CN111763725A - Kit for 21-trisomy risk detection - Google Patents
Kit for 21-trisomy risk detection Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 61
- 208000037280 Trisomy Diseases 0.000 title claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 34
- 201000010374 Down Syndrome Diseases 0.000 claims abstract description 12
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- 108010042407 Endonucleases Proteins 0.000 claims abstract description 11
- 102000004533 Endonucleases Human genes 0.000 claims abstract description 11
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- 210000000349 chromosome Anatomy 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
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- 239000000203 mixture Substances 0.000 claims description 3
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000012165 high-throughput sequencing Methods 0.000 description 2
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- 208000032170 Congenital Abnormalities Diseases 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 201000006347 Intellectual Disability Diseases 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
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Abstract
The invention discloses a kit for detecting a 21-trisomy risk, which comprises an endonuclease buffer solution system and a PCR reaction system, wherein the PCR reaction system comprises a primer group aiming at 8 detection regions EP1-EP 8. The kit removes unmethylated loci in the peripheral blood of the pregnant woman through the optimized restriction endonuclease to realize the separation of fetal DNA, detects 8 regions which can prompt the fetal trisomy risk, and then combines with mature fluorescent quantitative PCR to reflect the fetal trisomy 21-risk. The kit disclosed by the invention is used for detecting by screening a plurality of sites which are related to the risk of trisomy and have relatively stable levels, and 2 control sites are arranged, so that the accuracy of a detection result is improved; the invention can predict the fetal trisomy risk by only detecting a plurality of gene loci, and can overcome the defects of high cost and long detection period caused by high-throughput gene sequencing, thereby becoming a potential new product for noninvasive prenatal diagnosis.
Description
Technical Field
The invention relates to the field of reagents for medical detection, in particular to a kit for 21-trisomy risk detection.
Background
The 21-trisomy (T-21) syndrome, also known as Down syndrome, syndrome of congenital insipidus or Down syndrome, is the most common chromosomal aneuploidy disease in clinical settings. The incidence rates are about 1/700 respectively, and most children suffer from serious intellectual disability and organ malformation. To date, there is no effective treatment for this disease. Therefore, the best way to reduce the risk of children with reproductive chromosomal disorders is to pass prenatal genetic counseling as early as possible along with prenatal detection, diagnosis, discovery and resolution of the problem. Therefore, the T21 test has been the focus of the current clinical diagnosis for controlling and preventing birth defects.
Currently, prenatal diagnosis of fetal chromosomal aneuploidy is mainly performed by screening with down's method based on serology detection, and a pregnant woman with a high risk is subjected to amniotic fluid puncture to obtain cells, and then is subjected to karyotype analysis by using a cytobiology method. In addition, cytogenetic diagnosis is also disadvantageous in that it takes time (generally two weeks or more), culture failure is easy, and minor structural aberration of chromosome cannot be detected. Since 2007, noninvasive prenatal diagnosis technology based on high-throughput sequencing is beginning to be applied clinically, and is now widely used for trisomy screening and genetic disease detection of elderly parturients. However, this method relies on high throughput sequencing and a large number of statistical calculations, and therefore is costly, and requires a detection period of 2 weeks or more and a detection price of about 1200 yuan.
Based on the above research background, establishing a safe, fast, accurate and noninvasive prenatal diagnosis method is still necessary for future clinical technical development.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide a kit for detecting risk of trisomy 21, aiming at the above-mentioned deficiencies in the prior art.
In order to solve the technical problems, the invention adopts the technical scheme that: a kit for detecting the risk of trisomy 21 comprises an endonuclease buffer solution system and a PCR reaction system, wherein the PCR reaction system comprises the following primer groups:
upstream primer for the detection region EP 1: 5'-TCGTTATATGGATGCCTTG-3', downstream primer: 5'-AACTGTTGGGCTGAACTGA-3', respectively;
upstream primer for the detection region EP 2: 5'-TCATTTTCCTCTCGCTCTGC-3', downstream primer: 5'-CACTCAGGGTCCTCAGTTCG-3', respectively;
upstream primer for the detection region EP 3: 5'-GCGCCTAGCACAGACTCTTC-3', downstream primer: 5'-TCAAATACCCGCACAGTTCA-3', respectively;
upstream primer for the detection region EP 4: 5'-TTCTGTGCAACTTTCGCTTG-3', downstream primer: 5'-CGTCTCTCACTCCCTGCACT-3', respectively;
upstream primer for the detection region EP 5: 5'-CCGTGTGGCCAGAGGTG-3', downstream primer: 5'-AAAGGGCCAGGTCGGGA-3', respectively;
upstream primer for the detection region EP 6: 5'-GACCCAGACGATACCTGGAA-3', downstream primer: 5'-GCTGAACAAAACTCGGCTTC-3', respectively;
upstream primer for the detection region EP 7: 5'-TAGCTGGCACCCGCTGG-3', downstream primer: 5'-GTGTGGGGTTGCACGCGC-3', respectively;
upstream primer for the detection region EP 8: 5'-AGACCTCCGGTTTCTGGTTT-3', downstream primer: 5'-TTCCAGAACTTCCCAGCAAC-3', respectively;
wherein, the detection regions are all on chromosome 21, and the gene positions of EP1 are as follows: 42357214 and 42357340bp, the gene position of EP2 is: 38506854-38507163bp, and the gene position of EP3 is: 36706480-36706894bp, the gene position of EP4 is: 44284020-44284209bp, and the gene position of EP5 is as follows: 36980806 and 36980885bp, the gene position of EP6 is: 29136735-29136844bp, the gene position of EP7 is: 50340771-50340692bp, EP8 has the following gene positions: 160711942 and 160711789 bp.
Preferably, the endonuclease buffer system comprises a Cutsmart buffer, an HpaII enzyme, and sterile water.
Preferably, the PCR reaction system further includes a Premix solution and sterile double distilled water.
Preferably, the use method of the kit is as follows:
1) mixing the extracted peripheral blood of the pregnant woman with the gestational period of more than 12 weeks with the endonuclease buffer solution system, culturing in a water bath at 37 ℃ for 4h, and then putting the mixture in a water bath kettle at 80 ℃ for heat inactivation for 20 min;
2) mixing the mixed system obtained in the step 2) with the PCR reaction system to perform PCR amplification reaction;
3) and calculating the expression abundance of EP1-EP6 relative to EP7 and EP8 after the PCR reaction is completed, and judging that the 21-trisomy risk exists when the gene factor of the expression abundance in any group is more than 1.4 and is more than or equal to 4.
The invention has the beneficial effects that:
1. the kit disclosed by the invention is used for detecting by screening a plurality of sites which are related to the risk of trisomy and have relatively stable levels, and 2 control sites are arranged, so that the accuracy of a detection result is improved;
2. the kit can predict the fetal trisomy risk by only detecting a plurality of gene loci, has low cost and short detection time, and can overcome the defects of high cost and long detection period caused by high-throughput gene sequencing, thereby becoming a potential new product for noninvasive prenatal diagnosis;
3. the reagent of the method can be combined with the mature fluorescent quantitative PCR detection technology, can be further expanded, and can be combined with high-sensitivity detection technologies such as digital PCR and electrochemical detection, so that the detection accuracy and sensitivity are further improved.
Drawings
FIG. 1 shows the results of detection by the kit of the present invention;
FIG. 2 shows the results of the test using the amniotic fluid puncture method.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The kit for detecting the risk of trisomy 21 comprises an endonuclease buffer solution system and a PCR reaction system, wherein the PCR reaction system comprises the following primer groups:
upstream primer for the detection region EP 1: 5'-TCGTTATATGGATGCCTTG-3', downstream primer: 5'-AACTGTTGGGCTGAACTGA-3', respectively;
upstream primer for the detection region EP 2: 5'-TCATTTTCCTCTCGCTCTGC-3', downstream primer: 5'-CACTCAGGGTCCTCAGTTCG-3', respectively;
upstream primer for the detection region EP 3: 5'-GCGCCTAGCACAGACTCTTC-3', downstream primer: 5'-TCAAATACCCGCACAGTTCA-3', respectively;
upstream primer for the detection region EP 4: 5'-TTCTGTGCAACTTTCGCTTG-3', downstream primer: 5'-CGTCTCTCACTCCCTGCACT-3', respectively;
upstream primer for the detection region EP 5: 5'-CCGTGTGGCCAGAGGTG-3', downstream primer: 5'-AAAGGGCCAGGTCGGGA-3', respectively;
upstream primer for the detection region EP 6: 5'-GACCCAGACGATACCTGGAA-3', downstream primer: 5'-GCTGAACAAAACTCGGCTTC-3', respectively;
upstream primer for the detection region EP 7: 5'-TAGCTGGCACCCGCTGG-3', downstream primer: 5'-GTGTGGGGTTGCACGCGC-3', respectively;
upstream primer for the detection region EP 8: 5'-AGACCTCCGGTTTCTGGTTT-3', downstream primer: 5'-TTCCAGAACTTCCCAGCAAC-3', respectively;
wherein, the detection regions are all on chromosome 21, and the gene positions of EP1 are as follows: 42357214 and 42357340bp, the gene position of EP2 is: 38506854-38507163bp, and the gene position of EP3 is: 36706480-36706894bp, the gene position of EP4 is: 44284020-44284209bp, and the gene position of EP5 is as follows: 36980806 and 36980885bp, the gene position of EP6 is: 29136735-29136844bp, the gene position of EP7 is: 50340771-50340692bp, EP8 has the following gene positions: 160711942 and 160711789 bp.
Wherein the endonuclease buffer solution system comprises a Cutsmart buffer solution, HpaII enzyme and sterile water. The PCR reaction system also comprises a Premix solution and sterile double distilled water.
The use method of the kit comprises the following steps:
1) mixing the extracted peripheral blood of the pregnant woman with the gestational period of more than 12 with the endonuclease buffer solution system, then culturing in a water bath at 37 ℃ for 4h, and then putting the mixture in a water bath kettle at 80 ℃ for heat inactivation for 20 min;
2) mixing the mixed system obtained in the step 2) with the PCR reaction system to perform PCR amplification reaction;
3) and calculating the expression abundance of EP1-EP6 relative to EP7 and EP8 after the PCR reaction is completed, and judging that the 21-trisomy risk exists when the gene factor of the expression abundance in any group is more than 1.4 and is more than or equal to 4.
The kit removes unmethylated loci in the peripheral blood of the pregnant woman through the optimized restriction endonuclease to realize the separation of fetal DNA, detects 8 regions which can prompt the fetal trisomy risk, and then combines with mature fluorescent quantitative PCR to reflect the fetal trisomy 21-risk. Specific examples are provided below to further illustrate the present invention.
Example 1
The primer group, the concentration and the position of the primer group and the size of a product after PCR reaction in the kit are shown in the following table 1:
TABLE 1
The endonuclease buffer system in the kit comprises 5-fold Cutsmart buffer, HpaII enzyme and sterile water. The PCR reaction system also comprises a Premix solution and sterile double distilled water.
The kit comprises the following steps:
1. extracting peripheral blood of pregnant women with the pregnancy week number of 12, wherein the volume is 4-8mL, extracting free DNA by using a free DNA extraction kit (Sigma, B518253-0050), and detecting the purity and concentration of the obtained DNA: the DNA concentration was measured at 260nm, the protein concentration at 280nm and the salt concentration at 230 nm. The concentration is ensured to be more than or equal to 20 ng/mL; the absorbance at 260 nm/the absorbance at 280nm is between 1.6 and 2.0; meanwhile, the absorbance at 260 nm/the absorbance at 230nm is more than 1.5, so that the salt concentration is not overproof.
2. Then 200ng of the obtained DNA was taken and mixed with 5X of Cutsmart buffer, HpaII enzyme, sterile water to a 50. mu.l system, wherein each final concentration was: DNA 4 ng/. mu.l, HpaII enzyme 0.2U/. mu.l, Cutsmart 1;, the remainder was filled with sterile water.
3. Culturing the obtained mixed system in a 37-degree water bath for 4h, and then thermally inactivating in an 80-degree water bath for 20 min.
4. Mu.l of the resulting mixed system was mixed with a PCR reaction system (commercially available PCR kit: e.g., Takara, DRR036A, used in combination with the primer set of the present invention) in which Premix 10. mu.l, primers 2. mu.l, and sterile double distilled water 6. mu.l were used. The primers involved, the optimized concentrations and the product sizes are shown in table 1.
5. Performing PCR amplification, wherein the amplification program comprises the following steps: 95 degrees 30s, 95 degrees 5s of 40 cycles, 60 degrees 34s, and the matched instrument is an ABI7500PCR instrument.
6. And analyzing the concentration of the DNA template by adopting a PCR instrument with software, and calculating the expression abundance of EP1-EP6 relative to EP7 and EP8, wherein when the base factor of the expression abundance of any group, which is more than 1.4, is more than or equal to 4, the 21-trisome risk is judged to exist. According to the invention, a certain number of clinical trisome samples are counted in advance, the relative abundance of the genes is prompted to reach more than 1.5, and in order to ensure the accuracy, the relative abundance standard is set to be more than 1.4.
Referring to fig. 1 and table 2 below, the test results of the kit of the present invention are:
TABLE 2
| Gene | /EP7 | Error range | /EP8 | Error range |
| EP1 | 2.15 | 0.31 | 2.365 | 0.341 |
| EP2 | 1.84 | 0.21 | 2.024 | 0.231 |
| EP3 | 1.62 | 0.22 | 1.782 | 0.242 |
| EP4 | 1.21 | 0.18 | 1.331 | 0.198 |
| EP5 | 1.51 | 0.22 | 1.661 | 0.242 |
| EP6 | 1.19 | 0.15 | 1.309 | 0.165 |
As can be seen from FIG. 1 and Table 2, the relative expression levels of EP1, EP2, EP3 and EP5 were all greater than 1.4, indicating the risk of trisomy 21.
The pregnant woman is also detected by a amniotic fluid puncture method in a hospital: 20ml of amniotic fluid is extracted, cells are cultured, then slice staining, karyotype detection and manual analysis are carried out (the method is a hospital standard process), the obtained result is shown in figure 2, the result shown in figure 2 shows that the result shows 21-trisomy (T-21) syndrome, and the accuracy of the detection result of the kit can be shown by comparing the gold standard with the detection result of the kit.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
The invention name is as follows: kit for 21-trisomy risk detection
The applicant: jinan national medical science and technology development Co., Ltd
Upstream primer for the detection region EP 1: 5'-TCGTTATATGGATGCCTTG-3' the flow of the air in the air conditioner,
a downstream primer: 5'-AACTGTTGGGCTGAACTGA-3', respectively;
upstream primer for the detection region EP 2: 5'-TCATTTTCCTCTCGCTCTGC-3' the flow of the air in the air conditioner,
a downstream primer: 5'-CACTCAGGGTCCTCAGTTCG-3', respectively;
upstream primer for the detection region EP 3: 5'-GCGCCTAGCACAGACTCTTC-3' the flow of the air in the air conditioner,
a downstream primer: 5'-TCAAATACCCGCACAGTTCA-3', respectively;
upstream primer for the detection region EP 4: 5'-TTCTGTGCAACTTTCGCTTG-3' the flow of the air in the air conditioner,
a downstream primer: 5'-CGTCTCTCACTCCCTGCACT-3', respectively;
upstream primer for the detection region EP 5: 5'-CCGTGTGGCCAGAGGTG-3' the flow of the air in the air conditioner,
a downstream primer: 5'-AAAGGGCCAGGTCGGGA-3', respectively;
upstream primer for the detection region EP 6: 5'-GACCCAGACGATACCTGGAA-3' the flow of the air in the air conditioner,
a downstream primer: 5'-GCTGAACAAAACTCGGCTTC-3', respectively;
upstream primer for the detection region EP 7: 5'-TAGCTGGCACCCGCTGG-3' the flow of the air in the air conditioner,
a downstream primer: 5'-GTGTGGGGTTGCACGCGC-3', respectively;
upstream primer for the detection region EP 8: 5'-AGACCTCCGGTTTCTGGTTT-3' the flow of the air in the air conditioner,
a downstream primer: 5'-TTCCAGAACTTCCCAGCAAC-3' are provided.
Claims (4)
1. The kit for detecting the risk of trisomy 21 is characterized by comprising an endonuclease buffer solution system and a PCR reaction system, wherein the PCR reaction system comprises the following primer groups:
upstream primer for the detection region EP 1: 5'-TCGTTATATGGATGCCTTG-3', downstream primer: 5'-AACTGTTGGGCTGAACTGA-3', respectively;
upstream primer for the detection region EP 2: 5'-TCATTTTCCTCTCGCTCTGC-3', downstream primer: 5'-CACTCAGGGTCCTCAGTTCG-3', respectively;
upstream primer for the detection region EP 3: 5'-GCGCCTAGCACAGACTCTTC-3', downstream primer: 5'-TCAAATACCCGCACAGTTCA-3', respectively;
upstream primer for the detection region EP 4: 5'-TTCTGTGCAACTTTCGCTTG-3', downstream primer: 5'-CGTCTCTCACTCCCTGCACT-3', respectively;
upstream primer for the detection region EP 5: 5'-CCGTGTGGCCAGAGGTG-3', downstream primer: 5'-AAAGGGCCAGGTCGGGA-3', respectively;
upstream primer for the detection region EP 6: 5'-GACCCAGACGATACCTGGAA-3', downstream primer: 5'-GCTGAACAAAACTCGGCTTC-3', respectively;
upstream primer for the detection region EP 7: 5'-TAGCTGGCACCCGCTGG-3', downstream primer: 5'-GTGTGGGGTTGCACGCGC-3', respectively;
upstream primer for the detection region EP 8: 5'-AGACCTCCGGTTTCTGGTTT-3', downstream primer: 5'-TTCCAGAACTTCCCAGCAAC-3', respectively;
wherein, the detection regions are all on chromosome 21, and the gene positions of EP1 are as follows: 42357214 and 42357340bp, the gene position of EP2 is: 38506854-38507163bp, and the gene position of EP3 is: 36706480-36706894bp, the gene position of EP4 is: 44284020-44284209bp, and the gene position of EP5 is as follows: 36980806 and 36980885bp, the gene position of EP6 is: 29136735-29136844bp, the gene position of EP7 is: 50340771-50340692bp, EP8 has the following gene positions: 160711942 and 160711789 bp.
2. The kit for trisomy 21 risk detection according to claim 1, wherein the endonuclease buffer system comprises cutmarst buffer, HpaII enzyme, and sterile water.
3. The kit for trisomy 21 risk detection according to claim 2, wherein the PCR reaction system further comprises a Premix solution and sterile double distilled water.
4. The kit for trisomy 21 risk detection according to claim 3, wherein the kit is used in a method comprising:
1) mixing the extracted peripheral blood of the pregnant woman with the gestational period of more than 12 with the endonuclease buffer solution system, then culturing in a water bath at 37 ℃ for 4h, and then putting the mixture in a water bath kettle at 80 ℃ for heat inactivation for 20 min;
2) mixing the mixed system obtained in the step 2) with the PCR reaction system to perform PCR amplification reaction;
3) and calculating the expression abundance of EP1-EP6 relative to EP7 and EP8 after the PCR reaction is completed, and judging that the 21-trisomy risk exists when the gene factor of the expression abundance in any group is more than 1.4 and is more than or equal to 4.
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| CN107223159A (en) * | 2014-05-09 | 2017-09-29 | 科戴克斯生命股份公司 | The detection of DNA from particular cell types and correlation technique |
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| CN107223159A (en) * | 2014-05-09 | 2017-09-29 | 科戴克斯生命股份公司 | The detection of DNA from particular cell types and correlation technique |
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| RONG ZHANG等: "Application of Differentially Methylated Loci in Clinical Diagnosis of Trisomy 21 Syndrome", 《GENETIC TESTING MOLECULAR》 * |
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