CN116287412B - 一种基于二代测序开发用于三七药材真伪检测鉴定的引物、探针及其检测方法 - Google Patents
一种基于二代测序开发用于三七药材真伪检测鉴定的引物、探针及其检测方法 Download PDFInfo
- Publication number
- CN116287412B CN116287412B CN202310322085.4A CN202310322085A CN116287412B CN 116287412 B CN116287412 B CN 116287412B CN 202310322085 A CN202310322085 A CN 202310322085A CN 116287412 B CN116287412 B CN 116287412B
- Authority
- CN
- China
- Prior art keywords
- pseudo
- ginseng
- probe
- primer
- notog
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000000523 sample Substances 0.000 title claims abstract description 64
- 244000131316 Panax pseudoginseng Species 0.000 title claims abstract description 43
- 235000003181 Panax pseudoginseng Nutrition 0.000 title claims abstract description 43
- 238000001514 detection method Methods 0.000 title claims abstract description 29
- 239000000463 material Substances 0.000 title claims abstract description 10
- 238000012163 sequencing technique Methods 0.000 title claims abstract description 10
- 238000011161 development Methods 0.000 title description 4
- 238000003753 real-time PCR Methods 0.000 claims abstract description 13
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 20
- 230000003321 amplification Effects 0.000 claims description 16
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 16
- 239000012634 fragment Substances 0.000 claims description 6
- 241000196324 Embryophyta Species 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 238000011529 RT qPCR Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000007400 DNA extraction Methods 0.000 claims description 2
- 238000000137 annealing Methods 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 11
- 241000180649 Panax notoginseng Species 0.000 description 14
- 235000003143 Panax notoginseng Nutrition 0.000 description 14
- 239000013612 plasmid Substances 0.000 description 14
- 108020004414 DNA Proteins 0.000 description 11
- 238000012795 verification Methods 0.000 description 11
- 235000003140 Panax quinquefolius Nutrition 0.000 description 9
- 241000208340 Araliaceae Species 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 6
- 235000008434 ginseng Nutrition 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 240000005373 Panax quinquefolius Species 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 208000002193 Pain Diseases 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 210000003763 chloroplast Anatomy 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000003147 molecular marker Substances 0.000 description 2
- 230000036407 pain Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 235000002791 Panax Nutrition 0.000 description 1
- 241000208343 Panax Species 0.000 description 1
- 206010042674 Swelling Diseases 0.000 description 1
- 208000005652 acute fatty liver of pregnancy Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 208000031169 hemorrhagic disease Diseases 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 1
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 230000008736 traumatic injury Effects 0.000 description 1
Classifications
-
- 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
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- 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
- C12Q1/6869—Methods for sequencing
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Botany (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及一种基于二代测序开发用于三七药材真伪检测鉴定的引物、探针及其检测方法,用于三七检测的上下游引物序列如SEQ ID NO.1和2所示,探针序列如SEQ ID NO.3所示,探针的5’端修饰VIC,3’端修饰MGB。用于三七的实时荧光定量PCR检测,本发明具有特异性强,灵敏度高等优点。
Description
技术领域
本发明属于生物技术技术领域,具体的说,涉及一种基于二代测序开发用于三七药材真伪检测鉴定的引物、探针及其检测方法。
背景技术
三七(Panax notoginseng),为五加科人参属植物。具有化瘀止血,活血定痛的功效。主治出血症,跌打损伤,瘀血肿痛。
三七类中药的传统鉴定方法为基原鉴定、性状鉴定、显微鉴定和理化鉴定。传统鉴定方法的理论基础建立于分类群的性状特征分析,这些性状特征是与环境紧密相关的表现型。从分子遗传学角度来看,物种表现型的差异归根结底应追溯到基因型的差异,即在DNA序列上的差异。因此,对基因组序列差异的比较研究无疑为植物分类和鉴定提供了本质依据。随着生命科学不断取得重大突破,分子生物学鉴定方法应运而生,此类方法应用DNA分子标记技术鉴定中药原植物及其药材和饮片,取得了快速发展。DNA分子标记鉴别应用于参类药材鉴别已有不少的报道,RFLP、SSR、RAPD、AP-PCR、AFLP、ISSR、SNP等技术相继应用于参类药材的鉴别鉴定。然而,这些技术由于存在着操作步骤繁琐、工作量大、实验结果重复性差等弱点,难以在实际中得到应用而逐渐退出舞台。随着分子生物学的发展和基因组时代的到来,一些新的技术为参类药材的鉴定提供了新的方法。
实时荧光PCR技术在物种鉴定上已经是非常成熟与成功的技术,使用一组或多组特异性的引物探针,通过监测荧光强度变化达到鉴定的目的,该方法学不仅具备特异性强、灵敏度高,操作简单等优势,还具有较高的检测效率。运用实时荧光PCR技术鉴定三七,不仅特异性强,灵敏度高,结果准确快速,更具有良好的市场应用前景和推广应用价值。
发明内容
为了克服背景技术中存在的问题,本发明提供了一种基于二代测序开发用于三七药材真伪检测鉴定的引物、探针及其检测方法。
为实现上述目的,本发明是通过如下技术方案实现的:
一种基于二代测序开发用于三七检测的引物、探针,用于三七检测的上下游引物序列如SEQ ID NO.1和2所示,探针序列如SEQ ID NO.3所示,探针的5’端修饰VIC,3’端修饰MGB。
本发明还提供一种基于二代测序开发用于三七检测的引物、探针的检测方法,包含以下步骤:
S1,使用植物基因组DNA提取试剂盒提取待测样品的基因组DNA;
S2,使用S1步骤提取的待测样品的基因组DNA作为待测模板,并使用如权利要求1所示的引物、探针对待测模板进行实时荧光定量PCR扩增反应;
S3,待测样品中是否含有三七基因组特有DNA片段的判断方法如下:依据CT值来判断待测样品中是否含有三七基因组特有DNA片段,CT值小于40则判断为阳性,样品CT值大于40或无任何数值则判断为阴性。
进一步的,所述S2步骤中实时荧光定量PCR扩增的反应体系为:2xT5 Fast qPCRMix 10μL、10uM的上游引物0.7μL、10uM的下游引物0.7μL、10uM的探针0.6μL、待测模板1μL,使用ddH2O补足至20μL反应体系。
进一步的,所述S2步骤中实时荧光定量PCR扩增程序为:95℃1min,一个循环;95℃15s,60℃1min,40个循环;在60℃退火延伸阶段采集对应荧光1min。
本发明还提供用于三七检测的引物、探针在制备三七检测试剂盒中的应用。
本发明的有益效果:本发明通过对三七叶绿体基因组的序列比对分析,筛选出具有一定特异性和灵敏度的TaqMan-MGB荧光探针,使探针可以特异性的检测目的基因片段的CT值,从而实现对三七的快速鉴定。
附图说明
图1是本发明中实时荧光定量PCR的标准曲线和相关扩增曲线图;
图2是本发明中引物探针特异性验证试验扩增曲线图;
图3是本发明中引物探针灵敏度验证试验扩增曲线图。
具体实施方式
为了使本发明的目的、技术方案和有益效果更加清楚,下面将对本发明的优选实施例进行详细的说明,以方便技术人员理解。
实施例一、引物对和探针的设计
设计:基于三七叶绿体基因组DNA,利用分子生物学软件进行引物和探针设计。
引物和探针序列如下表1所示:
表1Panax notoginseng三七的引物探针序列
反应体系:
扩增程序:
实施例二、三七标准质粒的制备和探针标准曲线的创建
1、申请人委托生物公司提取三七的基因组DNA,根据实施例一的引物序列、反应体系和扩增程序,进行PCR扩增,获得PCR产物。
2、根据PCR产物制备阳性克隆样品,然后进行测序,选择测序结果中序列完全正确的阳性克隆样品,使用质粒提取试剂盒完成质粒的提取。
3、运用公式:C=A·B-1×6.02×1014(其中A代表质粒浓度ng·μL-1,B代表质粒DNA分子量,C代表copies·μL-1)计算出质粒浓度拷贝数,获得母液浓度:三七标准质粒(2.53*1010copies·μL-1)作为实验质粒标准品使用。
4、使用溶剂将三七质粒标准品稀释至2.53*108copies·μL-1,再按10倍浓度将其稀释为5个梯度。分别以稀释过的质粒标准品为模板,在前述反应条件下进行TaqMan RT-qPCR检测,每组试验重复3次,制定标准曲线。三七质粒标准品检测结果如表2所示,标准曲线和相关扩增曲线如图1所示。
三七标准曲线方程:y=-3.8916x+49.666,R2=0.9996;
且标准曲线的相关系数(R2)大于0.98。
表2Panax notoginseng三七质粒标准品检测结果
标准质粒 | 碱基数 | 分子量 | 浓度(ng/μL) | 拷贝数(copies/μL) |
6-P.notog | 2038 | 1345080 | 56.555 | 2.53E+10 |
稀释倍数 | 基因 | 拷贝数 | 拷贝数log值 | CT值平均值 |
10*2 | 6-P.notog | 2.53E+08 | 8.403319383 | 16.965 |
10*2 | 6-P.notog | 2.53E+08 | 8.403319383 | 16.965 |
10*2 | 6-P.notog | 2.53E+08 | 8.403319383 | 16.965 |
10*3 | 6-P.notog | 2.53E+07 | 7.403319383 | 20.727 |
10*3 | 6-P.notog | 2.53E+07 | 7.403319383 | 20.727 |
10*3 | 6-P.notog | 2.53E+07 | 7.403319383 | 20.727 |
10*4 | 6-P.notog | 2.53E+06 | 6.403319383 | 24.911 |
10*4 | 6-P.notog | 2.53E+06 | 6.403319383 | 24.911 |
10*4 | 6-P.notog | 2.53E+06 | 6.403319383 | 24.911 |
10*5 | 6-P.notog | 2.53E+05 | 5.403319383 | 28.689 |
10*5 | 6-P.notog | 2.53E+05 | 5.403319383 | 28.689 |
10*5 | 6-P.notog | 2.53E+05 | 5.403319383 | 28.689 |
10*6 | 6-P.notog | 2.53E+04 | 4.403319383 | 32.442 |
10*6 | 6-P.notog | 2.53E+04 | 4.403319383 | 32.442 |
10*6 | 6-P.notog | 2.53E+04 | 4.403319383 | 32.442 |
实施例三、实时荧光PCR检测三七样品的方法
1、根据实施例一配制反应体系和设置扩增程序。
2、检测过程除了样品外,设置阳性对照、阴性对照,阳性对照为:质粒标准品,阴性对照为:ddH2O。
3、在阳性对照、阴性对照结果均正常的情况下,依据CT值来判断待测样品中是否含有三七基因组特有DNA片段,CT值小于40则判断为阳性,样品CT值大于40或无任何数值则判断为阴性。
实施例四、引物探针特异性验证
验证内容:特定引物探针分别对七个近缘种进行扩增,每个样本重复3次;结果如表3所示,扩增曲线如图2所示。
验证目的:验证引物探针的特异性;
表3Panaxnotoginseng三七引物探针特异性检测结果
Sample Name | Target Name | Reporter | Quencher | CT | Ct Mean | Ct SD |
NC | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
NC | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
NC | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
人参 | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
人参 | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
人参 | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
西洋参 | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
西洋参 | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
西洋参 | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
姜状三七 | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
姜状三七 | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
姜状三七 | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
狭叶竹节参 | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
狭叶竹节参 | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
狭叶竹节参 | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
珠子参 | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
珠子参 | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
珠子参 | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
三七 | 6-P.notog | VIC | NFQ-MGB | 21.679 | 21.627 | 0.084 |
三七 | 6-P.notog | VIC | NFQ-MGB | 21.531 | 21.627 | 0.084 |
三七 | 6-P.notog | VIC | NFQ-MGB | 21.672 | 21.627 | 0.084 |
屏边三七 | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
屏边三七 | 6-P.notog | VIC | NFQ-MGB | Undetermined | ||
屏边三七 | 6-P.notog | VIC | NFQ-MGB | Undetermined |
结果:如上表3所示,Panax notoginseng三七引物探针只与三七种有扩增与其它种无扩增反应,特异性较好,3个重复CT值接近,重复性较好。
实施例五、引物探针灵敏度验证
验证内容:对应样本DNA浓度从0.00001ng/μL、0.0001ng/μL、0.001ng/μL、0.01ng/μL、0.1ng/μL、1ng/μL,6个浓度梯度测试对应引物探针的灵敏度,每个样本重复三次,并使用实时荧光定量PCR仪进行测定;检测结果如表4和图3所示。
验证目的:验证在反应体系下Panax notoginseng三七种引物探针的最低检测下限即灵敏度。
QuantStudio Dx检测结果(灵敏度)
表4 Panax notoginseng三七种引物探针灵敏度:0.0001ng/μL
Sample Name | Target Name | Task | Reporter | Quencher | Cт | CтMean | CтSD |
1 | 6-P.notog | UNKNOWN | VIC | NFQ-MGB | 21.901 | 21.925 | 0.110 |
1 | 6-P.notog | UNKNOWN | VIC | NFQ-MGB | 22.046 | 21.925 | 0.110 |
1 | 6-P.notog | UNKNOWN | VIC | NFQ-MGB | 21.829 | 21.925 | 0.110 |
0.1 | 6-P.notog | UNKNOWN | VIC | NFQ-MGB | 25.904 | 25.736 | 0.209 |
0.1 | 6-P.notog | UNKNOWN | VIC | NFQ-MGB | 25.802 | 25.736 | 0.209 |
0.1 | 6-P.notog | UNKNOWN | VIC | NFQ-MGB | 25.503 | 25.736 | 0.209 |
0.01 | 6-P.notog | UNKNOWN | VIC | NFQ-MGB | 29.516 | 29.666 | 0.154 |
0.01 | 6-P.notog | UNKNOWN | VIC | NFQ-MGB | 29.659 | 29.666 | 0.154 |
0.01 | 6-P.notog | UNKNOWN | VIC | NFQ-MGB | 29.823 | 29.666 | 0.154 |
0.001 | 6-P.notog | UNKNOWN | VIC | NFQ-MGB | 33.556 | 33.649 | 0.355 |
0.001 | 6-P.notog | UNKNOWN | VIC | NFQ-MGB | 33.349 | 33.649 | 0.355 |
0.001 | 6-P.notog | UNKNOWN | VIC | NFQ-MGB | 34.040 | 33.649 | 0.355 |
0.0001 | 6-P.notog | UNKNOWN | VIC | NFQ-MGB | 35.678 | 36.857 | 1.023 |
0.0001 | 6-P.notog | UNKNOWN | VIC | NFQ-MGB | 37.505 | 36.857 | 1.023 |
0.0001 | 6-P.notog | UNKNOWN | VIC | NFQ-MGB | 37.388 | 36.857 | 1.023 |
0.00001 | 6-P.notog | UNKNOWN | VIC | NFQ-MGB | 37.449 | 38.417 | 1.369 |
0.00001 | 6-P.notog | UNKNOWN | VIC | NFQ-MGB | 39.386 | 38.417 | 1.369 |
0.00001 | 6-P.notog | UNKNOWN | VIC | NFQ-MGB | Undetermined | 38.417 | 1.369 |
如表4和图3所示,本发明记载的引物探针灵敏度较高。
实施例六、引物探针重复性验证
分别从以下两个方面验证:
1.同实验员同实时荧光定量PCR仪(QuantStudio Dx)不同批次酶之间比较;
2.同批次酶同实时荧光定量PCR仪(QuantStudio Dx)不同人员操作;
验证内容:每个样本重复3次,通过计算变异系数(CV%)值来进行重复性评价。计算公式:cv=sd(标准偏差)/mean(平均值)×100%。
验证目的:验证重复性、人员操作性、不同批次聚合酶的稳定性。
表5 同实验员同QPCR仪(QuantStudio Dx)不同批次酶结果
表6 同批次酶同QPCR仪(QuantStudio Dx)不同人员操作
结果:据表5-6所示,同实验员同实时荧光定量PCR仪(QuantStudio Dx)不同批次酶之间比较;同批次酶同实时荧光定量PCR仪(QuantStudio Dx)不同人员操作;实验结果:重复性CV%均小于3%,实验重复性好,不同批次聚合酶稳定性优良。
最后说明的是,以上优选实施例仅用于说明本发明的技术方案而非限制,尽管通过上述优选实施例已经对本发明进行了详细的描述,但本领域技术人员应当理解,可以在形式上和细节上对其做出各种各样的改变,而不偏离本发明权利要求书所限定的范围。
Claims (5)
1.一种基于二代测序开发用于三七药材真伪检测鉴定的引物探针组合物,其特征在于:用于三七检测的上下游引物序列如SEQ ID NO.1和2所示,探针序列如SEQ ID NO.3所示,探针的5’端修饰VIC,3’端修饰MGB。
2.使用如权利要求1所述的引物探针组合物检测鉴定三七真伪的方法,其特征在于:包含以下步骤:
S1,使用植物基因组DNA提取试剂盒提取待测样品的基因组DNA;
S2,使用S1步骤提取的待测样品的基因组DNA作为待测模板,并使用如权利要求1所示的引物、探针对待测模板进行实时荧光定量PCR扩增反应;
S3,待测样品中是否含有三七基因组特有DNA片段的判断方法如下:依据CT值来判断待测样品中是否含有三七基因组特有DNA片段,CT值小于40则判断为阳性,样品CT值大于40或无任何数值则判断为阴性。
3.根据权利要求2所述的方法,其特征在于:所述S2步骤中实时荧光定量PCR扩增的反应体系为:2xT5 Fast qPCR Mix 10μL、10uM的上游引物0.7μL、10uM的下游引物0.7μL、10uM的探针0.6μL、待测模板1μL,使用ddH2O补足至20μL反应体系。
4.根据权利要求2所述的方法,其特征在于:所述S2步骤中实时荧光定量PCR扩增程序为:95℃1min,一个循环;95℃15s,60℃1min,40个循环;在60℃退火延伸阶段采集对应荧光1min。
5.如权利要求1所述的引物探针组合物在制备三七检测试剂盒中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310322085.4A CN116287412B (zh) | 2023-03-29 | 2023-03-29 | 一种基于二代测序开发用于三七药材真伪检测鉴定的引物、探针及其检测方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310322085.4A CN116287412B (zh) | 2023-03-29 | 2023-03-29 | 一种基于二代测序开发用于三七药材真伪检测鉴定的引物、探针及其检测方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116287412A CN116287412A (zh) | 2023-06-23 |
CN116287412B true CN116287412B (zh) | 2023-11-03 |
Family
ID=86834174
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310322085.4A Active CN116287412B (zh) | 2023-03-29 | 2023-03-29 | 一种基于二代测序开发用于三七药材真伪检测鉴定的引物、探针及其检测方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116287412B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116287148B (zh) * | 2023-05-24 | 2023-08-15 | 云南珩柯生物科技有限公司 | 鉴别珠子参的方法、引物、探针及其应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103484558A (zh) * | 2013-10-16 | 2014-01-01 | 云南农业大学 | 滇重楼的分子鉴定方法 |
KR101919605B1 (ko) * | 2017-10-16 | 2018-11-19 | 서울대학교산학협력단 | 인삼 속 5종의 엽록체 게놈 서열의 완전 해독 기반 종간 구별 마커와 프라이머 세트 및 이의 용도 |
-
2023
- 2023-03-29 CN CN202310322085.4A patent/CN116287412B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103484558A (zh) * | 2013-10-16 | 2014-01-01 | 云南农业大学 | 滇重楼的分子鉴定方法 |
KR101919605B1 (ko) * | 2017-10-16 | 2018-11-19 | 서울대학교산학협력단 | 인삼 속 5종의 엽록체 게놈 서열의 완전 해독 기반 종간 구별 마커와 프라이머 세트 및 이의 용도 |
Non-Patent Citations (5)
Title |
---|
A chloroplast genomic strategy for designing taxon specific DNA mini-barcodes: a case study on ginsengs;Wenpan Dong et al.;《BMC Genetics》;文献号:138 * |
Diversity and evolution of major Panax species revealed by scanning the entire chloroplast intergenic spacer sequences;Jun Ha Kim et al.;《Genetic Resources and Crop Evolution》;第60卷;图3,补充数据表1 * |
Panax notoginseng chloroplast, complete genome, REGION: 1757..6355;GenBank: KP036468.1;《GenBank》;"gene=trnK-UUU","gene=rps16"部分 * |
Phylogenetic relationship in the genus Panax: inferred from chloroplast trnK gene and nuclear 18S rRNA gene sequences;Shu Zhu et al.;《Planta Med》;第647-653页 * |
基于cpDNA分子标记的三七居群遗传多样性研究;韩岩;《中国优秀硕士学位论文全文数据库 农业科技辑》;第D047-774页 * |
Also Published As
Publication number | Publication date |
---|---|
CN116287412A (zh) | 2023-06-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN116287412B (zh) | 一种基于二代测序开发用于三七药材真伪检测鉴定的引物、探针及其检测方法 | |
CN112029891A (zh) | 一种快速鉴定伊犁贝母的特异性核酸探针、方法及用途 | |
CN116426680A (zh) | 一种基于二代测序开发用于人参药材真伪检测的引物、探针及其检测方法 | |
CN111057782A (zh) | 一种基于实时荧光定量pcr检测土壤细菌数量的方法 | |
CN116334188B (zh) | 鉴别屏边三七的方法、引物、探针及其应用 | |
CN116287149B (zh) | 鉴别狭叶竹节参的方法、引物、探针及其应用 | |
CN117448430A (zh) | 一种用于鉴别川牛膝、麻牛膝及其自然杂交种的hrm方法及鉴别方法 | |
CN105603081B (zh) | 一种非诊断目的的肠道微生物定性与定量的检测方法 | |
CN116287424B (zh) | 一种用于姜状三七检测的引物、探针及其检测方法 | |
CN117925904B (zh) | 鉴别滇重楼遗传纯合个体的方法、引物、探针及应用 | |
CN117925902B (zh) | 鉴别李氏重楼的方法、引物、探针及应用 | |
CN117965799A (zh) | 鉴别滇重楼杂交个体的方法、引物、探针及应用 | |
CN106755392B (zh) | 藻类培养中腔轮虫的快速定量检测qPCR方法 | |
CN116287148B (zh) | 鉴别珠子参的方法、引物、探针及其应用 | |
CN116004902B (zh) | 检测三七根腐病致病菌茄腐镰刀菌的引物组 | |
CN111073994A (zh) | 一种快速鉴别柴胡属种子的as-pcr方法及应用 | |
CN117230247B (zh) | 鉴别人参属属级植物的方法、试剂和应用 | |
CN105002166A (zh) | 褐斑大蠊分子标准样品及其制备方法 | |
CN114634992B (zh) | 一种暗紫贝母的Indel标记及其应用 | |
CN116287411A (zh) | 一种基于二代测序开发用于西洋参药材真伪检测的引物、探针及其检测方法 | |
CN117265177B (zh) | 一种鉴别蜀羊泉的引物探针组及实时荧光pcr鉴别方法 | |
CN117757979B (zh) | 一种用于鉴定大豆品种的引物组、试剂盒及鉴定方法 | |
CN114182002A (zh) | 一种检测中成药中三七的方法及应用 | |
CN116200526A (zh) | 基于分子生物学方法鉴别亳菊的特异性引物及亳菊的鉴定方法 | |
CN116334283A (zh) | 鉴定姜黄属植物的引物、试剂盒和鉴定方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
OL01 | Intention to license declared | ||
OL01 | Intention to license declared |