CN106591463A - Fluorescence labelled multiplex amplification primer set containing human genome DNA 22 loci, kit and application thereof - Google Patents
Fluorescence labelled multiplex amplification primer set containing human genome DNA 22 loci, kit and application thereof Download PDFInfo
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Abstract
The invention provides a fluorescence labelled multiplex amplification primer set containing human genome DNA 22 loci, a kit and application thereof. The kit cooperates with a new hot start Taq enzyme and a corresponding premixed system of a PCR buffer solution to enhance the resistant ability of a PCR system to an inhibitor. At the same time, the PCR sensitivity is enhanced, all the 22 genome loci can be detected under a DNA template amount of 50pg, and the kit can be stored at 4DEG C for one year. The kit is suitable for forensic STR typing detection, parentage identification, individual recognition and other multiplex PCR application fields.
Description
Technical field
The invention belongs to the technology of field of nucleic acid amplification, and in particular to a kind of comprising 22 locus of human gene group DNA
The primer sets of fluorescence labeling composite amplification, test kit and its application, the system can be applicable to the detection of legal medical expert STR typings, relationship mirror
Fixed and individual identification.
Background technology
(Polymerase Chain Reaction, PCR) is that a kind of external rapid amplifying is specific for polymerase chain reaction
The technology of DNA fragmentation, it is the foundation stone of modern molecular biology, is also one of most important means, is widely used in medical science inspection
Test, Basic of Biology research, animals and plants inspection etc. field.Multiplex PCR is one kind of round pcr classification, refers to more than 2 pairs primers
The composite amplification technology of composition.Multiple PCR technique used in practical application, typically refers to 10 more than i.e. 10 pairs primers answering again
Close amplification system.Due to being combined with each other between primer and act on, using without special modification and the Taq enzyme for configuring and buffering
Liquid, is easily caused the deflection of non-specific generation or amplification and cannot obtain complete amplification.
The difference of thermal starting DNA Taq enzymes and general T aq enzyme is, only in the case of high temperature (more than 90 DEG C), its chemistry
Modification just can be opened, and so as to enzyme activity starts release, at normal temperatures hot start Taq polymerase is not active.Therefore, only 90
More than DEG C, primer and template are in one with respect in the case of specific bond, and PCR reactions just can start, it is ensured that whole process
Specificity, high efficiency and accuracy.
PCR buffer provides the PCR buffer under suitable reaction condition, regular situation for Taq enzyme and only includes three groups
Point, i.e. Mg2+、Tris-HCl、KCl。Mg2+Used as Taq catalyst, its concentration plays critical effect to enzymatic activity；Tris-
HCl ensure that the pH of PCR reaction whole process stablizes；KCl can affect the secondary structure of template, play the work for improving specificity
With.
When template is special construction (such as high GC) or multiplex PCR, above-mentioned Standard PCR buffer just cannot be suitable for, generally
Need to increase PCR reinforcing agents (PCR enhancer).PCR reinforcing agents are guaranteeing reaction typically by the following aspects just
Often carry out：1st, to the protection of Taq：As BSA (Ralser etc, 2006), glycerol (Nagai etc, 1998), trehalose (Hor á
kováetc,2011)；2nd, template DNA melting temperature is changed：As DMSO (Chakrabarti etc, 2001), Methanamide
(Sarkar etc,1990)；3rd, ionic environment is changed：As ammonium sulfate (Ahmad etc, 2007).Add in PCR buffer systems
Reinforcing agent species is different with concentration, may obtain diverse amplification.
About 10% is tandem repetitive sequence in human genome DNA, referred to as satellite DNA.Again may be used by the length of recurring units
To be divided into large satellite, Satellite, moonlet and microsatellite, microsatellite is only by what 2-7 base pair was constituted by wherein recurring units,
It is tandem repeat loci (Short Tandem Repeat, abbreviation STR) also known as it, what this tandem sequence repeats were formed
DNA sequence can produce hundreds of millions of genotype combinations, and the frequency that each combination occurs in colony is all very low,
That is, str locus seat tool has high Individual identification ability, so being used for DNA analysis technology frequently as genetic marker
Middle legal medical expert's individual identification, Relationship iden- tification, while being also the mainstream technology of DNA Databases.Meanwhile, str locus seat
Fragment is little, easy amplification, is suitable for checking micro and degraded sample, and the amplification condition of each locus is similar and can be combined
Amplification, thus have the advantages that it is sensitive, accurate, quick, contain much information.Due to these advantage str locus seats typing research with
Screening is at home and abroad widely used in antropology, medical genetics and prudence and each association area.Especially
In terms of DNA data bases are set up, compared with the technologies such as conventional RFLP, STR composite amplification technologies have great superiority.Therefore
U.S. FBI has carried out numerous studies, have selected 13 str locus seats for setting up DNA data bases --- CODIS (Combined
DNA Index System)：CSF1PO、D3S1358、D5S818、D7S820、D8S1179、D13S317、D16S539、
D18S51, D21S11, FGA, TH01, TPOX, vWA, these str locus seats are commonly known as 13 core gene seats.
Just because of the application prospect that STR possesses, method medical circles and some companies have carried out large-scale exploitation to it
Journal of Sex Research.The nineties, middle and late stage was set up with the poly-talented company that American AB I (Applied Biosystems, USA) is representative
Composite amplification and fluorescent detection system, and be born it is most representational be ABI companies IdentifilerTMAnd Promega
(USA) the PowerPlex-16 fluorescence detection reagent kits of company..With the continuous development of technology, current euchromosome STR fluorescence
Detection kit can detect the locus of more than 20, and based on ABI, Promega, domestic manufacturers have in Wuxi for foreign vendor
Dolantin joins, Beijing basic point is cognitive, Ningbo Haier applies, Suzhou reads micro- etc..
From the mid-90, the Ministry of Public Security proposes the DNA data of " unified planning, seek unity of standard, implement step by step, developing at a progressive speed "
Storehouse construction principle starts, and the DNA data bases of China have the history of 15 years.Over more than 10 years, DNA data bases gathered 30,000,000 with
Upper STR data, have played effect in the case played more than 1,000,000, have tentatively realized the construction of polyvoltine application spanning space-time
Target, has played the big effect of tool in precisely fighting crime.
However, during actually detected, the multiformity and the restriction of other various conditions due to sample occurs in that quantity
More difficulty picks up material, i.e., cannot obtain the sample of genotyping result.Live sample is mostly micro, and precious, DNA content is non-in sample
Often few, the DNA content of generally only several cells, it is impossible to reach the minimum detectability of conventional STR test kits causes without amplification knot
Fruit or only part amplification.Criminal-scene sample wide material sources, it is understood that there may be suppression composition it is various, test kit is resisted
Rejection ability has higher requirements.Bloodstain in the special such as soil in source of some samples, the step of even across DNA extraction,
Still there is the presence of substantial amounts of PCR inhibitor such as haemachrome, humic acid etc., destroy the amplification procedure of PCR, lead to not obtain complete
Genotyping result.Chelex etc. slightly carries the extracting method of DNA and can also remain more inhibitor composition.
China, at the DNA database establishment initial stages, is by external product, mainly ABI company monopolizings using STR test kits
, as middle dolantin joins 17+1, EX20；The cognitive 20A of basic point；Two typer15 development ＆ production, just progressively break foreign countries
Monopolization, at present in DNA database establishments, domestic reagent box occupation rate is more than 50%.But due to the complexity of criminal-scene sample
Property and STR test kits are had high demands, temporarily using foreign vendor's test kit is, main flow is 2010 years bases of ABI
IdentifilerTMThe Identifiler that optimization is releasedTM plus。
The content of the invention
The technical problem of solution：The invention provides a kind of while analyzing the fluorescence mark of 22 locus of human gene group DNA
Remember primer sets, test kit and the application of composite amplification, can be used for the detection of legal medical expert STR typings, relationship identification and individual identification.For
From scene of a crime is micro, the detection containing the difficulty sample such as inhibitor, can simultaneously accomplish quick, high sensitivity and strong anti-suppression
Ability processed；By the optimization of PCR buffer and fluorescent dye system, amplification system sensitivity is improve, 29,50pg samples are followed
Ring can obtain complete typing.
Technical scheme：It is a kind of at the same analyze 22 locus of human gene group DNA fluorescence labeling composite amplification primer
Group, includes the primer pair for expanding following 22 locus：D3S1358、D13S317、D7S820、D16S539、
D1S1656、Penta E、DYS391、TPOX、TH01、D2S1338、CSF1PO、Penta D、D19S433、vWA、D21S11、
D18S51, D6S1043, D8S1179, D5S818, D12S391, FGA and Amelogenin.
The sequence of above-mentioned primer pair and the concentration in amplification system are as shown in table 1：
The each locus primer sequence of table 1 and concentration
5 ' ends of at least one primer carry out fluorochrome label in each described primer pair.
Described primer pair is through packet marking.Specifically it is grouped into：
D3S1358, D13S317, D7S820, D16S539, D1S1656, Penta E is first group；
DYS391, TPOX, TH01, D2S1338, CSF1PO, Penta D is second group；
D19S433, vWA, D21S11, D18S51, D6S1043 are the 3rd group；
Amelogenin, D8S1179, D5S818, D12S391, FGA are the 4th group.
It is using blue-fluorescence dye 6-FAM, Green fluorescent dye HEX, Yellow fluorochrome when being marked per group
Any one in VIG558, red fluorescence dyestuff labelling VIG598 is dyeed, and is different from per between group.
According to another aspect of the present invention, the detection kit for including above-mentioned primer sets is additionally provided.
In described test kit, molecular weight internal standard is also included.
Described molecular weight internal standard selects fluorescent orange SIZ.
Described test kit is by constituting as follows：The μ L of 2.5 × mixed solution special 10；The μ L of template DNA 1, DNA total amounts exist
0.05ng-2ng；The μ L of composite primer 5；sdH2O 9μL；
Wherein, described mixed solution special is：10 μ L 5U/ μ L thermal starting AGCU A-Taq, 75 μ L 500mM Tris-
HCl、12.5μL 1M KCl、12.5μL 25mM dNTPs、12.5μL 250mM MgCl2、75μL 20mg/mL BSA、25μL
Glycerol, 5 μ L NP40,50 μ L Tween20,50 μ L 0.5%NaN3With 172.5 μ L sdH2O。
Beneficial effect：
1st, can accomplish that DNA masterplates amount all detects 22 locus under 50pg, 29 cyclic amplifications can obtain complete
Typing.Sensitivity requirement of the sensitivity far above the 125pg that rower GA/T 815-2009 are formulated, also above the current country
Legal medical expert STR detection kit disclosed in Jing, such as patent CN103451311A, CN 105018597A etc..
2nd, using high performance PCR buffer systems and the thermal circulation parameters of optimization, downtrod case can be significantly improved
The amplification capability of sample, to the indivisible active component of trace sample and mixture bigger peak height is produced, and is easy to knot
Fruit is understood.
3rd, the present invention be Taq enzyme, PCR buffer, the mixture of various reinforcing agents, improve whole system specificity,
High efficiency and accuracy, and the step of simplify Standard PCR system configurations process, it is ensured that the accuracy of system configurations, reduce dirty
The possibility of dye.
4th, the present invention can be preserved in 4 DEG C of placements different from -20 DEG C of Standard PCR reagent, it is to avoid frozen-thaw process.
Description of the drawings
Fig. 1 is the STR typings knot of the gradient dilution of DNA standard substance 9948 to 50pg quantitative to qubit3.0 in embodiment 1
Really；
Fig. 2 is the genotyping result of EX21 allelic ladders in embodiment 1；
Fig. 3 is the stub AFLP system in embodiment 2；
Fig. 4 is the bloodstain AFLP system in embodiment 2；
Fig. 5 is that the high concentration sample that the costicartilage in embodiment 2 is extracted expands EX21 amplification figures；
Fig. 6 is that the high concentration sample that the costicartilage in embodiment 2 is extracted expands ID plus increasing figures；
Fig. 7 is ID plus amplification figures after the high concentration Sample Dilution that the costicartilage in embodiment 2 is extracted；
Fig. 8 is that certain the criminal-scene right mouse button in embodiment 2 wipes swab AFLP system；
Fig. 9 is that certain the criminal-scene door handle in embodiment 2 wipes swab AFLP system EX21；
Figure 10 is that certain the criminal-scene door handle in embodiment 2 wipes swab AFLP system IDplus.
Specific embodiment
The present invention is described in further detail below by specific embodiment.But those skilled in the art will manage
Solution, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted concrete skill in embodiment
Art or condition person, (for example write, Huang Pei according to the technology or condition described by document in the art with reference to J. Pehanorm Brookers etc.
What hall etc. was translated《Molecular Cloning:A Laboratory guide》, the third edition, Science Press) or carry out according to product description.Agents useful for same
Or the unreceipted production firm person of instrument, be can pass through city available from conventional products.
The invention provides a kind of while analyzing drawing for the fluorescence labeling composite amplification of 22 locus of human gene group DNA
Thing group, test kit and application, can be used for the detection of legal medical expert STR typings, relationship identification and individual identification.
First, the determination of locus
With reference to 18 autosomal genes required in GB/T21679-2008 forensic science database establishments specification -2008
Seat D3S1358, D13S317, D7S820, D16S539, Penta E, TPOX, TH01, D2S1338, CSF1PO, Penta D,
D19S433, vWA, D21S11, D18S51, D6S1043, D8S1179, D5S818, FGA, and added according to domestic practical application
D1S1656, D12S391 and Sexual discriminating locus Amelogenin inside FBI Expanded U.S Core Loci.
Due to the particularity of gender-specific genes Amelogenin, there is the possibility of Y allelic losses, therefore in locus choosing
When selecting, new Y chromosome locus DYS391 is added, as sex auxiliary judgement, if male's sample, should at the locus
Have typing, if women sample then without.
2nd, the locus assembled scheme design of fluorescence labeling composite amplification system
This research has been carried out differentiating, selected to fluorescent dye, has selected blue, green, yellow, red, orange five kinds of fluorescent markers, excellent
5 color fluorescence assembled schemes are selected.
According to the scope of each locus allele, the different fluorochrome label group of reasonable combination, design primer is examined
Consider mutational site, design in conserved region as far as possible, while considering the design of primers principles such as Tm, length, G/C content, dimer.Arrangement
After the completion of, each (there is fluorescent dye mark at the 5 ' ends that each pair primer only has a primer to the primer of design correspondence fluorochrome label
Note).A kind of preferred compositions mode therein of illustrating is：D3S1358, D13S317, D7S820, D16S539, D1S1656, Penta
E locus are labeled as blue dyess；DYS391, TPOX, TH01, D2S1338, CSF1PO, Penta D locus are labeled as green
Dyestuff；D19S433, vWA, D21S11, D18S51, D6S1043 locus are labeled as black dyes；Amelogenin、
D8S1179, D5S818, D12S391, FGA locus are labeled as orchil.
3rd, the optimization and foundation of florescence labeling STR multiplex system
1st, the determination of primer and amplification program
The complete conservative region of Primer selection in test kit site, avoids the high saltation zone that other like products may be included
Domain.Through design of primers, first test is adjusted repeatedly to the individual gene seat primer in 22 sites, annealing temperature specificity reaches
To after standard；And be grouped according to four colors, the test repeatedly that the system that carries out to per group of primer expands again after reaching standard, carries out 22
Big multiple expansion to primer, often walks and changes adjustment primer repeatedly according to practical situation.To 22 site composite amplification PCR reactions of research
Condition, the parameters in composite amplification are determined by substantial amounts of experiment repeatedly, e.g., loop parameter, annealing temperature, compound expansion
Increase change and template DNA amount of reaction volume etc., make amplified production reach balance, special requirement, it is established that composite amplification
System, while amplifying 22 sites.Consider that composite amplification is very high to the efficiency of primer, specific requirements, needs between each group primer
Balance, this needs that design of primers is repeated, debug, system is expanded again in final determination.
2nd, the determination of assorted dyestuff
According to the rower GA/T 815-2009 that STR composite amplifications are detected《The fluorescence marked STR of forensic science people is compound to be expanded
Increase detection kit quality basic demand》In the harmony between different fluorescent markers is little to be required to the harmony of test kit
In 30%.It is respectively 488nm and 505nm and the excitation wavelength of genetic analyzer ABI3130 or 3500 is fixed wave length,
Efficiency between different dyes has very big difference.The excitation wavelength of such as blue common dyes FAM is and red in 495nm or so
The conventional dyestuff ROX excitation wavelengths in chrominance channel are in 588nm, therefore dyestuff launching efficiency has very big difference.The present invention is using new
Dyestuff VIG558, the VIG598 of type, improves harmonious this index between test kit color.
3rd, enzyme screening
The anti-rejection ability and specific amplification of Taq are directly related with the performance for entirely expanding system again, for common sample,
Most of Taq enzyme can obtain preferable amplification, due to there may be micro and high inhibitor in live case sample
Sample, there is high requirement to Taq enzyme.The present invention tens kinds of Taq enzymes of screening, it is determined that final enzyme has stability strong, special
Property good, anti-rejection ability it is strong the characteristics of, coordinate targetedly mix, greatly improve test kit performance.
4th, the determination of mix components
The requirement of high specific, high sensitivity, strong anti-rejection ability, Jing many experiments is needed to increased for the present invention
The PCR reinforcing agents of particular types and concentration, research and development are adapted to the mixed solution special of the system, greatly improve the spy of PCR system
The opposite sex and sensitivity and anti-rejection ability.Simultaneously 4 DEG C of preservations can be positioned over Taq enzyme premix, it is to avoid frozen-thaw process, raising is matched somebody with somebody
Put the efficiency of process.It is final to determine formula：10 μ L 5U/ μ L thermal starting AGCU A-Taq, 75 μ L 500mM Tris-HCl, 12.5
μ L1M KCl, 12.5 μ L 25mM dNTPs, 12.5 μ L 250mM MgCl2, 75 μ L 20mg/mL BSA, 25 μ L glycerols, 5 μ
LNP40,50 μ L Tween20,50 μ L 0.5%NaN3, 172.5 μ L sdH2O。
Embodiment 1
Test kit sensitivity is tested, 50pg Control DNA 9948 (dilute after ubit3.0 plasmid standards for quantitation
To 50pg)/10 μ L systems, 29 cyclic amplifications.
It is a kind of at the same analyze 22 locus of human gene group DNA fluorescence labeling composite amplification test kit, by such as the following group
Into：The corresponding primer of wherein 22 locus and its primer concentration are：
Above-mentioned primer pair is that, through packet marking, having 5 ' ends of a primer in each primer pair carries out fluorescent dye mark
Note.The compound mode that the present embodiment is adopted for：D3S1358, D13S317, D7S820, D16S539, D1S1656, Penta E bases
Because coordinate is designated as blue dyess；DYS391, TPOX, TH01, D2S1338, CSF1PO, Penta D locus are labeled as green dye
Material；D19S433, vWA, D21S11, D18S51, D6S1043 locus are labeled as black dyes；Amelogenin、D8S1179、
D5S818, D12S391, FGA locus are labeled as orchil.
In test kit, molecular weight internal standard is also included, molecular weight internal standard selects fluorescent orange SIZ.
A, amplification system are：
2.5 wherein described × MasterMix is：10 μ L 5U/ μ L thermal starting AGCU A-Taq, 75 μ L 500mM
Tris-HCl, 12.5 μ L 1M KCl, 12.5 μ L 25mM dNTPs, 12.5 μ L 250mM MgCl2, 75 μ L 20mg/mL BSA,
25 μ L glycerols, 5 μ L NP40,50 μ L Tween20,50 μ L 0.5%NaN3, 172.5 μ L sdH2O.4 DEG C are put in after the completion of configuration
Preserve.
B, amplification thermal cycle
L () is placed in PCR amplification pipes on thermal cycler；
(2) program recommended below is selected to be expanded；
(3) sample after expanding should keep in dark place；
The amplification program of thermal cycler
C, the amplified production fluoroscopic examination on genetic analyzer
Loading mixture is constituted by deionized formamide and system middle-molecular-weihydroxyethyl internal standard AGCU Marker SIZ-500
((0.5 μ LAGCU Marker SIZ-500 (Zhongde Meilian Biotech Co., Ltd. Wuxi)) × (sample introduction number)+(12 μ L go from
Sub- Methanamide) × (sample introduction number)).By 12.5 μ L loadings mixture and 1 μ L amplified productions or system allelic analytical standard
Thing mixes, it is to avoid produce bubble.95 DEG C of degeneration 3 minutes, ice bath 3 minutes, and electrophoresis as early as possible；Tested and analyzed with genetic analyzer.
D, phenotypic analysis
The data collected with genetic analyzer detection in fragment analysis software GeneMapper ID-X analytical procedures C, by sample
The genotyping result of product analytical data and allelic ladder is compared, and obtains the typing data of actual sample.To mark
Quasi- product 9948 (U.S. of Promega companies) gradient dilution is expanded to 50pg, and genotyping result such as Fig. 1 shows.To allele
The genotyping result of parting standard thing such as Fig. 2 shows.It can be seen that the testing result and its genotype one to standard substance 9948
Cause, the genotyping result of parting standard thing is also consistent with the typing of each allele, 3500 electrophoresis can detect full gene seat point
Type, this test kit is minimum can to detect 50pg minim DNA masterplates.
Embodiment 2
The identification of the micro sample of criminal-scene is carried out using the fluorescence labeling composite amplification checking system of 22 locus.Together
Batch adopts the STR test kit Identifiler of ABI companiesTMPlus carries out contrast test.566 parts are provided by certain public security bureau to come
From the sample of Different Individual：250 parts of bloodstain, stub, seminal fluid, costicartilage sample, 316 wipe swab.The extraction ginseng of genomic DNA
Examine《GA/T 383-2014 forensic DNA profiling laboratory inspection specifications》Carry out.
It is a kind of while analyze the test kit of the fluorescence labeling composite amplification of 22 locus of human gene group DNA, by such as the following group
Into：The corresponding primer of wherein 22 locus and its primer concentration are：
Above-mentioned primer pair is that, through packet marking, having 5 ' ends of a primer in each primer pair carries out fluorescent dye mark
Note.The compound mode that the present embodiment is adopted for：D3S1358, D13S317, D7S820, D16S539, D1S1656, Penta E bases
Because coordinate is designated as blue dyess；DYS391, TPOX, TH01, D2S1338, CSF1PO, Penta D locus are labeled as green dye
Material；D19S433, vWA, D21S11, D18S51, D6S1043 locus are labeled as black dyes；Amelogenin、D8S1179、
D5S818, D12S391, FGA locus are labeled as orchil.
In test kit, molecular weight internal standard is also included, molecular weight internal standard selects fluorescent orange SIZ.
A, amplification system are：
| Component | Volume |
| 2.5×MasterMix | 10.0μL |
| DNA masterplate 0.05-2ng | 1-6μL |
| As above the corresponding primer of 22 locus | 5.0μL |
| sdH2O | Complement to 25.0 μ L |
2.5 wherein described × MasterMix is：10 μ L 5U/ μ L thermal starting AGCU A-Taq, 75 μ L 500mM
Tris-HCl, 12.5 μ L 1M KCl, 12.5 μ L 25mM dNTPs, 12.5 μ L 250mM MgCl2, 75 μ L 20mg/mL BSA,
25 μ L glycerols, 5 μ L NP40,50 μ L Tween20,50 μ L 0.5%NaN3, 172.5 μ L sdH2O.4 DEG C are put in after the completion of configuration
Preserve.
B, amplification thermal cycle
L () is placed in PCR amplification pipes on thermal cycler；
(2) program recommended below is selected to be expanded；
(3) sample after expanding should keep in dark place；
The amplification program of thermal cycler
The present invention
ID Plus
C, the amplified production fluoroscopic examination on genetic analyzer
Loading mixture is constituted by deionized formamide and system middle-molecular-weihydroxyethyl internal standard AGCU Marker SIZ-500
((0.5 μ LAGCU Marker SIZ-500 (Zhongde Meilian Biotech Co., Ltd. Wuxi)) × (sample introduction number)+(12 μ L go from
Sub- Methanamide) × (sample introduction number)).By 12.5 μ L loadings mixture and 1 μ L amplified productions or system allelic analytical standard
Thing mixes, it is to avoid produce bubble.95 DEG C of degeneration 3 minutes, ice bath 3 minutes, and electrophoresis as early as possible；Tested and analyzed with genetic analyzer.
D, phenotypic analysis and result
The data collected with genetic analyzer detection in fragment analysis software GeneMapper ID-X analytical procedures C, by sample
The genotyping result of product analytical data and allelic ladder is compared, and obtains the typing data of actual sample.According to
Embodiment 1 enters performing PCR amplification, genetic analyzer detection and finally obtains genotyping result, includes in 566 parts of live case samples
250 parts of bloodstain, stub, seminal fluid, costicartilage samples.The recall rate of the present invention also exists in the recall rate of 97.2%, IDplus
97.2%.The universal DNA masterplates content of this kind of sample is higher, is extracted using chelex, needs stronger anti-rejection ability, such as Fig. 3,
Shown in Fig. 4.Its middle and high concentration sample, IDplus amplifications occur low after front height and add the infull phenomenons of A as shown in Figure 6
The phenomenon that D8S1179, D3S1358, TH01, vWA, D5S818 this five locus occur plus A is not complete, small fragment amplicon with
Be present obvious unbalanced phenomena in large fragment amplicon, expand after dilution normal as shown in Figure 7.And same sample EX21 shows phase
To more excellent, there is no such situation, without substantially plus A problems and it is unbalanced as shown in Figure 5.
The 316 wiping swabs included in 566 parts of live case samples, the recall rate of the present invention is concrete to expand 24%
Figure is shown in：Fig. 8-Fig. 9, the ID plus compareed with same batch are also 24%, and both are in terms of low-copy pattern detection without substantially poor
Away from.As shown in Fig. 9, Figure 10, same door handle sample, two test kit homologous genes seat amplification typings are as shown in the table.Two
There is any discrepancy in the locus typing of D2S1338, D21S11, FGA for individual test kit, deletes these three locus warehouse-ins and compares, than
Middle suspect Lee.D2S1338, D21S11 of EX21 are identical with true typing, and the FGA typings of ID plus are identical with true.
Occur such case, to amplification the close test kit detectable limit of masterplate content it is related, occur in that under extremely low masterplate concentration with
The phenomenon of machine amplification, also side reacted PCR amplification system masterplate concentration it is extremely low when, it should plus large sample repeat number and increase
Contrast agent box, it is ensured that sample typing correctness.
| Locus | EX21 | ID plus | The real typing of suspect than in |
| D3S1358 | 15,17 | 15,17 | 15,17 |
| D13S317 | 9 | 9 | 9 |
| D7S820 | 11,12 | 11,12 | 11,12 |
| D16S539 | 9,11 | 9,11 | 9,11 |
| D2S1338 | 20,24 | 24 | 20,24 |
| TH01 | 8,9.3 | 8,9.3 | 8,9.3 |
| D18S51 | 18 | 18 | 18 |
| Amel | X | X | X |
| TPOX | 11 | 11 | 11 |
| vWA | 16,19 | 16,19 | 16,19 |
| D21S11 | 30,30.2 | 30 | 30,30.2 |
| DYS391 | - | - | - |
| D8S1179 | 14,15 | 14,15 | 14,15 |
| D5S818 | 12 | 12 | 12 |
| CSF1PO | 10,11 | 10,11 | 10,11 |
| D19S433 | 13 | 13 | 13 |
| FGA | 21 | 21,23 | 21,23 |
| PentaE | 11 | - | 11 |
| PentaD | 9 | - | 9 |
| D6S1043 | - | - | 16 |
As seen from the above table, test kit of the invention is for micro, containing the difficulty sample such as inhibitor from scene of a crime
Detection, can simultaneously accomplish quick, high sensitivity and strong anti-rejection ability；By the excellent of PCR buffer and fluorescent dye system
Change, improve amplification system sensitivity, 29 circulations of 50pg samples can obtain complete typing.
SEQUENCE LISTING
<110>Zhongde Meilian Biotech Co., Ltd. Wuxi
<120>One kind includes human gene group DNA
The primer sets of the fluorescence labeling composite amplification of 22 locus, test kit and application
<130> 2016
<160> 44
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence
<400> 1
gggcatctct tatactcatg aa 22
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
caatctgggt gacagagcaa 20
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence
<400> 3
gacagacaga aagatagata gat 23
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<400> 4
ggcatccgtg actctctgga c 21
<210> 5
<211> 27
<212> DNA
<213>Artificial sequence
<400> 5
gagttatttt aaggttaata tatataa 27
<210> 6
<211> 25
<212> DNA
<213>Artificial sequence
<400> 6
tatatattct taagaattat aacga 25
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence
<400> 7
ttgtgcacaa atctaaatgc 20
<210> 8
<211> 27
<212> DNA
<213>Artificial sequence
<400> 8
tatactcaat aactcattaa tttagta 27
<210> 9
<211> 23
<212> DNA
<213>Artificial sequence
<400> 9
ggaaagagag aaaccatgtg att 23
<210> 10
<211> 19
<212> DNA
<213>Artificial sequence
<400> 10
ttgctgtctc aggggtggt 19
<210> 11
<211> 30
<212> DNA
<213>Artificial sequence
<400> 11
tatcatgatt gatacatgga aagaattctc 30
<210> 12
<211> 18
<212> DNA
<213>Artificial sequence
<400> 12
tgattcacgc ctgcaatc 18
<210> 13
<211> 21
<212> DNA
<213>Artificial sequence
<400> 13
tcgactggca cagaacaggc a 21
<210> 14
<211> 19
<212> DNA
<213>Artificial sequence
<400> 14
cttactcctg ttcccttcc 19
<210> 15
<211> 18
<212> DNA
<213>Artificial sequence
<400> 15
ccgattatcc agcctggc 18
<210> 16
<211> 20
<212> DNA
<213>Artificial sequence
<400> 16
ctggggtgat tcccattggc 20
<210> 17
<211> 23
<212> DNA
<213>Artificial sequence
<400> 17
tcaccccttt tcctaccaga atg 23
<210> 18
<211> 20
<212> DNA
<213>Artificial sequence
<400> 18
ggaaacagaa atggcttggc 20
<210> 19
<211> 23
<212> DNA
<213>Artificial sequence
<400> 19
tccacacacc actggccatc ttc 23
<210> 20
<211> 21
<212> DNA
<213>Artificial sequence
<400> 20
gtctcagttt tcctacctgt a 21
<210> 21
<211> 19
<212> DNA
<213>Artificial sequence
<400> 21
aaatagccag gcatggtga 19
<210> 22
<211> 25
<212> DNA
<213>Artificial sequence
<400> 22
tatctaagaa atatttgcct aacct 25
<210> 23
<211> 21
<212> DNA
<213>Artificial sequence
<400> 23
ctccagcctg ggcaacagaa t 21
<210> 24
<211> 22
<212> DNA
<213>Artificial sequence
<400> 24
ttctctcttt cttcctctct cc 22
<210> 25
<211> 23
<212> DNA
<213>Artificial sequence
<400> 25
gttgtgaaag ccctagtgga tga 23
<210> 26
<211> 27
<212> DNA
<213>Artificial sequence
<400> 26
aatagataca taggttagat agagata 27
<210> 27
<211> 28
<212> DNA
<213>Artificial sequence
<400> 27
cttttctcag tctccataaa tatgtgag 28
<210> 28
<211> 25
<212> DNA
<213>Artificial sequence
<400> 28
taaagatgtt gtattagtca atgtt 25
<210> 29
<211> 24
<212> DNA
<213>Artificial sequence
<400> 29
tgcacttcac tctgagtgac aaat 24
<210> 30
<211> 20
<212> DNA
<213>Artificial sequence
<400> 30
ggcttctctc tgttcttaac 20
<210> 31
<211> 25
<212> DNA
<213>Artificial sequence
<400> 31
agcaatagtg tgcaaggatg ggtgg 25
<210> 32
<211> 22
<212> DNA
<213>Artificial sequence
<400> 32
gagagagaga aaaggaaagg tg 22
<210> 33
<211> 22
<212> DNA
<213>Artificial sequence
<400> 33
aatgccctgg gctctgtaaa ga 22
<210> 34
<211> 20
<212> DNA
<213>Artificial sequence
<400> 34
agcttaaact gggaagctgg 20
<210> 35
<211> 22
<212> DNA
<213>Artificial sequence
<400> 35
gcctggcaac ttatatgtat tt 22
<210> 36
<211> 22
<212> DNA
<213>Artificial sequence
<400> 36
ccattgcgtg aatatgcctt aa 22
<210> 37
<211> 28
<212> DNA
<213>Artificial sequence
<400> 37
tcctctttgg tatccttacg taatattt 28
<210> 38
<211> 22
<212> DNA
<213>Artificial sequence
<400> 38
ctaagcaaaa aagtaattgt ct 22
<210> 39
<211> 23
<212> DNA
<213>Artificial sequence
<400> 39
atgaaatcag agaaactcaa att 23
<210> 40
<211> 23
<212> DNA
<213>Artificial sequence
<400> 40
taattcctct aataaatccc ctc 23
<210> 41
<211> 23
<212> DNA
<213>Artificial sequence
<400> 41
tgaactcaca gattaaactg taa 23
<210> 42
<211> 27
<212> DNA
<213>Artificial sequence
<400> 42
tcatatcaac caactgagct ctaacat 27
<210> 43
<211> 24
<212> DNA
<213>Artificial sequence
<400> 43
tgtctgtcca tttagctatc tatt 24
<210> 44
<211> 27
<212> DNA
<213>Artificial sequence
<400> 44
aattgttgtc agacatagcc aaatatc 27
Claims (10)
1. a kind of primer sets of the fluorescence labeling composite amplification comprising 22 locus of human gene group DNA, it is characterised in that：Bag
Include the primer pair for expanding following 22 locus：D3S1358、D13S317、D7S820、D16S539、D1S1656、
Penta E、DYS391、TPOX、TH01、D2S1338、CSF1PO、Penta D、D19S433、vWA、D21S11、D18S51、
D6S1043, D8S1179, D5S818, D12S391, FGA and Amelogenin.
2. the primer of the fluorescence labeling composite amplification comprising 22 locus of human gene group DNA according to claim 1
Group, it is characterised in that：The sequence of described primer pair is as follows：D3S1358 SEQ ID NO.1～2, D13S317 SEQ
ID NO.3～4, D7S820 SEQ ID NO.5～6, D16S539 SEQ ID NO.7～8, D1S1656 SEQ ID NO.9～
10th, Penta E SEQ ID NO.11～12, TPOX SEQ ID NO.13～14, TH01 SEQ ID NO.15～16,
D2S1338 SEQ ID NO.17～18, CSF1PO SEQ ID NO.19～20, Penta D SEQ ID NO.21～22,
D19S433 SEQ ID NO.23～24, vWA SEQ ID NO.25～26, D21S11 SEQ ID NO.27～28, D18S51
SEQ ID NO.29～30, D6S1043 SEQ ID NO.31～32, Amelogenin SEQ ID NO.33～34, D8S1179
SEQ ID NO.35～36, D5S818 SEQ ID NO.37～38, D12S391 SEQ ID NO.39～40, FGA SEQ ID
NO.41～42, DYS391 SEQ ID NO.43～44.
3. the primer of the fluorescence labeling composite amplification comprising 22 locus of human gene group DNA according to claim 1
Group, it is characterised in that：Final concentration of the described primer pair in amplification volume is as follows：SEQ ID NO.1～2,0.25 μM,
SEQ ID NO.3～4,0.3 μM, SEQ ID NO.5～6,0.4 μM, SEQ ID NO.7～8,0.35 μM, SEQ ID NO.9～
10,0.5 μM, SEQ ID NO.11～12,1.3 μM, SEQ ID NO.13～14,0.3M, SEQ ID NO.15～16,0.35 μ
M, SEQ ID NO.17～18,0.5 μM, SEQ ID NO.19～20,0.7 μM, SEQ ID NO.21～22,1.6 μM, SEQ ID
NO.23～24,0.6 μM, SEQ ID NO.25～26,0.7 μM, SEQ ID NO.27～28,0.8 μM, SEQ ID NO.29～
30,1.2 μM, SEQ ID NO.31～32,1.5 μM, SEQ ID NO.33～34,0.8 μM, SEQ ID NO.37～38,2.0 μ
M, SEQ ID NO.39～40,2.5 μM, SEQ ID NO.41～42,3.0 μM, SEQ ID NO.43～44,0.8 μM.
4. the primer of the fluorescence labeling composite amplification comprising 22 locus of human gene group DNA according to claim 1
Group, it is characterised in that：5 ' ends of at least one primer carry out fluorochrome label in each described primer pair.
5. the primer of the fluorescence labeling composite amplification comprising 22 locus of human gene group DNA according to claim 4
Group, it is characterised in that：Described primer pair is through packet marking.
6. the primer of the fluorescence labeling composite amplification comprising 22 locus of human gene group DNA according to claim 5
Group, it is characterised in that：D3S1358, D13S317, D7S820, D16S539, D1S1656, Penta E is first group；DYS391、
TPOX, TH01, D2S1338, CSF1PO, Penta D is second group；D19S433, vWA, D21S11, D18S51, D6S1043 are
3rd group；Amelogenin, D8S1179, D5S818, D12S391, FGA are the 4th group.
7. the primer of the fluorescence labeling composite amplification comprising 22 locus of human gene group DNA according to claim 6
Group, it is characterised in that：It is using blue-fluorescence dye 6-FAM, Green fluorescent dye HEX, yellow fluorescence dye when being marked per group
Any one in material VIG558, red fluorescence dyestuff labelling VIG598 is dyeed, and is different from per between group.
8. include the fluorescent labeling comprising 22 locus of human gene group DNA described in any one of claim 1～7 to be combined
The test kit of the primer sets of amplification.
9. test kit according to claim 8, it is characterised in that：In described test kit, molecular weight internal standard is also included；
Described molecular weight internal standard selects fluorescent orange SIZ；Described test kit is by constituting as follows：2.5 the μ L of × mixed solution special 10；Mould
The μ L of plate DNA 1, DNA total amounts are in 0.05ng-2ng；The μ L of composite primer 5；sdH2O 9μL；Wherein, described mixed solution special is：
10 μ L 5U/ μ L thermal starting AGCU A-Taq, 75 μ L 500mM Tris-HCl, 12.5 μ L 1M KCl, 12.5 μ L 25mM
dNTPs、12.5μL 250mM MgCl2, 75 μ L 20mg/mL BSA, 25 μ L glycerols, 5 μ L NP40,50 μ L Tween20,50 μ L
0.5%NaN3With 172.5 μ L sdH2O。
10. application of the test kit described in claim 8 in the detection of legal medical expert STR typings, relationship identification and individual identification.
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| CN201611247192.1A CN106591463A (en) | 2016-12-29 | 2016-12-29 | Fluorescence labelled multiplex amplification primer set containing human genome DNA 22 loci, kit and application thereof |
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| CN201611247192.1A CN106591463A (en) | 2016-12-29 | 2016-12-29 | Fluorescence labelled multiplex amplification primer set containing human genome DNA 22 loci, kit and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107267643A (en) * | 2017-07-28 | 2017-10-20 | 公安部物证鉴定中心 | Detect reagent set and the application of human sample |
| CN114703291A (en) * | 2022-02-17 | 2022-07-05 | 苏州市公安局 | Eight-color fluorescence composite amplification kit for detecting micro-degradation DNA and application thereof |
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| CN103451311A (en) * | 2013-09-24 | 2013-12-18 | 无锡中德美联生物技术有限公司 | Kit for simultaneous analysis of fluorescent mark composite amplification of 26 loca of human genome DNA and using method and application of kit |
| CN104745691A (en) * | 2015-03-02 | 2015-07-01 | 无锡中德美联生物技术有限公司 | Primer group complexly amplified by fluorescent mark for synchronously analyzing 27 genetic locus of human genomic DNA, kit and application |
| CN105018597A (en) * | 2015-05-27 | 2015-11-04 | 宁波海尔施基因科技有限公司 | Kit for multiplex amplification of 34 loci of human genomic DNA |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451311A (en) * | 2013-09-24 | 2013-12-18 | 无锡中德美联生物技术有限公司 | Kit for simultaneous analysis of fluorescent mark composite amplification of 26 loca of human genome DNA and using method and application of kit |
| CN104745691A (en) * | 2015-03-02 | 2015-07-01 | 无锡中德美联生物技术有限公司 | Primer group complexly amplified by fluorescent mark for synchronously analyzing 27 genetic locus of human genomic DNA, kit and application |
| CN105018597A (en) * | 2015-05-27 | 2015-11-04 | 宁波海尔施基因科技有限公司 | Kit for multiplex amplification of 34 loci of human genomic DNA |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107267643A (en) * | 2017-07-28 | 2017-10-20 | 公安部物证鉴定中心 | Detect reagent set and the application of human sample |
| CN114703291A (en) * | 2022-02-17 | 2022-07-05 | 苏州市公安局 | Eight-color fluorescence composite amplification kit for detecting micro-degradation DNA and application thereof |
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