CN113046430B - Amplification composition for chromosome aneuploid number abnormality and application thereof - Google Patents
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- 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
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Abstract
The invention relates to an amplification composition for chromosome aneuploidy number abnormality, a kit and application thereof.
Description
Technical Field
The invention belongs to the field of gene detection, and particularly relates to an amplification composition for chromosome aneuploidy number abnormality and application thereof.
Background
Common autosomal aneuploidy diseases include trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), trisomy 13 (Patau syndrome), and some sex chromosome aberrations such as Klinefelter syndrome (47, XXY) and Turner syndrome (45, X). Currently, karyotyping is the diagnostic gold standard in the clinic, however, this technique is very limited. Amniocentesis is required to take amniotic fluid, then fetal cells in the amniotic fluid are cultured, and then karyotype analysis is carried out. The required amniotic fluid sample amount is large, about 20mL of amniotic fluid sample is required for one-time amniotic fluid cell culture, the analysis period time is long, generally about 2-3 weeks, great mental stress is brought to the pregnant woman and the family in the result waiting process, the operation is complex, and the operation is required by experienced detection personnel. In addition, Fluorescence In Situ Hybridization (FISH), which is a relatively popular technique used in recent years, is a method that is slightly faster and does not require cell culture, and is performed by using a known nucleic acid sequence as a probe, labeling the probe with fluorescein directly or labeling the probe with a non-radioactive substance, and then hybridizing the probe with a target DNA. And connecting the fluorescein marker through an immunocytochemistry process, and finally observing a hybridization signal under a fluorescence microscope so as to perform qualitative, positioning and quantitative analysis on the nucleic acid to be detected in the sample. The method has the characteristics of high cost, long period and high requirement on operators, and meanwhile, the result is a picture, manual interpretation or intervention is needed, and automation and high-throughput analysis are difficult to realize.
The QF-PCR technology is characterized in that different regions of sample DNA are subjected to PCR amplification through fluorescent primers, amplified fragments are separated through capillary electrophoresis, the length of the amplified fragments is determined through a fluorescent detection system, polymorphism sites are classified, amplified products of expected sizes are quantified in an equal area correspondingly through scanning software, and the original template is quantified through experiments, so that the QF-PCR technology can be applied to aneuploid disease detection. In QF-PCR, multiple Short Tandem Repeat (STR) loci on 13, 18, 21, X and Y chromosomes are amplified simultaneously, and the number of chromosomes is judged by using polymorphism of STR loci. The abnormal aneuploidy number is a disease caused by the increase or decrease of one chromosome number, so that the STR locus on the chromosome can obtain 1:1:1 or 2: 1, wherein the first two cases are characteristic of a trisomy syndrome sample relative to a normal sample. The higher the STR locus polymorphism, the lower the probability of single peak occurrence, and the greater the ratio of the first two occurrences. And detecting STR loci on a plurality of specific chromosomes, and comprehensively considering the number and area of peaks of each locus to judge whether the detected sample has trisomy syndrome. And the QF-PCR technology has short detection period, and the result can be obtained 5 hours after the sample is obtained. The genomic DNA extracted from the amniotic fluid cells is used for amplification, and the amplification time and the detection time are 5 hours in total, so that the speed is greatly increased.
In the prior art, the amplification of 13, 18, 21, X and Y chromosome aneuploid number abnormality based on QF-PCR is provided, for example, in the applicant's early patent CN104651488A, but in practice, the method has certain problems of detection efficiency and sensitivity, and cannot meet some practical applications.
The invention is provided in view of the above.
Disclosure of Invention
The primary object of the present invention is to find a method for amplifying or detecting the abnormal number of chromosome aneuploidies, which has significantly improved detection efficiency and sensitivity.
Another object of the present invention is to find an amplification composition for chromosomal aneuploidy number abnormality with significantly improved detection efficiency and sensitivity and a detection kit thereof.
In order to achieve the above purpose, the present invention is based on the amplification composition and the rapid detection kit for detecting the chromosome aneuploid number abnormality in the applicant's earlier patent CN104651488A, and the technical improvement is as follows: the STR locus arrangement and site selection are improved, the improved kit not only has higher amplification detection efficiency, but also integrates a Uracil DNA Glycosylase (UDG) anti-pollution system, and can digest the amplification product possibly containing uracil before PCR amplification, thereby avoiding product pollution and having higher detection result accuracy.
Specifically, the invention firstly provides an amplification composition for amplifying or detecting the abnormal number of chromosome aneuploidies, which is characterized by comprising the following primers:
the primers are directed against D13S256, D13S797, DXS6809, DXS9895, D21S2052, TAF9L, D18S535, D13S317, D21S1411, D18S1002, D13S305, AMEL, D18S877, LFG21, ZFXY, D18S851, D21S1435, D18S391, D13S800, D21S1246, XHPRT, D13S325, GATA165B12, D21S1446, D21S11, and SRY, respectively.
In some embodiments, the primer sequence is set forth in SEQ ID NO. 1-52.
Further, the concentrations or amounts of primers for D13S256, D13S797, DXS6809, DXS9895, D21S2052, TAF9L, D18S535, D13S317, D21S1411, D18S1002, D13S305, AMEL, D18S877, LFG21, ZFXY, D18S851, D21S1435, D18S391, D13S800, D21S1246, XHPRT, D13S325, GATA165B12, D21S1446, D21S11, and SRY are as shown in table 5 or as follows: 1: 0.35: 0.65: 1.2: 0.35: 0.3: 3.1: 0.35: 1.5: 1.3: 0.65: 0.75: 0.85: 0.7: 0.75: 1.15: 0.5: 1.65: 0.35: 3: 0.45: 0.65: 0.6: 0.6: 0.9: 0.4.
further, the primers can be divided into five groups:
the first group is D13S256, D13S797, DXS6809, DXS9895, D21S 2052; the second group is TAF9L, D18S535, D13S317, D21S1411, D18S1002, D13S 305; the third group is AMEL, D18S877, LFG21, ZFXY, D18S851, D21S 1435; the fourth group is D18S391, D13S800, D21S1246, XHPRT, D13S 325; the fifth group is GATA165B12, D21S1446, D21S11, SRY; in some embodiments, different sets of primers carry different fluorescent labels;
preferably, the first set is labeled with FAM, the second set with HEX, the third set with L552, the fourth set with LR600, and the fifth set with TET 592.
The invention also provides any one of the following uses of the amplification composition:
1) use in detecting abnormal numbers of chromosomal aneuploidies;
2) the application in preparing a reagent or a kit for detecting the chromosome aneuploid number abnormality;
3) use in the diagnosis of a disease associated with chromosomal aneuploidy abnormalities;
4) the application in preparing a diagnostic reagent or a kit for diagnosing diseases related to chromosome aneuploid abnormality.
In some embodiments, in a particular detection or diagnostic use, the analysis of results is performed by the following criteria:
and analyzing the data of the amplification detection product after capillary electrophoresis, wherein the judgment standard of the analysis result is as follows:
the normal locus is shown as a double peak, and the peak area ratio is between 0.8 and 1.4; the abnormal site is represented as a trimodal point, the peak area ratio is close to 1:1:1, or is represented as a bimodal point, and the peak area ratio is 0.45-0.65 or 1.8-2.4; the peak area ratio of the invalid site is between the normal site and the abnormal site;
in some preferred embodiments, the specific criteria are as follows:
1) and (4) judging a normal result:
at least two STR sites are normal sites and are shown as double peaks, the ratio of the front peak area to the rear peak area is 0.8-1.4, and the rest sites are invalid sites (the ratio of the peak areas is between the normal sites and the abnormal sites);
2)13, 18 and 21 chromosome aneuploidy result judgment:
13. at least two STR genetic loci on chromosome 18 or 21 are abnormal loci and are represented as three peaks, the peak area ratio is close to 1:1:1, or are represented as two peaks, the peak area ratio is between 0.45-0.65 or 1.8-2.4, and the peak area ratio of the rest loci are invalid loci and is out of the interval between normal loci and abnormal loci.
The invention also provides a kit for detecting the chromosome aneuploid number abnormality, which is characterized by comprising the amplification composition for detecting the chromosome aneuploid number abnormality.
In some embodiments, the kit further comprises a PCR reaction solution, an enzyme mixture solution and a quality control product;
in some embodiments, the enzyme cocktail comprises a DNA polymerase and a UDG enzyme;
in some embodiments, the PCR reaction solution comprises dUTP, preferably, dUTP/(dTTP + dUTP) is 0.1 to 0.5;
in other embodiments, the quality control product further comprises a trisomy 21 positive quality control product and the like.
The invention also provides an amplification or detection method for the chromosome aneuploid number abnormality, which is characterized by comprising the step of carrying out amplification detection by using the amplification composition or the kit.
Further, the amplification detection product is subjected to data analysis after capillary electrophoresis, and the judgment standard of the analysis result is as follows:
the normal locus is shown as a double peak, and the peak area ratio is between 0.8 and 1.4; the abnormal site is represented as a trimodal point, the peak area ratio is close to 1:1:1, or is represented as a bimodal point, and the peak area ratio is 0.45-0.65 or 1.8-2.4; the peak area ratio of the invalid site is between the normal site and the abnormal site;
in some preferred embodiments, the specific criteria are as follows:
1) and (4) judging a normal result:
at least two STR sites are normal sites and are shown as double peaks, the ratio of the front peak area to the rear peak area is 0.8-1.4, and the rest sites are invalid sites (the ratio of the peak areas is between the normal sites and the abnormal sites);
2)13, 18 and 21 chromosome aneuploidy result judgment:
13. at least two STR genetic loci on chromosome 18 or 21 are abnormal loci and are represented as three peaks, the peak area ratio is close to 1:1:1, or are represented as two peaks, the peak area ratio is between 0.45-0.65 or 1.8-2.4, and the peak area ratio of the rest loci are invalid loci and is out of the interval between normal loci and abnormal loci.
Compared with the prior art, the invention at least has the following beneficial technical effects:
(1) compared with the early patents of the applicant, the invention obviously improves the efficiency, sensitivity and accuracy of amplification detection by optimizing and adjusting detection sites, primer sequences, groups, dosage, reaction systems and the like. The small PCR product is easier to be effectively amplified than the large PCR product, the PCR amplification products are all within 400bp, and the energy transfer primer is used, so that the amplification detection efficiency is higher.
(2) Compared with the traditional method, the method has higher sensitivity, only nanogram-level DNA is needed for each detection, the required sample amount is small, and 0.5-1mL of amniotic fluid can meet the requirement; the method has high accuracy, and compared with Fluorescence In Situ Hybridization (FISH), the method has low requirements on operator operation and has less pollution probability. Because the reliable PCR technology and the detection of the genetic analyzer are adopted, the automatic and digital data analysis can be realized, the manual intervention is not needed, and the result is more objective and accurate; the detection period is short, and the result can be obtained after the sample is obtained and the amplification detection time is fastest 5 hours.
(3) The invention also integrates an anti-pollution system of Uracil DNA Glycosylase (UDG), and can digest the amplification product possibly existing and containing uracil before PCR amplification, thereby avoiding product pollution and having higher accuracy of detection result; the pollution condition of the maternal cells of the sample can be clearly judged.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph of test results for normal males;
FIG. 2 is a graph of test results for normal females;
FIG. 313 is a graph showing the results of trisomy syndrome detection;
FIG. 418 is a graph showing the results of trisomy syndrome detection;
FIG. 521 is a graph showing the results of detection of trisomy syndrome;
FIG. 6 is a diagram of the results of syndrome XXY;
FIG. 7 is a graph of the results of a XYY syndrome test;
FIG. 8X is a graph of the results of monomeric syndrome testing;
FIG. 9 is a graph of XXX syndrome detection results;
FIG. 10 is a chart showing the results of 1 ng/. mu.L of trisomy syndrome CN104651488A 13
FIG. 11 is a graph of 2 ng/. mu.L of trisomy syndrome CN104651488A 13 in patent CN 8913
FIG. 12 is a diagram showing the results of the 1 ng/. mu.L assay for trisomy 13 syndrome according to the invention
FIG. 13 is a graph showing the results of 2 ng/. mu.L detection of trisomy 13 syndrome according to the present invention
FIG. 14 is a comparison of the peak height of trisomy syndrome CN104651488A 13, wherein the upper graph is a peak of CN104651488A, and the lower graph is a peak of trisomy syndrome.
FIG. 15 is a comparison of the peak height of the syndrome of CN104651488A XXY in the present invention, wherein the upper graph is the peak graph of CN104651488A, and the lower graph is the peak graph of the present patent.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The following terms or definitions are provided only to aid in understanding the present invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.
Unless defined otherwise below, all technical and scientific terms used in the detailed description of the present invention are intended to have the same meaning as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.
As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If in the following a certain group is defined to comprise at least a certain number of embodiments, this should also be understood as disclosing a group which preferably only consists of these embodiments.
Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun.
The terms "about" and "substantially" in the present invention denote an interval of accuracy that can be understood by a person skilled in the art, which still guarantees the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.
Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
The term "nucleic acid" or "nucleic acid sequence" in the present invention refers to any molecule, preferably polymeric molecule, comprising units of ribonucleic acid, deoxyribonucleic acid, or analogues thereof. The nucleic acid may be single-stranded or double-stranded. The single-stranded nucleic acid may be a nucleic acid that denatures one strand of a double-stranded DNA. Alternatively, the single-stranded nucleic acid may be a single-stranded nucleic acid not derived from any double-stranded DNA.
The term "complementary" as used herein relates to hydrogen bonding base pairing between nucleotide bases G, A, T, C and U, such that when two given polynucleotides or polynucleotide sequences anneal to each other, a pairs with T, G pairs with C in DNA, G pairs with C, and a pairs with U in RNA.
Example 1 optimization of the reaction System of the invention
The present embodiment is improved by adjusting and optimizing parameters in relation to the earlier patent CN 104651488A.
1) Site selection optimization
The D18S51 site repeat sequence in the patent CN104651488A is (AGAA), and the repeat number is 7-27. There are more than 50 alleles reported for this locus. Penta D site repeat sequence (AAAGA), repeat number 2.2-17. The repetition times of the D18S51 and PentaD sites are greatly different, the maximum distance between two allelic gene peaks at the D18S51 site can reach 80bp, the maximum distance between two allelic gene peaks at the PentaD site can reach 71.5bp, the distance is large, the detection of the chromosome aneuploidy number abnormality needs to depend on the ratio of the peak areas of the allelic gene peaks, and the dominant amplification of small segments can lead the situation of allelic gene imbalance in normal people to interfere with judgment. Therefore, the present invention attempts to delete the D18S51 and PentaD sites and adjust the system to seek an increase in effect.
In addition, in patent CN104651488A, there are only 3 specific STR loci on the X chromosome, and in view of the accuracy of detection, the present invention further attempts to increase the specific STR loci on the X chromosome. The patent investigates the genetic polymorphism of STR loci on 16X chromosomes in people, and determines that GATA165B12 loci are added in a system for detecting the abnormal number of the X chromosomes through optimized screening, so that the effect is optimal.
2) Primer sequence optimization
Considering that when a DNA sample contains inhibitors such as hemoglobin, the amplification result shows that large STR alleles are lost, and even all loci fail to amplify. Seeking to reduce the size of STR amplicons may help to obtain information from a suppressed sample.
Therefore, the primer modification is carried out on the site of the CN104651488A PCR amplification product which is larger than 400bp, in the primer modification process, due to the change of the primer in the amplification system, the new primer interacts with the primer of other sites, so that the amplification efficiency of the primer is reduced or non-specific amplification is caused, and partial sites of the PCR amplification product which are smaller than 400bp also need to be modified. The sequence of the primer designed initially in the primer modification and optimization process is shown in table 1:
TABLE 1 primer sequences during primer modification
Primers in the above table were amplified at a single site to confirm primer specificity, and the results of primer-specific amplification are shown in table 2:
TABLE 2 primer-specific amplification results
According to the specific amplification result of the primers, primers with specific primers, PCR amplification products of less than 400bp and good amplification efficiency (peak height) of the primers are comprehensively selected to carry out composite amplification primers of 26 sites, and finally, the comprehensively selected primer sequences are shown as SEQ ID NO. 1-52.
Grouping the primers again according to the difficulty of sequences before and after the STR locus repetitive structure in primer design under the condition of ensuring the original locus primer sequence unchanged as much as possible, finally, dividing the optimized primers into five groups, wherein each group of primers is provided with different fluorescent markers, and the first group is D13S256, D13S797, DXS6809, DXS9895 and D21S2052 and is marked by FAM; the second group is TAF9L, D18S535, D13S317, D21S1411, D18S1002, D13S305, labeled HEX; the third group is AMEL, D18S877, LFG21, ZFXY, D18S851, D21S1435, marked with L552; the fourth group is D18S391, D13S800, D21S1246, XHPRT, D13S325, marked with LR 600; the fifth group was GATA165B12, D21S1446, D21S11, SRY, labeled with TET 592. Third, fourth and fifth sets enhanced PCR using energy transfer primers.
3) Primer dosage optimization
The primer ratio affects the peak area and the ratio of the peak areas, thereby affecting the detection result, and therefore, the amount of the primer used in the amplification system needs to be optimized. And 3, a primer mixed solution final concentration group after preliminary optimization is obtained, PCR amplification is carried out after the primer mixed solution is prepared according to the final concentration of the primers in the system in the table, adjustment is carried out according to an amplification result, and the final adjustment result meets the requirements of 30-100% of the balance in the same color group and 20-100% of the balance among color groups.
TABLE 3 final concentration of primer mixture
Taking 1 sample in the experiment as an example, the balance of the amplification samples with the final concentration of the primer mixture solution of 3 proportions is shown in table 4, the final concentration 3 of the primer mixture solution meets the balance of 30-100% in the same color group, and the balance of 20-100% among the color groups.
TABLE 4 equality
The primer sequences and the primer dosage ratios are determined by optimizing the primer sequences and the primer dosages as shown in Table 5
TABLE 5 primer sequences and dosage ratios
4) Improved amplification system
The invention further integrates a Uracil DNA Glycosylase (UDG) anti-pollution system, and digests the amplification product possibly existing and containing uracil before PCR amplification, thereby avoiding product pollution. The use amount of dUTP, the use amount of UDG enzyme and the incubation time in the anti-pollution system are searched, and the result shows that better anti-pollution effect can be obtained when dUTP/(dTTP + dUTP) is 0.1-0.5 and the incubation is carried out for 5-10min at 37 ℃.
Example 2 preparation of the detection kit of the invention and establishment of the detection method
According to the optimized parameter system, primers, enzyme, buffer solution and the like are assembled into the kit, specifically, the kit comprises PCR reaction solution, primer mixed solution, enzyme mixed solution, quality control products and the like, and specifically, the kit comprises the following components:
the detection method of the kit is established as follows:
DNA extraction
Extracting DNA by using a genome DNA extraction kit, carrying out the operation steps according to the kit specification, quantifying by using an ultraviolet spectrophotometer after the DNA extraction is finished, and diluting to 2-10 ng/mu l
2. Multiplex PCR amplification
2.1 PCR reaction system preparation is carried out according to the following table, vortex oscillation and mixing are carried out, and then centrifugation is carried out for a short time, so that liquid is gathered at the bottom of the tube.
2.2PCR reaction conditions:
2.3 capillary electrophoresis detection
Before detection, the product is denatured and mixed with the molecular weight internal standard, 1 mul of the product +0.5 mul of the molecular weight internal standard +8.5 mul of HIDI (deionized formamide) is mixed uniformly and centrifuged for a short time, and after denaturation at 95 ℃ for 5min, the mixture is immediately subjected to ice bath for 10min, and is subjected to electrophoresis detection by an ABI series genetic analyzer.
2.4 quality control Standard
(1) The detection result of the nuclease-free pure water is that no PCR product is amplified.
(2) The detection result of the trisomy 21 positive quality control product is trisomy 21 positive.
When the above (1) and (2) are satisfied simultaneously, the PCR amplification is successful, otherwise, re-experiment is required.
3. Analysis of results
Data obtained by capillary electrophoresis is analyzed by using GeneMapper software, the amount of the amplified product can be represented by the peak area due to the fact that the fluorescence is marked by the amplification primers, and whether the number of 21, 18 and 13 chromosomes of the sample is abnormal or not is judged by the peak area ratio of the number of peaks appearing at STR sites. The criteria are as follows: normal sites (showing double peaks with peak area ratio between 0.8-1.4); abnormal sites (appearing as three peaks with peak area ratio close to 1:1:1, or appearing as two peaks with peak area ratio between 0.45-0.65 or 1.8-2.4); null sites (peak area ratios outside the interval between normal and abnormal sites).
3.1 judging a normal result:
at least two STR sites are normal sites (showing double peaks, and the ratio of the front peak area to the rear peak area is between 0.8 and 1.4), and the rest sites are invalid sites (the ratio of the peak areas is between the normal sites and the abnormal sites). As shown in fig. 1-2.
3.213, 18, 21 chromosome aneuploidy result judgment:
13. at least two STR genetic loci on chromosome 18 or 21 are abnormal loci (shown as three peaks, the peak area ratio is close to 1:1:1, or shown as two peaks, the peak area ratio is between 0.45-0.65 or 1.8-2.4), and the rest loci are invalid loci (the peak area ratio is outside the interval of normal loci and abnormal loci), as shown in figure 3-figure 5.
3.3 sex chromosome outcome determination: as shown in fig. 6-9.
Example 3 comparison of the Effect of the invention with CN104651488A
Samples of 1 example of trisomy 21, trisomy 18, trisomy 13, monosomy X, XXX, XXY and XYY were tested and compared using CN104651488A and the amplification system of the present invention.
Compared with patent CN104651488A, the invention has significant advantages, which are as follows:
1) the detection sensitivity is higher
1 example 13 trisomy syndrome genome DNA is diluted to 1 ng/muL, 2 ng/muL and 4 muL/muL in gradient, the invention and patent CN104651488A detect the DNA diluted in gradient simultaneously, and the detection is repeated for 3 times. The results are shown in Table 6.
TABLE 6 results of sensitivity detection
The detection sensitivity of the method disclosed by the patent CN104651488A is 2 ng/mu L, the detection sensitivity of the method disclosed by the invention is 1 ng/mu L, and the detection sensitivity of the method disclosed by the invention is higher. The detection result maps of the patent CN104651488A and the invention at 1 ng/. mu.L and 2 ng/. mu.L are shown in figures 10-13.
2) The amplification detection efficiency is better
Samples of 1 example trisomy 21 syndrome, 1 example trisomy 18 syndrome, 1 example trisomy 13 syndrome, 1 example monosomy X syndrome, 1 example XXX syndrome, 1 example XXY syndrome and 1 example XYY syndrome were diluted to 2 ng/. mu.l and 4. mu.l/. mu.l. The invention and patent CN104651488A detect 2 ng/muL and 4 muL/muL DNA template at the same time, compare the average value of the peak height, see Table 7.
TABLE 7 mean values of measured peak heights
The detection rate of the invention and the detection rate of the patent CN104651488A are both 100%, the higher the detection peak height value is, the better the detection efficiency is, and the average value of the detection peak height of the invention is superior to that of the patent CN104651488A when the detection is carried out on the DNA templates of 2 ng/muL and 4 muL/muL, and the detection efficiency of the invention is better. Taking 1 color group of 13 trisomy syndrome and XXY syndrome sample 2 ng/. mu.L as an example, the ordinate represents the peak height value, and the ordinate is adjusted to the same value and compared by the peak height. The peak height chart of the invention compared with patent CN104651488A is shown in figures 14-15.
3) The accuracy is higher: the detection sensitivity and the detection efficiency of the invention are superior to those of the CN104651488A patent, and when the detection template amount is low, the invention can better obtain the detection result, thereby ensuring the accuracy of the detection result.
The comparative experiment shows that the invention is obviously superior to the patent CN104651488A in the aspects of detection sensitivity, detection efficiency and accuracy, and is more suitable for industrial application.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Sequence listing
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Claims (3)
1. An amplification composition for detecting abnormal numbers of chromosomal aneuploidies, said amplification composition comprising primers for:
the primers are primers for D13S256, D13S797, DXS6809, DXS9895, D21S2052, TAF9L, D18S535, D13S317, D21S1411, D18S1002, D13S305, AMEL, D18S877, LFG21, ZFXY, D18S851, D21S1435, D18S391, D13S800, D21S1246, XHPRT, D13S325, GATA165B12, D21S1446, D21S11, and SRY, respectively;
the primer sequence is shown in SEQ ID NO. 1-52;
the primer usage ratios for D13S256, D13S797, DXS6809, DXS9895, D21S2052, TAF9L, D18S535, D13S317, D21S1411, D18S1002, D13S305, AMEL, D18S877, LFG21, ZFXY, D18S851, D21S1435, D18S391, D13S800, D21S1246, XHPRT, D13S325, GATA165B12, D21S1446, D21S11 and SRY are:
1 :0.35:0.65 :1.2 :0.35 :0.3 :3.1 :0.35 :1.5 :1.3 :0.65 :0.75 :0.85 :0.7 :0.75 :1.15 :0.5 :1.65 :0.35 :3 :0.45 :0.65 :0.6 :0.6 :0.9 :0.4;
the primers are further divided into five groups:
the first group is for D13S256, D13S797, DXS6809, DXS9895, and D21S 2052;
the second set is for TAF9L, D18S535, D13S317, D21S1411, D18S1002 and D13S 305;
the third group is for AMEL, D18S877, LFG21, ZFXY, D18S851 and D21S 1435;
the fourth group is for D18S391, D13S800, D21S1246, XHPRT and D13S 325;
the fifth group is for GATA165B12, D21S1446, D21S11, and SRY;
the first set of primers was labeled with FAM, the second set of primers was labeled with HEX, the third set of primers was labeled with L552, the fourth set of primers was labeled with LR600, and the fifth set of primers was labeled with TET 592.
2. A kit for detecting a numerical abnormality of chromosomal aneuploidy, comprising the amplification composition for detecting a numerical abnormality of chromosomal aneuploidy of claim 1.
3. Use of the amplification composition of claim 1, wherein the use comprises:
1) the application in preparing a reagent or a kit for detecting the chromosome aneuploid number abnormality;
2) the application of the compound in preparing a diagnostic reagent or a kit for diagnosing diseases related to chromosome aneuploidy abnormality.
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Denomination of invention: An amplification composition with abnormal number of chromosomal aneuploidy and its application Granted publication date: 20220201 Pledgee: Zhongguancun Branch of Bank of Beijing Co.,Ltd. Pledgor: Beijing Yuewei Gene Technology Co.,Ltd. Registration number: Y2024110000027 |