained by mixing DNA extracted from amniocentesis cells of fetuses with trisomy 21 and blood cell DNA obtained from peripheral blood of healthy adults in a ratio of 1:20, with a concentration of 50ng/μl; The negative control is blood cell DNA obtained from the peripheral blood of healthy female adults, with a concentration of 50ng/μl; the DNA content of each group during the methylated DNA isolation process is 0.8μg; in the final elution of methylated DNA Add 10 μl ddH2 O to dissolve the eluted methylated DNA.
(3)酶切：(3) Enzyme digestion:
按照HpaII酶切反应体系处理，将待测DNA样本、阳性对照、阴性对照的甲基化DNA配制酶切反应体系，酶切反应体系配比如表1所示：According to the HpaII enzyme digestion reaction system, prepare the enzyme digestion reaction system from the DNA sample to be tested, the methylated DNA of the positive control, and the negative control. The enzyme digestion reaction system ratio is shown in Table 1:
表1：Table 1:
配制好的酶切反应体系在37℃水浴2h进行酶切反应，反应后在80-90℃水浴20-30min灭活HpaII酶，得到cut待测DNA样本、cut阳性对照、cut阴性对照；每个样本配制的酶切反应体系体积分别为50μl；其中10×Buffer是酶公司提供的，为现有的商品化试剂，目的是使酶活性最大。The prepared enzyme digestion reaction system is carried out in a 37°C water bath for 2 hours. After the reaction, the HpaII enzyme is inactivated in a 80-90°C water bath for 20-30 minutes to obtain the cut DNA sample to be tested, the cut positive control, and the cut negative control; each The volume of the enzyme digestion reaction system prepared for the sample is 50 μl; the 10×Buffer is provided by the enzyme company and is an existing commercial reagent, with the purpose of maximizing the enzyme activity.
(4)配制PCR反应体系：(4) Prepare PCR reaction system:
将获得的cut待测DNA样本、cut阳性对照、cut阴性对照分别使用9组特异性引物配制PCR反应体系，每个样品每组引物配置3-6个体系重复，PCR反应体系配比如表2所示：The obtained cut DNA samples, cut positive controls, and cut negative controls were prepared using 9 sets of specific primers to prepare a PCR reaction system. Each sample was configured with 3-6 system repeats for each set of primers. The PCR reaction system configuration is as shown in Table 2. Show:
表2：Table 2:
其中，2xUniversal SYBR qPCR Master Mix为荧光定量PCR 2×预混液，购自南京诺唯赞生物科技股份有限公司，为现有的商品化试剂。Primer F为上游引物，PrimerR为下游引物。Among them, 2x Universal SYBR qPCR Master Mix is a fluorescence quantitative PCR 2× master mix, purchased from Nanjing Novezan Biotechnology Co., Ltd., and is an existing commercial reagent. Primer F is the upstream primer, and PrimerR is the downstream primer.
所述的9组特异性引物中包括1组常规染色体位点检测引物和8组21号染色体位点检测引物，具体如下：The 9 sets of specific primers include 1 set of conventional chromosome site detection primers and 8 sets of chromosome 21 site detection primers, as follows:
第1组用于3号常规染色体位点的检测：Group 1 is used for the detection of conventional chromosome 3 loci:
上游引物Ch3-F：AGCTGGCACCCGCTGGUpstream primer Ch3-F: AGCTGGCACCCGCTGG
下游引物Ch3-R：GTGTGGGGTTGCACGCGDownstream primer Ch3-R: GTGTGGGGTTGCACCGG
第2组用于21号染色体位点1的检测：Group 2 is used for the detection of chromosome 21 locus 1:
上游引物ERG-F：CAGGAGGCTGAGGCAGGGUpstream primer ERG-F: CAGGAGGCTGAGGCAGGG
下游引物ERG-R：GGTACAGGTTGGGAGTTTGGGDownstream primer ERG-R: GGTACAGGTTGGGAGTTTGGG
第3组用于21号染色体位点2的检测：Group 3 is used for the detection of chromosome 21 locus 2:
上游引物AIRE-F：TAGTAAGGGAGGGCCGAGAAUpstream primer AIRE-F: TAGTAAGGGAGGGCCGAGAA
下游引物AIRE-R：CAGAGCCAGAACGCACAGAGDownstream primer AIRE-R: CAGAGCCAGAACGCACAGAG
第4组用于21号染色体位点3的检测：Group 4 is used for the detection of chromosome 21 locus 3:
上游引物SIM-F：ACCGGGCCTTCTGTCTGTCUpstream primer SIM-F: ACCGGGCCTTCTGTCTGTC
下游引物SIM-R：TCTGCACGCTTCAATCCTTCDownstream primer SIM-R: TCTGCACGCTTCAATCCTTC
第5组用于21号染色体位点4的检测：Group 5 is used for the detection of chromosome 21 locus 4:
上游引物NIPK4-F：GAGCCAGGTCTGTTCTCCACGUpstream primer NIPK4-F: GAGCCAGGTCTGTTTCTCCACG
下游引物NIPK4-R：AGCCGATGCCCTTGTTGCDownstream primer NIPK4-R: AGCCGATGCCCTTGTTGC
第6组用于21号染色体位点5的检测：Group 6 is used for the detection of chromosome 21 locus 5:
上游引物YBEY-1F：GGTTCGTGCACTCGCTGAGUpstream primer YBEY-1F: GGTTCGTGCACTCGCTGAG
下游引物YBEY-1R：GCCTTCTCCTTCTGGAACATCTDownstream primer YBEY-1R: GCCTTCTCCTTCTGGAACATCT
第7组用于21号染色体位点6的检测：Group 7 is used for the detection of chromosome 21 locus 6:
上游引物JAM2-F：TTTTGGGCCACATCATTCTUpstream primer JAM2-F:TTTTGGGCCACATCATTCT
下游引物JAM2-R：GCTGGGATTACAGGCGTGAGDownstream primer JAM2-R: GCTGGGATTACAGGCGTGAG
第8组用于21号染色体位点7的检测：Group 8 is used for the detection of chromosome 21 locus 7:
上游引物DSCAM-F：CCTAAGACCTTTGCCTAGCTTCTUpstream primer DSCAM-F: CCTAAGACCTTTGCCTAGCTTCT
下游引物DSCAM-R：TCCTTTATGGGATTTCTGTTGTGDownstream primer DSCAM-R: TCCTTTATGGGATTTCTGTTGTG
第9组用于21号染色体位点8的检测：Group 9 is used for the detection of chromosome 21 locus 8:
上游引物PCNT-F：AGCAGACACGGGCTCGGTUpstream primer PCNT-F: AGCAGACACGGGCTCGGT
下游引物PCNT-R：CGGTGCTCAGCAACCACCDownstream primer PCNT-R: CGGTGCTCAGCAACCACC
每组上游引物和下游引物的浓度分别为10μM。The concentrations of each set of upstream primers and downstream primers were 10 μM respectively.
(5)PCR反应：(5)PCR reaction:
使用LightCycler96实时荧光定量PCR仪进行PCR反应和荧光测定，PCR过程如下：Use LightCycler96 real-time fluorescence quantitative PCR instrument for PCR reaction and fluorescence measurement. The PCR process is as follows:
第一阶段、预变性：PCR反应体系在95℃下反应5min；The first stage, pre-denaturation: the PCR reaction system reacts at 95°C for 5 minutes;
第二阶段、循环反应：PCR反应体系在95℃下反应10sec后在60℃下反应30sec；The second stage, cycle reaction: the PCR reaction system reacts at 95°C for 10 seconds and then at 60°C for 30 seconds;
第三阶段、获得溶解曲线：PCR反应体系在95℃下反应15sec后，在60℃下反应30sec；再升温至95℃反应15sec，得到溶解曲线；The third stage is to obtain the dissolution curve: after the PCR reaction system reacts at 95°C for 15 seconds, react at 60°C for 30 seconds; then increase the temperature to 95°C and react for 15 seconds to obtain the dissolution curve;
LightCycler96实时荧光定量PCR仪为现有设备。The LightCycler96 real-time fluorescence quantitative PCR instrument is an existing equipment.
(6)分析：(6)Analysis:
使用LightCycler96实时荧光定量PCR仪自带的分析软件分析实验结果获得Cq值，将获得的Cq值进行分析，Cq值包括待测DNA样本Cq值：Cq样本，阳性对照Cq值：Cq阳性对照，阴性对照Cq值：Cq阴性对照；各自将21号染色体上8个位点的Cq值与常规染色体的Cq值相比，获得比值R；若4个以上位点R阳性对照≈R样本＞R阴性对照，则说明胎儿21三体概率极高，建议进行羊水穿刺做核型分析确认；若1-4个位点R阳性对照≈R样本＞R阴性对照，则建议复查；若所有位点R阳性对照＞R样本≈R阴性对照，则说明胎儿21三体概率极低，可放心生育。Use the analysis software that comes with the LightCycler96 real-time fluorescence quantitative PCR instrument to analyze the experimental results to obtain the Cq value. Analyze the obtained Cq value. The Cq value includes the Cq value of the DNA sample to be tested: Cqsample , the positive control Cq value: Cqpositive control , negative Control Cq value: Cqnegative control ; compare the Cq values of 8 sites on chromosome 21 with the Cq values of conventional chromosomes to obtain the ratio R; if there are more than 4 sites Rpositive control ≈ Rsample > Rnegative control , it means that the probability of fetal trisomy 21 is extremely high, and it is recommended to perform amniocentesis for karyotyping confirmation; if 1-4 sites are Rpositive control ≈ Rsample > Rnegative control , it is recommended to reexamine; if all sites are Rpositive control > Rsample ≈ Rnegative control , which means that the probability of fetal trisomy 21 is extremely low and you can have a baby with confidence.
利用上述实施例的方法实际检测了六个胎儿案例，结果如下：Six fetal cases were actually detected using the method of the above embodiment, and the results are as follows:
如图1-2所示，案例一、二的检测结果显示：4个以上位点R阳性对照≈R样本＞R阴性对照，说明胎儿大概率为三体胎儿，后续经羊水穿刺检验结果显示两例均为三体胎儿；As shown in Figure 1-2, the test results of Cases 1 and 2 show: Rpositive control ≈ Rsample > Rnegative control at more than 4 sites, indicating that the fetus is likely to have a trisomic fetus. The subsequent amniocentesis test results show two All cases were trisomic fetuses;
如图3-4所示，案例三、四的检测结果显示：1-4个位点R阳性对照≈R样本＞R阴性对照，建议复查，后续经羊水穿刺检验结果显示一例为三体胎儿，一例为正常胎儿；As shown in Figure 3-4, the test results of Cases 3 and 4 showed: 1-4 sites Rpositive control ≈ Rsample > Rnegative control . It is recommended to review. The subsequent amniocentesis test results showed that one case was a trisomic fetus. One case was a normal fetus;
如图5-6所示，案例五、六的检测结果显示：所有位点R阳性对照＞R样本≈R阴性对照，说明胎儿三体概率极低，后续经羊水穿刺检验结果显示两例均为正常胎儿。As shown in Figure 5-6, the test results of Cases 5 and 6 show: Rpositive control > Rsample ≈ Rnegative control at all sites, indicating that the probability of fetal trisomy is extremely low. The subsequent amniocentesis test results showed that both cases were Normal fetus.
序列表 sequence list
<110> 吉林大学第一医院<110> Jilin University First Hospital
<120> 一种用于产前胎儿21-三体综合征的检测方法<120> A detection method for prenatal fetal trisomy 21
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| CN202110527026.1ACN113046435B (en) | 2021-05-14 | 2021-05-14 | Specific primer for preparing PCR reaction system for detecting prenatal fetal 21-trisomy syndrome |
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| CN202110527026.1ACN113046435B (en) | 2021-05-14 | 2021-05-14 | Specific primer for preparing PCR reaction system for detecting prenatal fetal 21-trisomy syndrome |
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