CN105176977A - Primer, kit and method for identifying sex of human individuals and human cell strains - Google Patents
Primer, kit and method for identifying sex of human individuals and human cell strains Download PDFInfo
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Abstract
The invention provides a primer pair for identifying the sex of human individuals and human cell strains, an application of the primer pair, a kit for identifying the sex of human individuals and human cell strains, and an identification method. By utilizing the provided primer, kit, and method, the sex of human individuals and human cell strains can be precisely identified. The false judgment on a female sample can be effectively avoided. Moreover, the operation is convenient, the sensitivity and specificity are high, and the application prospect is good.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind ofly identify human individual, the primer of human archeocyte strain sex, test kit and method.
Background technology
The sex identification of human individual judges at medical jurisprudence, archaeological research, multiple field such as athlete's is significant, in addition, in Science Explorations field, the performance of cell in many cell characteristics in different sexes source is also inconsistent, such as QiuP finds [QiuP, etal.Genderdependedpotentialityofdifferentiationofhumanu mbilicalcordmesenchymalstemcellsintooocyte-Likecellsinvi tro.CellBiochemFunct.2013, 31 (5): 365-373]: be people source umbilical cord mesenchymal stem cells (hUC-MSCs) equally, from the hUC-MSCs of female individual when differentiation-inducing to female sex cell, relative to the hUC-MSCs from male individual, the marker gene expressing female reproduction differentiation is faster, the class ovum individuality formed is larger, in culture supernatant, estrogenic generation is more, research [the Ghasemzadeh-HasankolaeiMetal.Maleandfemaleratbonemarrow-derivedmesenchymalstemcellsaredifferentintermsoftheexpre ssionofgermcellspecificgenes.AnatSciInt.2015 of Ghasemzadeh-HasankolaeiM, 90 (3): 187-196] show: the mescenchymal stem cell from female individual more trends towards to female reproductive cell differentiation, and more trends towards breaking up to male germ cell from the mescenchymal stem cell of male individual, our study group also finds in an experiment: the hUC-MSCs growth velocity from different sexes individuality there are differences.Therefore, in order to full appreciation individual character, advance science exploration, and sex identification is most important.
Utilizing round pcr to carry out sex identification is current most widely used method, comprises regular-PCR method, labelled by nested-PCR method, fluorescence quantitative PCR method.Wherein, fluorescence quantifying PCR method (qPCR), because it is easy, directly perceived, sensitivity is higher, has broad application prospects.Above-mentioned detection method is all by detecting in sample whether judge gender containing fragment on Y chromosome, bring distinct issues thus, namely in operating process, women's sample may pollute a small amount of male sex DNA and cause Tomboyishness, affects the accuracy of judged result.
Summary of the invention
The object of the present invention is to provide a kind of can precise Identification human individual, the primer of human archeocyte strain sex, test kit and method.
The invention provides the primer pair shown in the primer pair shown in SEQIDNO:1-2 or SEQIDNO:3-4.
Present invention also offers the primer pair 1 shown in SEQIDNO:1-2 and the purposes of the primer pair shown in SEQIDNO:3-4 2 in discriminating human individual, human archeocyte strain sex.
Present invention also offers the primer pair 1 shown in SEQIDNO:1-2 and the purposes of the primer pair shown in SEQIDNO:3-4 2 in the reagent of preparation discriminating human individual, human archeocyte strain sex.
The present invention identifies the test kit of human individual, human archeocyte strain sex, comprises the primer pair 1 shown in SEQIDNO:1-2 and the primer pair shown in SEQIDNO:3-4 2, for NROB1 gene and the Sry gene of increasing respectively.
The invention provides the purposes of mentioned reagent box in qualification human individual, human archeocyte strain sex.
The present invention identifies human individual, human archeocyte strain method for distinguishing, comprises the steps:
A, extract the DNA of sample to be checked;
B, with the DNA of sample to be checked for template, carry out quantitative pcr amplification NROB1 and Sry gene respectively with the primer pair 2 shown in the primer pair 1 shown in SEQIDNO:1-2 and SEQIDNO:3-4;
C, interpretation of result: analyze amplification, according to the CT value of NROB1, Sry gene amplification, calculate the relative expression quantity ratio NROB1/Sry of NROB1, Sry gene in sample to be checked: if ratio is greater than 10 for women, and ratio is less than 10 for the male sex.
CT value: English Cyclethreshold, Chinese cycle threshold by name, implication for: the cycle number experienced when the fluorescent signal in each reaction tubes arrives setting threshold value, the template original bulk of CT value and qPCR reaction system is negative correlation.
Wherein: the sample to be checked described in step a is human individual or human archeocyte strain.
Crossed contamination slight unavoidably in experimental implementation, if pollute a small amount of male sex's sample in women's sample, when using ordinary method to detect, have the signal from the slight chromogene of male sex's sample, at this moment, because seeing this signal, operator probably thinks that this is male sex's sample by mistake, thus cause women's sample Tomboyishness phenomenon.But if use the inventive method, a small amount of male sex's sample polluted essence can not change the large general layout of ratio, still can much larger than 10, and at this moment, according to ratio, operator still judges that it is women's sample, thus avoids the Tomboyishness phenomenon of women's sample.
In addition, use the inventive method, the false women's phenomenon of male sex's sample can also be reduced.If male sex sample extraction DNA is not smooth, have lost more DNA, this is that pcr template amount can corresponding reduction, if by ordinary method, gene signal on y karyomit(e) can be very weak, easily thought by mistake there is no signal by operator, and judge that it is women, cause the false women's phenomenon of male sex's sample, if but use present method, the signal of ratio from an x chromogene and the signal of a y chromogene, even if DNA profiling amount is low, from the signal of x chromogene with reduce from the signal of y chromogene simultaneously, ratio still changes not quite, like this according to ratio, still the male sex is judged as, thus avoid the false women's phenomenon of male sex's sample.
Primer provided by the invention, test kit and method, calculated the relative expression quantity ratio NROB1/Sry of NROB1, Sry gene in sample to be checked by quantitative fluorescent PCR, ratio is greater than 10 and is judged as women, and ratio is less than 10 and is judged as the male sex; Primer provided by the invention is highly sensitive, high specificity; Utilize primer of the present invention, test kit and method can precise Identification human individual, human archeocyte strain sex, effectively reduce women's sample Tomboyishness phenomenon, have a good application prospect.
Obviously, according to foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, not departing under the present invention's above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
Accompanying drawing explanation
Fig. 1 is to the chromosome analysis result of the sampling male sex.
Fig. 2 qPCR reaction conditions.
Fig. 3 qPCR amplification figure; Wherein: A is the negative amplification curve diagram for NROB1 gene when not adding DNA profiling, visible without clear signal; B is the negative amplification for Sry gene when not adding DNA profiling, as seen also without clear signal; C is the amplification curve diagram for NROB1 gene; D is the amplification curve diagram for Sry gene; E is the melt curve analysis figure for NROB1 gene; F is the melt curve analysis figure for Sry gene.
Fig. 4 is to the chromosome analysis result of sampling women.
Embodiment
Be described further with embodiment below, but the present invention is not limited to these embodiments.
The present invention's experiment reagent used, instrument are as follows:
Whole Blood Genomic DNA rapid extraction test kit (solution-type) DP1101: capital hundred Tyke Bioisystech Co., Ltd;
iQSybrGreenSupermix：Bio-Rad，USA；
The high sugared nutrient solution of DMEM, cell cultures dual anti-, newborn calf serum, cell cultures trypsinase, cell cultures phosphoric acid buffer: all biological from Chengdu Harry;
CFX96qPCR instrument: Bio-Rad, USA.
The design of embodiment 1 primer of the present invention
1, the gene for quantitative fluorescent PCR is selected
The term single gene of people's X chromosome is chosen to be NROB1, and it is positioned at the Xp21 of X chromosome, 2 exons, 1 intron, a kind of orphan proteins matter of Codocyte nuclear receptor superfamily;
The term single gene of people's Y chromosome is chosen to be Sry, and it is positioned at the Yp11.3 of Y chromosome, and 1 exon, does not have intron, and coding determines the protein of biological male sex.
2, design of primers
For the primer information of NROB1 and Sry in table 1.
Table 1 primer information
3, primer carries out the checking of quantitative fluorescent PCR
1) DNA profiling is prepared:
Get the peripheral blood of healthy male, utilize Whole Blood Genomic DNA rapid extraction test kit (solution-type) to extract blood DNA, obtain male sex DNA.
Chromosome analysis is carried out to the male sex of sampling, the results are shown in Figure 1.
As seen from Figure 1, a male sex's sample obviously visible X sex chromosome and Y sex chromosome of sampling, confirms that the individuality in DNA source is the male sex on genomic sense really.
2) quantitative fluorescent PCR: with the male sex DNA extracted for template, adopts NROB1 primer and Sry primer respectively, carries out qPCR detection; Reaction is undertaken by the specification sheets of test kit iQSybrGreenSupermix: qPCR reacts employing 20 μ l system, wherein the DNA profiling amount of each reaction system is 5ng, the amount of forward and reverse primer is all 20pg, the amount of iQSybrGreenSupermix is 10 μ l, reaches 20 μ l with the pure water polishing volume without RNA enzyme and DNA enzymatic.QPCR reaction conditions is shown in Fig. 2.
3) primer amplification result
The present invention's two pairs of primers used carry out the amplification curve of qPCR reaction, melt curve analysis is shown in Fig. 3.
As seen from Figure 3, the present invention is for the primer pair of the NROB1 gene that increases and the primer pair for the Sry gene that increases, the two is all obvious to the expanding effect of template DNA, melt curve analysis is unimodal, and peak is obviously most advanced and sophisticated, therefore, fluorescence quantification PCR primer specificity provided by the invention is good, reliable to the amplification of NROB1 gene and Sry gene.
Embodiment 2 the present invention property identified method for distinguishing
1, the DNA of the blood middle leukocytes of the people to be detected or DNA of human archeocyte strain to be detected is extracted;
2, with the DNA extracted for template, adopt the primer of NROB1 gene of embodiment 1 and the primer of Sry gene respectively, carry out qPCR detection; Reaction is undertaken by the specification sheets of test kit iQSybrGreenSupermix: qPCR reacts employing 20 μ l system, wherein the DNA profiling amount of each reaction system is 5ng, the amount of forward and reverse primer is all 20pg, the amount of iQSybrGreenSupermix is 10 μ l, reaches 20 μ l with the pure water polishing volume without RNA enzyme and DNA enzymatic.QPCR reaction conditions is shown in Fig. 1.
3, after qPCR detects, amplification is analyzed, according to the amplification curve CT value of NROB1, Sry gene, calculates the relative expression quantity ratio NROB1/Sry of NROB1, Sry gene in sample to be checked: if ratio is greater than 10 for women, ratio is less than 10 for the male sex.
Below by the mode of test example, beneficial effect of the present invention is described:
Test example 1 verifies the inventive method with human individual
One, experiment material
Women DNA: the peripheral blood getting healthy women, utilizes Whole Blood Genomic DNA rapid extraction test kit (solution-type) to extract blood DNA, obtains women DNA.
Chromosome analysis is carried out to the women of sampling, the results are shown in Figure 4.
Fig. 4 is visible, women's sample obviously visible two X sex chromosome of sampling, confirms that the individuality in DNA source is the women on genomic sense really.
Male sex DNA: with the male sex DNA of embodiment 1.
Two, the inventive method
With the DNA extracted for template, adopt the primer of NROB1 gene and the primer of Sry gene of embodiment 1 respectively, carry out qPCR detection; QPCR reaction system, reaction conditions and amplification analytical procedure the results are shown in Table 2 with embodiment 2, qPCR.
The NROB1/Sry ratio of women and the male sex verified by table 2
From table 2, the ratio of women is greater than 10000, and the ratio of the male sex is less than 10, utilizes the inventive method can the ratio of sex of precise Identification human individual.
Two, the DNA consumption diluting male sex's sample verifies the inventive method
The male sex DNA of artificial dilution male sex sample, extension rate is from 5 times to 20 times, and except DNA profiling amount, qPCR reaction system, reaction conditions and amplification analytical procedure the results are shown in Table 3 with embodiment 2, qPCR.
Table 3 dilutes male sex's sample and verifies the inventive method
From table 3, male sex's Sample Dilution multiple is higher, and the CT value of NROB1 gene and Sry gene is larger, meets expection; When amount of dilution reaches 20 times (being reduction larger in actually operating), ratio, still near 1, is still less than 10, is still judged as the male sex, and can not cause the false women's phenomenon of male sex's sample, qualification result is accurate.
Be mixed into male sex DNA in test example 2 women sample and verify the inventive method
1, experiment material
Women DNA and male sex DNA: with test example 1.
2, experimental technique
In women's sample, people is for being mixed into a certain amount of male sex DNA, and mixed volume is from accounting for 2.5% to 10% of women's STb gene, and except DNA profiling amount, qPCR reaction system, reaction conditions and amplification analytical procedure the results are shown in Table 4 with embodiment 2, qPCR.
Tomboyishness phenomenon avoided by table 4 women sample
From table 4, the male sex DNA mixed volume be mixed in women's sample is more, and NROB1/Sry ratio is lower, meets expection; When mixed volume reaches 10% (being contamination level larger in actually operating), ratio is 38.07, is still greater than 10, is judged as women, and avoid the phenomenon of the Tomboyishness phenomenon of women's sample, qualification result is accurate.
To sum up, the NROB1/Sry ratioing technigue that the present invention sets up can identify women and male sex's sample, of the present invention
The inventive method is verified in test example 3 human archeocyte strain
1, experiment material
From umbilical cord mesenchymal stem cells (hUC-MSCs) prepared by the umbilical cord tissue of girl baby and boy baby, extract DNA respectively, obtain female's umbilical cord mesenchymal stem cells DNA (female-hUC-MSCs) and male umbilical cord mesenchymal stem cells DNA (man-hUC-MSCs);
From induced multi-potent stem cells (IPS) prepared by the peripheral blood of women, extract DNA, obtain women's induced multi-potent stem cells DNA (women IPS);
From the Hela cell (women's cervical cancer cell) of Sichuan University's cell bank, extract DNA from purchase, obtain HelaDNA (women Hela);
DNA sample all uses Whole Blood Genomic DNA rapid extraction test kit (solution-type) to extract.
2, experimental technique
QPCR reaction system, reaction conditions and amplification analytical procedure are with embodiment 2.
3, experimental result
QPCR result is respectively in table 5, table 6, table 7.
The sex identification of table 5 couple people hUC-MSCs
The sex identification of table 6 couple people women IPS
The sex identification of table 7 pair Hela cell
From table 5, table 6, table 7, to cell strain hUC-MSCs, IPS, Hela of human female origin, NROB1/Sry ratio is all greater than 10 (>110.41), and for male sex hUC-MSCs, ratio is only 0.68, is less than 10; Employ the accuracy that the known human archeocyte strain of four kinds of sexes can verify the inventive method, utilize the inventive method can the sex of precise Identification human archeocyte strain.
Test example 4 sex of the inventive method identifier 293 cell
1, experiment material
From 293 cells of Sichuan University's cell bank, extract DNA from purchase, obtain 293 cell DNAs; DNA sample uses Whole Blood Genomic DNA rapid extraction test kit (solution-type) to extract.
2, experimental technique
QPCR reaction system, reaction conditions and amplification analytical procedure the results are shown in Table 8 with embodiment 2, qPCR.
The sex identification of table 8 couple people 293 cell
From table 8, the NROB1/Sry ratio of 293 cells is 68.75, is greater than 10, so be judged to be women.People 293 cell (ATCC numbers: CRL-1573 is people's Embryonic Kidney cells) is widely used in scientific research, but the sex of this cell is failed to understand, make troubles to scientific research, the sex of qualification 293 cell that the sex identification method using the present invention to set up can be accurate and effective.
To sum up, primer provided by the invention, test kit and method, can precise Identification human individual, human archeocyte strain sex, effectively reduce women's sample Tomboyishness phenomenon, easy and simple to handle, highly sensitive, high specificity, has a good application prospect.
Claims (7)
- Primer pair shown in 1.SEQIDNO:1-2 or the primer pair shown in SEQIDNO:3-4.
- Primer pair 1 shown in 2.SEQIDNO:1-2 and the primer pair shown in SEQIDNO:3-4 2 are differentiating the purposes in human individual, human archeocyte strain sex.
- Primer pair 1 shown in 3.SEQIDNO:1-2 and the primer pair shown in SEQIDNO:3-4 2 differentiate the purposes in the reagent of human individual, human archeocyte strain sex in preparation.
- 4. identify a test kit for human individual, human archeocyte strain sex, it is characterized in that: it comprises the primer pair 1 shown in SEQIDNO:1-2 and the primer pair shown in SEQIDNO:3-4 2, for NROB1 gene and the Sry gene of increasing respectively.
- 5. the purposes of test kit according to claim 4 in qualification human individual, human archeocyte strain sex.
- 6. identify human individual, a human archeocyte strain method for distinguishing, it is characterized in that: comprise the steps:A, extract the DNA of sample to be checked;B, with the DNA of sample to be checked for template, carry out quantitative pcr amplification NROB1 and Sry gene respectively with the primer pair 2 shown in the primer pair 1 shown in SEQIDNO:1-2 and SEQIDNO:3-4;C, interpretation of result: analyze amplification, according to the CT value of NROB1, Sry gene amplification, calculate the relative expression quantity ratio NROB1/Sry of NROB1, Sry gene in sample to be checked: if ratio is greater than 10 for women, and ratio is less than 10 for the male sex.
- 7. method according to claim 6, is characterized in that: the sample to be checked described in step a is human individual or human archeocyte strain.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107119110A (en) * | 2017-03-20 | 2017-09-01 | 谱天福信（天津）分子诊断技术有限公司 | The primer of the TaqMan probe real-time PCR detection of human genome DNA's sry gene in trace sample |
| CN107365768A (en) * | 2017-07-24 | 2017-11-21 | 胡松 | Gene primer combines and unicellular gene order extraction liquid kit |
| CN107885975A (en) * | 2016-09-30 | 2018-04-06 | 有劲生物科技股份有限公司 | Non-intrusion type fetus sex character abnormality detection system |
| CN110438216A (en) * | 2019-09-06 | 2019-11-12 | 成都新生命霍普医学检验实验室有限公司 | It is a kind of to assist identifier box for the kit gene of sex identification and No. 21 syndromes |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107885975A (en) * | 2016-09-30 | 2018-04-06 | 有劲生物科技股份有限公司 | Non-intrusion type fetus sex character abnormality detection system |
| CN107119110A (en) * | 2017-03-20 | 2017-09-01 | 谱天福信（天津）分子诊断技术有限公司 | The primer of the TaqMan probe real-time PCR detection of human genome DNA's sry gene in trace sample |
| CN107365768A (en) * | 2017-07-24 | 2017-11-21 | 胡松 | Gene primer combines and unicellular gene order extraction liquid kit |
| CN110438216A (en) * | 2019-09-06 | 2019-11-12 | 成都新生命霍普医学检验实验室有限公司 | It is a kind of to assist identifier box for the kit gene of sex identification and No. 21 syndromes |
| CN110438216B (en) * | 2019-09-06 | 2023-01-24 | 成都新生命霍普医学检验实验室有限公司 | Gene kit for sex identification and kit for auxiliary identification of chromosome 21 syndrome |
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