CN105219864A - A kind of genotype tests method - Google Patents
A kind of genotype tests method Download PDFInfo
- Publication number
- CN105219864A CN105219864A CN201510698148.1A CN201510698148A CN105219864A CN 105219864 A CN105219864 A CN 105219864A CN 201510698148 A CN201510698148 A CN 201510698148A CN 105219864 A CN105219864 A CN 105219864A
- Authority
- CN
- China
- Prior art keywords
- pcr
- gene
- internal reference
- probe
- dabcyl3
- 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.)
- Pending
Links
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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of genotype tests method, comprise the steps: that testing gene adds in PCR reaction solution by (1) to form mixed solution and be enclosed in the enterprising performing PCR amplified reaction of PCR Sptting plate, amplimer and the specific probe of testing gene is comprised in described PCR reaction solution, and internal reference primer and internal reference probe, 5 ' end fluorophor of described specific probe and described internal reference probe marks and 3 ' end quenching group marks, the background fluorescence value of described mixed solution is read before described pcr amplification reaction, reacted fluorescent value is read after pcr amplification reaction terminates, (2) described reacted fluorescent value is deducted described background fluorescence value, if increment is more than or equal to 90%, then judge that described testing gene is as the positive, if increment is less than 90%, judge that described testing gene is as feminine gender.Method of the present invention simple, convenience very, the risk that PCR reacts after stain is rejected completely and is eliminated the possibility that SSP visual inspection produces error.
Description
Technical field
The present invention relates to gene engineering technology field, particularly relate to a kind of genotype tests method.
Background technology
The authentication method of the abo blood group that current clinical labororatory is the most frequently used is serology hemagglutination test and micro-column gel experiment, and the genotype detection of abo blood group is used for the clinical case of ABO typing discrepan-cy and the qualification etc. of difficult blood group in addition.Hemagglutination test refers to that macroscopic agglutination reaction occurs in liquid medium for antibody and red corpuscle.Slide method, test tube method and full-automatic microplate method is divided into again according to the difference of operation.Blood clotting method belongs to prescreening method, but often can not provide sentence read result to the ABO blood sample of general weak expression and the suspicious blood sample of aggegation.Micro-column gel agglutination assay (MGT) adopt gel technique to carry out sensitivity and specificity that antiglobulin test can improve test.But red blood cell concentration is too low or too high and centrifugal thoroughly, in serum cell " grumeleuse ", bacterial contamination etc. do not appear in fibre-bearing albumen, may cause MGT false negative or false positive.For ABO based on common at present mainly primer specificity amplification (SSP) of the gene test of molecular level then gel electrophoresis observe band, but the follow-up gel electrophoresis step of the method finally needs Visual observations, increase the risk of human error, and because electrophoresis of uncapping also increases the risk of laboratory pollution.
The mainly lymphocytotoxicity experiment of the allelic detection method of current HLA-B27, the methods such as primer specificity amplification and flow cytomery.The method of lymphocytotoxicity experiment due to affinity of antibody more weak, tire low, easily produce cross reaction and had a strong impact on the reliability of genotyping result.Primer specific amplification technique i.e. traditional SSP method, although be the typing method based on molecular level, because manipulation require electrophoresis observation bar brings judgement, complicated operation and personal errors is large.Adopt Flow Cytometry cost higher, need to wash plate etc., complicated operation, and need batch operation just meaningful.Be not suitable for the needs of disease quick diagnosis.
Summary of the invention
In order to make up above-mentioned the deficiencies in the prior art, the present invention proposes a kind of genotype tests method.
Technical problem of the present invention is solved by following technical scheme:
A kind of genotype tests method, comprises the steps:
(1) testing gene is added in PCR reaction solution and form mixed solution and be enclosed in the enterprising performing PCR amplified reaction of PCR Sptting plate, the amplimer of testing gene and specific probe and internal reference primer and internal reference probe is comprised in described PCR reaction solution, 5 ' end fluorophor of described specific probe and described internal reference probe marks and 3 ' end quenching group marks, before described pcr amplification reaction, read the background fluorescence value of described mixed solution, after pcr amplification reaction terminates, read reacted fluorescent value;
(2) described reacted fluorescent value is deducted described background fluorescence value, if increment is more than or equal to 90%, then judge that described testing gene is as the positive, if increment is less than 90%, judge that described testing gene is as feminine gender.
The beneficial effect that the present invention is compared with the prior art is: reaction of the present invention is enclosed on PCR Sptting plate to carry out, all carry out in this environment closed with the work of reacted fluorescent value digital independent before reaction, the risk that PCR reacts after stain is rejected completely, whole operation simple, convenience very, carry out somatotype by the fluorescent value increment before and after reaction to testing gene, such qualitative analysis eliminates the possibility that the visual inspection of SSP technology produces error.
Embodiment
Below in conjunction with preferred embodiment the invention will be further described.
The invention provides a kind of genotype tests method, comprise the steps: in a specific embodiment
(1) testing gene is added in PCR reaction solution and form mixed solution and be enclosed in the enterprising performing PCR amplified reaction of PCR Sptting plate, the amplimer of testing gene and specific probe and internal reference primer and internal reference probe is comprised in described PCR reaction solution, 5 ' end fluorophor of described specific probe and described internal reference probe marks and 3 ' end quenching group marks, before described pcr amplification reaction, read the background fluorescence value of described mixed solution, after pcr amplification reaction terminates, read reacted fluorescent value; (2) described reacted fluorescent value is deducted described background fluorescence value, if increment is more than or equal to 90%, then judge that described testing gene is as the positive, if increment is less than 90%, judge that described testing gene is as feminine gender.
Wherein, the fluorophor on the fluorophor on specific probe described in each and described internal reference probe is all different, and fluorophor can be selected from following group: FAM, HEX, TET, ROX, TAMRA and CY5; Quenching group can be Dabcyl.
Below by way of embodiment more specifically, the present invention is described in detail.
Embodiment one: testing gene is O1, O2, B, A, A2 genotype of ABO gene
A () extracts genomic dna in blood sample
Use the whole blood DNA of German inno-train company to extract test kit and extract DNA, specific operation process is undertaken by its specification sheets.
(b) design of amplification primers
ABO gene order to be detected is found out from GenBank, by the MegAlign function contrast sequence of Lasergene software, universal amplification primer is designed in the various parting gene of ABO, then Blast amplimer being put into NCBI carries out the specific assay increased, to guarantee that it carries out specific amplification to each somatotype of ABO.Design internal reference primer simultaneously.General amplimer and the internal reference primer of design see the following form 1-1, and wherein F represents forward primer, and R represents reverse primer.
(c) pcr amplification
In order to make PCR primer better hybridize with probe, the pcr amplification reaction in this example adopts asymmetric amplification, that is: the consumption of downstream primer is greater than upstream primer, makes pcr amplification product major part be minus strand product; Correspondingly, specific probe is designed to normal chain probe, just can hybridize with this minus strand product.
(d) designing probe
In the amplification region of primer, designing probe, should ensure the specificity of probe.The each genotypic specific probe of ABO and internal reference probe hold the fluorophor adopting different wave length to mark 5 ', and 3 ' end adopts quenching group mark, to read plate instrument reading respective fluorescent value respectively.The probe of design is as shown in following table 1-2:
Wherein: FAM, HEX, TET, ROX, TAMRA, CY5 are fluorophor, Dabcyl is quenching group.
The mixed solution of (e) configuration PCR reaction
Added by testing gene in PCR reaction solution and form mixed solution, mixed solution is divided on PCR Sptting plate with 40 μ l/ pipes, and the essentially consist of the mixed solution often in pipe is as shown in following table 1-3:
Table 1-3
| Reagent | Every person-portion consumption (μ L) |
| Distilled water | 4.8 |
| 10×PCR Buffer | 15 |
| 25mM MgCl 2 | 2 |
| The each dNTP of 2.5mM | 4 |
| 10 μMs of primer GF | 0.1 |
| 10 μMs of primer GR | 1 |
| 10 μMs of primer I F | 0.1 |
| 10 μMs of primer I R | 1 |
| 10 μMs of probe PO1 | 1 |
| 10 μMs of probe PO2 | 1 |
| 10 μMs of probe PB | 1 |
| 10 μMs of probe PA | 1 |
| 10 μMs of probe PA2 | 1 |
| 10 μMs of probe PI | 1 |
| 5U/ μ L HotstarTaq enzyme | 1 |
| The DNA sample of testing gene | 5 |
| Total amount | 40 |
By the adhesive transparent ParafilmTM of PCR Sptting plate one side, under this enclosed environment, read the background fluorescence value before reaction.
(f) PCR reaction process
96 DEG C of denaturations 15 minutes; 95 DEG C of sex change 15 seconds, 65 DEG C of renaturation 20 seconds, 72 DEG C extend 20 seconds, circulate 30 times; 72 DEG C extend 5 minutes again, and along with the increase of amplified production, the fluorophor discharged in system becomes positive correlation to increase, until reacted, after having reacted, read reacted fluorescent value.Reacted fluorescent value is deducted background fluorescence value, if increment is more than or equal to 90%, then judges that testing gene is as the positive, if increment is less than 90%, be judged to be feminine gender.
Adopt the method in above embodiment to carry out ABO gene type by different DNA sample, its result is as shown in following table 1-4:
Table 1-4
| Detect target | Fluorescent mark | Background fluorescence value before reaction | Reacted fluorescent value | Result judges |
| O1 | FAM | 61 | 209 | O1 |
| O2 | HEX | 56 | 190 | O2 |
| B | TET | 60 | 278 | B |
| A | TAMRA | 54 | 240 | A |
| A2 | ROX | 63 | 290 | A2 |
| Internal reference | CY5 | 54 | 230 | Normally |
Detect 435 increments originally with the SSP somatotype detected through gel electrophoresis decision method of existing maturation, coincidence rate is 100% (P<0.05), and this detected result by method of the present invention of this 435 increment is as shown in following table 1-5 simultaneously:
Table 1-5:
Based on the probe of the luminophore mark of the general ABO glycosyltransferase gene amplimer of particular design and improvement and six kinds of different wave lengths in embodiment one, the ABO genotype identification to sample is realized in a tube reaction system, the test kit that the method is made is a complete system, by primer, probe, the compositions such as reaction buffer, only need other sampling and water in systems in which, read fluorescence values respectively afterwards before the reaction, then carry out numerical value contrast conting and can realize genotype identification, the present embodiment eliminates the electrophoresis step needed for conventional molecular biological ABO genotype identification, make whole technological operation more accurate, convenient, fast, greatly simplifie manually-operated flow process, only about 90 minutes can be needed from collected specimens to result, and all carry out in the environment closed with the work of reacted fluorescent value digital independent before reaction, therefore the risk of PCR reaction after stain is rejected completely, result is also analyzed and is eliminated the possibility that SSP visual inspection produces error.
Embodiment two: testing gene is HLA-B27 gene
A () extracts genomic dna in blood sample
Use the whole blood DNA of German inno-train company to extract test kit and extract DNA, specific operation process is undertaken by its specification sheets.
(b) design of amplification primers
HLA-B27 gene order to be detected is found out from GenBank, by the MegAlign function contrast sequence of Lasergene software, design of amplification primers in the various parting gene of HLA-B27, then Blast amplimer being put into NCBI carries out the specific assay increased, to guarantee that it carries out specific amplification to HLA-B27.Design internal reference primer simultaneously.Amplimer and the internal reference primer of design see the following form 2-1, and wherein F represents forward primer, and R represents reverse primer.
(c) pcr amplification
In order to make PCR primer better hybridize with probe, the pcr amplification reaction in this example adopts asymmetric amplification, that is: the consumption of downstream primer is greater than upstream primer, makes pcr amplification product major part be minus strand product; Correspondingly, specific probe is designed to normal chain probe, just can hybridize with this minus strand product.
(d) designing probe
In the amplification region of primer, designing probe, should ensure the specificity of probe.The specific probe of HLA-B27 gene and internal reference probe hold the fluorophor adopting different wave length to mark 5 ', and 3 ' end adopts quenching group mark, to read plate instrument reading respective fluorescent value respectively.The probe of design is as shown in following table 2-2:
Wherein: FAM, HEX are fluorophor, Dabcyl is quenching group.
The mixed solution of (e) configuration PCR reaction
Added by testing gene in PCR reaction solution and form mixed solution, mixed solution is divided on PCR Sptting plate with 40 μ l/ pipes, and the essentially consist of the mixed solution often in pipe is as shown in following table 2-3:
Table 2-3
| Reagent | Every person-portion consumption (μ L) |
| Distilled water | 8.8 |
| 10×PCR Buffer | 15 |
| 25mM MgCl 2 | 2 |
| The each dNTP of 2.5mM | 4 |
| 10 μMs of primer SF | 0.1 |
| 10 μMs of primer SR | 1 |
| 10 μMs of primer I F | 0.1 |
| 10 μMs of primer I R | 1 |
| 10 μMs of probe PS | 1 |
| 10 μMs of probe PI | 1 |
| 5U/ μ L HotstarTaq enzyme | 1 |
| The DNA sample of testing gene | 5 |
| Total amount | 40 |
By the adhesive transparent ParafilmTM of PCR Sptting plate one side, under this enclosed environment, read the background fluorescence value before reaction.
(f) PCR reaction process
96 DEG C of denaturations 15 minutes; 95 DEG C of sex change 15 seconds, 55 DEG C of renaturation 20 seconds, 72 DEG C extend 20 seconds, circulate 30 times; 72 DEG C extend 5 minutes again, and along with the increase of amplified production, the fluorophor discharged in system becomes positive correlation to increase, until reacted, after having reacted, read reacted fluorescent value.Reacted fluorescent value is deducted background fluorescence value, if increment is more than or equal to 90%, then judges that testing gene is as the positive, if increment is less than 90%, be judged to be feminine gender.
Adopt the method in above embodiment to carry out HLA-B27 gene test by different DNA sample, its result is as shown in following table 2-4:
Table 2-4
| Detect target | Fluorescent mark | Background fluorescence value before reaction | Reacted fluorescent value | Result judges |
| HLA-B27 | FAM | 61 | 209 | Positive |
| Internal reference | HEX | 56 | 190 | Normally |
| HLA-B27 | FAM | 60 | 89 | Negative |
| Internal reference | HEX | 54 | 240 | Normally |
Detect 347 increments originally with the SSP somatotype detected through gel electrophoresis decision method of existing maturation, coincidence rate is 100% (P<0.05), and this detected result by method of the present invention of this 347 increment is as shown in following table 2-5 simultaneously:
Table 2-5:
In embodiment two, the primer of design is the special universal primer covering HLA-B27 gene, the specificity that the application of specific probe detects except enhanced system, read fluorescence values respectively afterwards before the reaction, then carry out numerical value contrast conting and can realize HLA-B27 gene test, the present embodiment eliminates electrophoresis step, make whole technological operation more accurate, convenient, fast, greatly simplifie manually-operated flow process, only about 90 minutes can be needed from sample to result, and all carry out in the environment closed with the work of reacted fluorescent value digital independent before reaction, therefore the risk of PCR reaction after stain is rejected completely, result is also analyzed and is eliminated the possibility that SSP visual inspection produces error.Method of the present invention, except above two embodiments, also can be applied at present equally based on other HLA somatotype of SSP technology, red corpuscle gene type and platelet surface antigen gene type etc.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For those skilled in the art, without departing from the inventive concept of the premise, some equivalent to substitute or obvious modification can also be made, and performance or purposes identical, all should be considered as belonging to protection scope of the present invention.
Claims (7)
1. a genotype tests method, is characterized in that, comprises the steps:
(1) testing gene is added in PCR reaction solution and form mixed solution and be enclosed in the enterprising performing PCR amplified reaction of PCR Sptting plate, the amplimer of testing gene and specific probe and internal reference primer and internal reference probe is comprised in described PCR reaction solution, 5 ' end fluorophor of described specific probe and described internal reference probe marks and 3 ' end quenching group marks, before described pcr amplification reaction, read the background fluorescence value of described mixed solution, after pcr amplification reaction terminates, read reacted fluorescent value;
(2) described reacted fluorescent value is deducted described background fluorescence value, if increment is more than or equal to 90%, then judge that described testing gene is as the positive, if increment is less than 90%, judge that described testing gene is as feminine gender.
2. genotype tests method as claimed in claim 1, is characterized in that: described pcr amplification reaction adopts asymmetric amplification.
3. genotype tests method as claimed in claim 1, is characterized in that: the fluorophor on the fluorophor on specific probe described in each and described internal reference probe is all different; Described fluorophor is selected from following group: FAM, HEX, TET, ROX, TAMRA and CY5; Described quenching group is Dabcyl.
4. the genotype tests method as described in claim 1-3 any one, it is characterized in that: described testing gene is O1, O2, B, A, A2 genotype of ABO gene, described amplimer is the primer being common to described O1, O2, B, A, A2 gene, and sequence is as follows:
GF：5’-ATATATATGGCAAACACAGTTAACCCAATG-3’
GR：5’-CCACTCACGGATTTCTGTTGTGTTTC-3’；
The sequence of described internal reference primer is:
IF：5’-CAGTGCCTTCCCAACCATTCCCTTA-3’
IR：5’-ATCCACTCACGGATTTCTGTTGTGTTTC-3’；
The specific probe of described O1, O2, B, A, A2 gene and described internal reference probe are distinguished as follows:
PO1：5’FAM-TAAGTGGAAGGATGTCCTCGTCGTG-Dabcyl3’
PO2：5’HEX-AGTGGACGTGGACATGGAGTTCC-Dabcyl3’
PB：5’TET-AGTGGACGTGGACATGGAGTTCC-Dabcyl3’
PA：5’TAMRA-CCGACCCCCCGAAGAGCC-Dabcyl3’
PA2：5’ROX-TGCCTTCCCAACCATTCCCTTA-Dabcyl3’
PI：5’CY5-CAGGCAGTACCCGTTCAAGC-Dabcyl3’。
5. the genotype tests method as described in claim 1-3 any one, is characterized in that: described testing gene is HLA-B27 gene, and the sequence of described amplimer is as follows:
SF：5’-GATCAGGACGAAGTCCCAGGCC-3’
SR：5’-CATCTCGGCGTCTGAGGAGA-3’；
The sequence of described internal reference primer is:
IF：5’-CAGTGCCTTCCCAACCATTCCCTTA-3’
IR：5’-ATCCACTCACGGATTTCTGTTGTGTTTC-3’；
The specific probe of described HLA-B27 gene and described internal reference probe are distinguished as follows:
PS：5’FAM-TGTCGGGTCCTTGTTCCAGGAT-Dabcyl3’
PI：5’HEX-CCCACATCAGGTACAGGCTT-Dabcyl3’。
6. genotype tests method as claimed in claim 4, is characterized in that: also comprise damping fluid, warm start enzyme, dNTP, water and magnesium chloride in described PCR reaction solution, the program of described pcr amplification reaction is as follows: 96 DEG C of denaturations 15 minutes; 95 DEG C of sex change 15 seconds, 65 DEG C of renaturation 20 seconds, 72 DEG C extend 20 seconds, circulate 30 times; 72 DEG C extend 5 minutes again.
7. genotype tests method as claimed in claim 5, is characterized in that: also comprise damping fluid, warm start enzyme, dNTP, water and magnesium chloride in described PCR reaction solution, the program of described pcr amplification reaction is as follows: 96 DEG C of denaturations 15 minutes; 95 DEG C of sex change 15 seconds, 55 DEG C of renaturation 20 seconds, 72 DEG C extend 20 seconds, circulate 30 times; 72 DEG C extend 5 minutes again.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510698148.1A CN105219864A (en) | 2015-10-23 | 2015-10-23 | A kind of genotype tests method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510698148.1A CN105219864A (en) | 2015-10-23 | 2015-10-23 | A kind of genotype tests method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105219864A true CN105219864A (en) | 2016-01-06 |
Family
ID=54989130
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510698148.1A Pending CN105219864A (en) | 2015-10-23 | 2015-10-23 | A kind of genotype tests method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105219864A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101403007A (en) * | 2008-11-03 | 2009-04-08 | 深圳市血液中心 | Detection agent for ABO blood type gene and shaping method |
| CN101892314A (en) * | 2010-06-22 | 2010-11-24 | 天津市秀鹏生物技术开发有限公司 | Primer group and kit for detecting human platelet alloantigen gene |
| CN103184296A (en) * | 2011-12-27 | 2013-07-03 | 上海复星医学科技发展有限公司 | One-step genotyping detection kit for hepatitis C virus |
| CN104745721A (en) * | 2013-12-27 | 2015-07-01 | 上海星耀医学科技发展有限公司 | A hepatitis C virus one-step-method nucleic acid quantification/genotype RT-PCR detection kit |
-
2015
- 2015-10-23 CN CN201510698148.1A patent/CN105219864A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101403007A (en) * | 2008-11-03 | 2009-04-08 | 深圳市血液中心 | Detection agent for ABO blood type gene and shaping method |
| CN101892314A (en) * | 2010-06-22 | 2010-11-24 | 天津市秀鹏生物技术开发有限公司 | Primer group and kit for detecting human platelet alloantigen gene |
| CN103184296A (en) * | 2011-12-27 | 2013-07-03 | 上海复星医学科技发展有限公司 | One-step genotyping detection kit for hepatitis C virus |
| CN104745721A (en) * | 2013-12-27 | 2015-07-01 | 上海星耀医学科技发展有限公司 | A hepatitis C virus one-step-method nucleic acid quantification/genotype RT-PCR detection kit |
Non-Patent Citations (7)
| Title |
|---|
| LI RUAN等: "Multicolor Real-Time PCR Genotyping of ABO System Using Displacing Probes", 《J FORENSIC SCI》 * |
| MARTIN L. OLSSON等: "A rapid and simple ABO genotype screening method using a novel B/O2 versus A/O2 discriminating nucleotide substitution at the ABO locus", 《VOX SANGUINIS》 * |
| 刘芳等: "HLA-DRB1*08/16等位基因与广西肝癌家族聚集性的相关性", 《世界华人消化杂志》 * |
| 戎奇吉等: "HLA-B27基因荧光定量方法的确立与多种方法比较研究", 《中华医院感染学杂志》 * |
| 李伟等主编: "《分子诊断学》", 30 September 2015 * |
| 聂咏梅等: "RhDel表型与RHEC表型的相关性研究", 《热带医学杂志》 * |
| 马红玲等: "TaqMan实时PCR与SYBR GreenⅠ实时PCR检测HLA-B*27的比较性分析", 《国际检验医学杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Loeffler et al. | Quantification of fungal DNA by using fluorescence resonance energy transfer and the light cycler system | |
| Jiang et al. | Rapid detection of Candida albicans by polymerase spiral reaction assay in clinical blood samples | |
| CN112501268B (en) | Nanopore sequencing-based primer group and kit for rapidly identifying respiratory microorganisms and application of primer group and kit | |
| CN102154498B (en) | Nucleic acid detecting method | |
| CN103397107B (en) | Bovine viral diarrhea virus (BVDV) fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit | |
| KR102245849B1 (en) | Composition for detecting Coronavirus and kit for detection thereof | |
| CN106520984A (en) | Pseudomonas aeruginosa nucleic acid fluorescent PCR (polymerase chain reaction) detection kit and detection method | |
| US8637251B2 (en) | Nucleic acid quantification method and microchip for nucleic acid amplification reaction | |
| CN106636390A (en) | Gene methylation detection method, detection kit and application thereof | |
| CN108588277A (en) | A kind of canine distemper virus visualization nucleic acid detection method | |
| CN111763768A (en) | COVID-19 rapid detection color development indication kit | |
| CN110791592A (en) | Primer and kit for rapidly detecting African swine fever virus | |
| CN107881261A (en) | Detect LAMP primer group, kit and the application of the type of pig circular ring virus 3 | |
| CN103966228A (en) | RH blood type DEL-type RHD93T>A allele and detection method thereof | |
| CN102251061A (en) | Nucleic acid dual fluorescence PCR (Polymerase Chain Reaction) detection kit for influenza A/B virus | |
| CN110878381A (en) | Primer composition, kit and method for detecting mycoplasma bovis and infectious bovine rhinotracheitis virus | |
| CN103937884A (en) | Loop-mediated isothermal amplification kit for detecting mycobacterium tuberculosis and application method of kit | |
| CN103103259A (en) | Method, kit and primers for determining whether two predetermined loci of nucleic acid sample mutate or not | |
| CN107365684B (en) | A kind of paper micro-fluidic chip and its method for extracting nucleic acid and isothermal amplification method | |
| CN102925560A (en) | Kit and method for detecting mutant alpha-Mediterranean anemia genes through HRM (high resolution melting) method | |
| Liu et al. | A novel microfluidic device that integrates nucleic acid extraction, amplification, and detection to identify an EGFR mutation in lung cancer tissues | |
| CN103937806A (en) | Rh blood group DEL phenotype RHD838>A allele and detection method thereof | |
| CN107513494A (en) | A kind of detection of nucleic acids card and its application method | |
| CN102146467A (en) | Reagent for detecting Yersinia pestis and method for carrying out fluorescence quantitative PCR (Polymerase Chain Reaction) detection on Yersinia pestis | |
| CN116064842A (en) | Composite amplification box for degradation material deducing biological geographical ancestor DIPs and sex identification |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160106 |
|
| RJ01 | Rejection of invention patent application after publication |