WO2020143044A1 - 一种双质控检测方法 - Google Patents

一种双质控检测方法 Download PDF

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WO2020143044A1
WO2020143044A1 PCT/CN2019/071419 CN2019071419W WO2020143044A1 WO 2020143044 A1 WO2020143044 A1 WO 2020143044A1 CN 2019071419 W CN2019071419 W CN 2019071419W WO 2020143044 A1 WO2020143044 A1 WO 2020143044A1
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quality control
tested
gene
detection method
site
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周双念
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深圳市双科生物科技有限公司
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Priority to PCT/CN2019/071419 priority Critical patent/WO2020143044A1/zh
Publication of WO2020143044A1 publication Critical patent/WO2020143044A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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

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  • the invention relates to the field of gene detection, in particular to a double quality control detection method.
  • the calibration and maintenance status of the testing equipment In the current genetic testing methods, the calibration and maintenance status of the testing equipment, the reliability of the testing methods in the experimental operation, the effectiveness of the reaction reagents, and the interference between the peaks after the nucleic acid reaction all affect the reliability of the testing results.
  • the object of the present invention is to provide a dual quality control detection method to ensure the qualification of the detection method and to eliminate the interference between the waveforms in the detection results.
  • a dual quality control detection method including:
  • judging whether the gene site to be tested is abnormal according to the expression state and the peak number of the primers at the quality control sites of each reaction well includes:
  • the quality control site primer is detected in each of the reaction well positions, it is determined whether the gene site to be tested is abnormal according to the number of peaks, otherwise, the current detection is ended.
  • the quality control site primer is a positive quality control specimen
  • the quality control site primer is detected at each of the reaction well positions includes: the detection result of each of the reaction well positions is positive.
  • determining whether the gene locus to be tested is abnormal according to the peak number includes:
  • the detection result is a peak, the gene site to be tested corresponding to the pore position is normal.
  • the two peaks corresponding to the abnormal gene locus are the quality control peak and the mutation peak, respectively, and the one peak corresponding to the normal gene locus is the quality control peak.
  • the number of primers at the quality control site is three.
  • the beneficial effect of the present invention lies in that the quality control site primer is used to judge whether the detection method is qualified according to the expression state of the quality control site primer, to avoid inaccurate results caused by the interference of various uncertain factors, and
  • the wild-type bases of multiple gene sites to be tested are cut off to facilitate the determination of the test results and ensure the reliability of the test results.
  • FIG. 1 is a flowchart of a dual quality control detection method provided by an embodiment of the present invention
  • FIG. 2 is a flowchart of determining whether a gene locus to be tested is abnormal according to the number of peaks provided by an embodiment of the present invention.
  • the dual quality control detection method provided by the embodiment of the present invention includes:
  • Step S101 Cut off the wild-type bases of multiple gene sites to be tested.
  • the wild type in the gene locus is removed so that the wild peak does not appear in the waveform of the detection result, to prevent misjudgment caused by the overlapping of the wild peak and the mutant peak in the detection result, and to avoid the risk of false positives.
  • Step S102 Configure a quality control site primer for each of the gene sites to be tested according to the sequence of each of the gene sites to be tested.
  • a quality control site primer is configured for each site to be tested to ensure that the sequence of the quality control site primer and the sequence of the gene site to be tested do not interfere with each other.
  • the quality control site primers are positive quality control specimens, and the number of quality control site primers is 3.
  • Step S103 Place a plurality of the gene sites to be tested and corresponding quality control point primers in corresponding reaction well positions, respectively, and perform a nucleic acid mass spectrometry reaction.
  • the gene site to be tested in each reaction well position is put into the corresponding reaction well position together with the corresponding quality control point primers to perform nucleic acid mass spectrometry reaction.
  • Step S104 judging whether the gene site to be tested is abnormal according to the expression state and the peak number of the primers at the quality control sites in each reaction well.
  • the detection method is qualified according to whether the quality control site primers at each well position are expressed. If a predetermined quality control site primer is not detected at a certain well, it means that the detection method is unqualified and the current test is ended . If the primers of preset quality control sites are detected in each well, that is, the test results of each well are positive, it means that the detection method is qualified, the nucleic acid mass spectrometry reaction equipment is normal and the operation method meets the standard, to avoid the detection results False negative risk. If the detection method is qualified, further determine whether the gene locus to be tested is abnormal according to the peak number.
  • Step S201 If the detection result is two peaks, the gene site to be tested corresponding to the pore position is abnormal.
  • abnormal gene loci will show wild peaks and mutant peaks. After removing the wild type from the gene locus to be tested, no wild peaks will appear.
  • the peak one of the peaks is the quality control peak that appears in the primers of the preset quality control site, and the other peak is the mutation peak, indicating that the gene site on the corresponding pore position is abnormal.
  • the genetic sample may be circulating tumor cell DNA extracted from blood or tissues, including deoxyribonucleic acid (DNA) and single nucleotide polymorphism (SNP).
  • DNA deoxyribonucleic acid
  • SNP single nucleotide polymorphism
  • Step S202 If the detection result is a peak, the gene site to be tested corresponding to the pore position is normal.
  • the peak number is used to determine whether the gene locus is abnormal, to avoid the false positive risk of the test result, and to ensure the accuracy and reliability of the test result.
  • the dual quality control detection method provided by the present invention judges whether the detection method is qualified according to the expression state of the quality control site primers through quality control site primers, avoids the inaccurate results caused by the interference of various uncertain factors, and simultaneously
  • the wild-type bases of the gene locus to be tested are clipped to facilitate the determination of the test results and ensure the reliability of the test results.

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  • Chemical & Material Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Wood Science & Technology (AREA)
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Abstract

提供一种双质控检测方法，用于检测基因位点，方法包括：将多个待测基因位点的野生型碱基剪除；根据各待测基因位点的序列为每个待测基因位点配置质控位点引物；将多个待测基因位点和对应的质控点引物分别放置在对应的反应孔位，并实施核酸质谱反应；根据各反应孔位质控位点引物的表达状态和峰值数量判断待测基因位点是否异常。提供的双质控检测方法，根据质控位点引物的表达状态判断检测方法是否合格，避免各种不确定因素的干扰导致的结果不准确，同时将多个待测基因位点的野生型碱基剪除，便于检测结果的判定，保证检测结果的可靠性。

Description

一种双质控检测方法 技术领域
本发明涉及基因检测领域，尤其涉及一种双质控检测方法。
背景技术
目前的基因检测方法中，检测设备的校准和维护状况、实验操作中检测方法的可靠性、反应试剂的有效性及核酸反应后波峰之间的干扰都会影响检测结果的可靠性。
发明内容
为了克服现有技术的不足，本发明的目的在于提供一种双质控检测方法，以保证检测方法的合格，且能排除检测结果中波形之间的干扰。
本发明的目的采用如下技术方案实现：
一种双质控检测方法，包括：
将多个待测基因位点的野生型碱基剪除；
根据各所述待测基因位点的序列为每个所述待测基因位点配置质控位点引物；
将多个所述待测基因位点和对应的质控点引物分别放置在对应的反应孔位，并实施核酸质谱反应；
根据各反应孔位质控位点引物的表达状态和峰值数量判断所述待测基因位点是否异常。
进一步地，根据各反应孔位质控位点引物的表达状态和峰值数量判断所述待测基因位点是否异常包括：
若每个所述反应孔位均检测到所述质控位点引物，则根据峰值数量判断待 测基因位点是否异常，否则，结束当前检测。
进一步地，所述质控位点引物为阳性质控标本，每个所述反应孔位均检测到所述质控位点引物包括：每个所述反应孔位的检测结果均为阳性。
进一步地，根据峰值数量判断待测基因位点是否异常包括：
若检测结果为两个波峰，则对应孔位的待测基因位点异常；
若检测结果为一个波峰，则对应孔位的待测基因位点正常。
进一步地，异常基因位点对应的两个波峰分别为质控峰和突变峰，正常基因位点对应的一个波峰为质控峰。
进一步地，所述质控位点引物的数量为3个。
相比现有技术，本发明的有益效果在于：通过质控位点引物，根据质控位点引物的表达状态判断检测方法是否合格，避免各种不确定因素的干扰导致的结果不准确，同时将多个待测基因位点的野生型碱基剪除，便于检测结果的判定，保证检测结果的可靠性。
附图说明
图1为本发明实施例提供的双质控检测方法流程图；
图2为本发明实施例提供的根据峰值数量判断待测基因位点是否异常流程图。
具体实施方式
下面，结合附图以及具体实施方式，对本发明做进一步描述，需要说明的是，在不相冲突的前提下，以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。
如图1所示，本发明实施例提供的双质控检测方法，包括：
步骤S101：将多个待测基因位点的野生型碱基剪除。
较佳的，去掉基因位点中的野生型，从而在检测结果的波形中不会出现野生峰，防止检测结果中野生峰与突变峰重叠造成的误判，避免假阳性风险。
步骤S102：根据各所述待测基因位点的序列为每个所述待测基因位点配置质控位点引物。
较佳的，根据每个待测基因位点的序列，为每个待测基于位点配置质控位点引物，确保质控位点引物的序列与待测基因位点的序列互不干扰。
较佳的，质控位点引物为阳性质控标本，质控位点引物的数量为3个。
步骤S103：将多个所述待测基因位点和对应的质控点引物分别放置在对应的反应孔位，并实施核酸质谱反应。
具体的，每个反应孔位的待测基因位点与对应的质控点引物一起放入对应的反应孔位，实施核酸质谱反应。
步骤S104：根据各反应孔位质控位点引物的表达状态和峰值数量判断所述待测基因位点是否异常。
较佳的，首先根据每个孔位的质控位点引物是否表达判断检测方法是否合格，若某个孔位没有检测到预设质控位点引物，则说明检测方法不合格，结束当前检测。若每个孔位均检测到预设质控位点引物，即每个孔位的检测结果均为阳性，则说明检测方法合格，核酸质谱反应设备正常且操作方法符合规范标准，避免检测结果出现假阴性风险。若检测方法合格，则进一步根据峰值数量判断待测基因位点是否异常。
根据峰值数量判断待测基因位点是否异常包括：
步骤S201：若检测结果为两个波峰，则对应孔位的待测基因位点异常。
具体的，由于正常的基因位点只会出现野生峰，异常的基因位点会出现野 生峰和突变峰，待测的基因位点去掉野生型后不会出现野生峰，若检测结果出现两个波峰，则其中一个波峰为预设质控位点引物出现的质控峰，另一个波峰为突变峰，则说明对应孔位上的基因位点异常。
若每个孔位的待测基因位点均来自同一个基因样本，若检测结果中有至少一个孔位出现两个波峰，则说明被分析的基因样本存在基因位点突变。基因样本可以是血液或组织中提取的循环肿瘤细胞DNA，包含脱氧核糖核酸(DNA)与单核苷酸多态性(SNP)。
步骤S202：若检测结果为一个波峰，则对应孔位的待测基因位点正常。
具体的，若只出现一个波峰，即为质控峰，说明对应孔位上的基因位点为正常基因。
剪除基因位点的野生型碱基后，根据峰值数量判断基因位点是否异常，避免检测结果出现假阳性风险，保证检测结果的准确性和可靠性。
本发明提供的双质控检测方法，通过质控位点引物，根据质控位点引物的表达状态判断检测方法是否合格，避免各种不确定因素的干扰导致的结果不准确，同时将多个待测基因位点的野生型碱基剪除，便于检测结果的判定，保证检测结果的可靠性。
上述实施方式仅为本发明的优选实施方式，不能以此来限定本发明保护的范围，本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。

Claims (6)

  1. 一种双质控检测方法，用于检测基因位点，其特征在于，方法包括：
    将多个待测基因位点的野生型碱基剪除；
    根据各所述待测基因位点的序列为每个所述待测基因位点配置质控位点引物；
    将多个所述待测基因位点和对应的质控点引物分别放置在对应的反应孔位，并实施核酸质谱反应；
    根据各反应孔位质控位点引物的表达状态和峰值数量判断所述待测基因位点是否异常。
  2. 根据权利要求1所述的双质控检测方法，其特征在于，根据各反应孔位质控位点引物的表达状态和峰值数量判断所述待测基因位点是否异常包括：
    若每个所述反应孔位均检测到所述质控位点引物，则根据峰值数量判断待测基因位点是否异常，否则，结束当前检测。
  3. 根据权利要求2所述的双质控检测方法，其特征在于，所述质控位点引物为阳性质控标本，每个所述反应孔位均检测到所述质控位点引物包括：每个所述反应孔位的检测结果均为阳性。
  4. 根据权利要求2所述的双质控检测方法，其特征在于，根据峰值数量判断待测基因位点是否异常包括：
    若检测结果为两个波峰，则对应孔位的待测基因位点异常；
    若检测结果为一个波峰，则对应孔位的待测基因位点正常。
  5. 根据权利要求4所述的双质控检测方法，其特征在于，异常基因位点对应的两个波峰分别为质控峰和突变峰，正常基因位点对应的一个波峰为质控峰。
  6. 根据权利要求1所述的双质控检测方法，其特征在于，所述质控位点引物 的数量为3个。
PCT/CN2019/071419 2019-01-11 2019-01-11 一种双质控检测方法 WO2020143044A1 (zh)

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