CN116479105A - Nucleic acid reagent, kit and multiplex fluorescence quantitative PCR detection method for human mitochondrial DNA copy number detection - Google Patents
Nucleic acid reagent, kit and multiplex fluorescence quantitative PCR detection method for human mitochondrial DNA copy number detection Download PDFInfo
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Abstract
The disclosure relates to the biotechnology field, in particular to a nucleic acid reagent, a kit and a multiplex fluorescence quantitative PCR detection method for detecting human mitochondrial DNA copy number. The nucleic acid reagent comprises a primer capable of specifically amplifying human mitochondrial DNA and a primer capable of specifically amplifying albumin genes. The nucleic acid reagent, the kit and the detection method provided by the disclosure take the albumin gene as an internal reference gene, are used for detecting the copy number of human mitochondrial DNA, are simple and convenient to operate, have small systematic errors, high accuracy and high sensitivity, and are suitable for rapid detection of large-sample-number and large-age-span people.
Description
Technical Field
The disclosure relates to the biotechnology field, in particular to a nucleic acid reagent, a kit and a multiplex fluorescence quantitative PCR detection method for detecting human mitochondrial DNA copy number.
Background
Mitochondria are metabolic organelles that exist independently outside the nucleus and have only DNA, and mitochondrial DNA (mtDNA) is a substance that is independent of the nuclear genome and carries the mitochondrial genetic code inside the cell. Mitochondrial DNA can participate in the synthesis, transcription and replication of proteins, plays an important role in the physiological and pathological processes of human bodies, and has higher research value.
It has been known so far that human mitochondrial DNA contains 37 genes in total, encoding 2 rRNAs (12S rRNA and 16S rRNA), 22 tRNAs, and 13 polypeptides. Mitochondrial DNA is more prone to mutation than the nuclear genome due to its specificity, e.g. lack of histone protection and imperfect repair system, resulting in high instability. Due to environmental changes, mitochondrial DNA copy numbers fluctuate, thereby affecting the transcriptional level of the constituent genes. Mitochondrial DNA copy number variation is closely related to the occurrence and development of many age-related diseases, including metabolic syndrome, cardiovascular disease, neurodegenerative disease, and cancer. Studies have shown that peripheral blood mitochondrial DNA copy number is related to visceral fat, and that mitochondrial DNA copy number is a potential predictor of metabolic disorders. In addition, in some cancers, particularly bladder, breast and kidney cancers, mitochondrial DNA tends to be depleted relative to the corresponding normal tissues. However, the molecular mechanism leading to abnormal mitochondrial DNA copy numbers in tumor tissue cells has not been well understood to date. Therefore, establishing a high throughput human mitochondrial DNA copy number assay is of great importance for revealing cellular aging processes, predicting metabolic disorders, cancer screening, diagnosis and prognosis, and assessing individual health levels.
At present, a common method for determining the copy number of mitochondrial DNA is a PCR fluorescent quantitative method, and the method can be widely applied to crowd research due to convenient operation and short determination time. However, most of the studies currently use methods for determining the relative copy number of mitochondrial DNA by quantifying single copy gene and mitochondrial gene respectively, in which multiple pipetting between different wells would generate a large systematic error, and in order to reduce this error, the scholars have proposed improved fluorescent quantitative PCR detection methods, i.e. collecting mitochondrial DNA fluorescent signal and single copy gene fluorescent signal simultaneously in a set of reactions, the same well. Even so, the accuracy of the detection results remains to be improved, and it is necessary to optimize the fluorescent quantitative PCR protocol for detecting mitochondrial DNA copy numbers from various aspects to significantly improve the detection accuracy thereof.
Disclosure of Invention
The invention aims to overcome the defect that the accuracy of detecting the human mitochondrial DNA copy number still needs to be improved in the prior art, and provides a nucleic acid reagent, a kit and a multiplex fluorescence quantitative PCR detection method for detecting the human mitochondrial DNA copy number.
In order to achieve the above object, the present disclosure provides a nucleic acid reagent for human mitochondrial DNA copy number detection, the nucleic acid reagent comprising a primer capable of specifically amplifying human mitochondrial DNA, and a primer capable of specifically amplifying an albumin gene.
Optionally, the primer capable of specifically amplifying human mitochondrial DNA comprises a primer shown as SEQ ID NO. 1-2;
and/or the primer capable of specifically amplifying the albumin gene comprises a primer shown as SEQ ID NO. 3-4.
Optionally, the molar ratio of the primer shown in SEQ ID NO.1, the primer shown in SEQ ID NO.2, the primer shown in SEQ ID NO.3 and the primer shown in SEQ ID NO.4 is (0.06-0.08): (0.06-0.08): (0.10-0.15): (0.10-0.15).
Optionally, the primer shown in SEQ ID NO.1 is an upstream primer (Mt-F) for specifically amplifying human mitochondrial DNA, the primer shown in SEQ ID NO.2 is a downstream primer (Mt-R) for specifically amplifying human mitochondrial DNA, the primer shown in SEQ ID NO.3 is an upstream primer (ALB-F) for specifically amplifying albumin gene, and the primer shown in SEQ ID NO.4 is a downstream primer (ALB-R) for specifically amplifying albumin gene.
Alternatively, the primer shown in SEQ ID NO.1 is CACCCAAGAACAGGGTTTGT; the primer shown in SEQ ID NO.2 is TGGCCATGGGTATGTTGTTA; the primer shown in SEQ ID NO.3 is CGGCGGCGGGCGGCGCGGGCTGGGCGGAAATGCTGCACAGAATCCTTG; the primer shown in SEQ ID NO.4 is GCCCGGCCCGCCGCGCCCGTCCCGCCGGCATGGTCGCCTGTTCAC.
The disclosure also provides a kit for detecting human mitochondrial DNA copy number, which contains the nucleic acid reagent of any one of the above.
Optionally, the kit further comprises a PCR amplification premix, a reference standard, a reference dye and enzyme-free water;
optionally, the PCR amplification premix comprises a reaction buffer,Green I、dNTPs、Mg 2+ 2 x premix of rTaq DNA polymerase and anti-DNA polymerase antibody;
optionally, the reference standard comprises a human whole blood DNA extract;
alternatively, the human whole blood DNA extract is obtained by mixing whole blood DNA of not less than 3000 persons; optionally, the age span of the population from which the human whole blood DNA extract is derived is 20 to 60 years;
optionally, the reference dye comprises ROX reference dye;
alternatively, the PCR amplification pre-mix is used in an amount of more than 100 times, preferably 500 times, the amount of the reference dye by volume.
When the kit provided by the disclosure is used for detecting the copy number of human mitochondrial DNA, a reference standard substance can be diluted and configured into a series of standard substance reagents with a concentration according to the concentration of the DNA of a sample to be detected to form a standard series concentration gradient, so that the concentration of the DNA of the sample to be detected is contained in the standard series concentration gradient range, and then four primers, a premixed solution for real-time quantitative PCR amplification, a reference dye, non-enzymatic water, the standard substance with each concentration and the sample to be detected are respectively mixed according to a given sample adding amount to form an amplification reaction system for PCR reaction.
The disclosure also provides a multiplex fluorescent quantitative PCR detection method for non-diagnostic human mitochondrial DNA copy number, comprising the steps of:
(1) Amplifying and fluorescence detecting a DNA sample of a sample to be detected by adopting a multiplex fluorescence quantitative PCR method, and determining a Ct value Ct corresponding to human mitochondrial DNA in the DNA sample 1 And Ct value Ct corresponding to the reference gene 2 Wherein the reference gene is an albumin gene;
(2) Based on Ct value C corresponding to the human mitochondrial DNA in the DNA samplet 1 Determining the relative content Mt of the human mitochondrial DNA in the DNA sample; ct value Ct based on the corresponding Ct value of the internal reference gene in the DNA sample 2 Determining the relative content S of the reference gene in the DNA sample according to a second preset standard curve;
wherein the first preset standard curve indicates a Ct value Ct corresponding to human mitochondrial DNA in the reference standard 1 ' correlation with the relative content of human mitochondrial DNA in the reference standard; the second preset standard curve indicates the Ct value Ct corresponding to the reference gene in the reference standard substance 2 ' correlation with the relative content of reference genes in the reference standard;
(3) Determining the relative copy number Mt/S of the human mitochondrial DNA and the reference gene in the DNA sample based on the relative content Mt of the human mitochondrial DNA and the relative content S of the reference gene in the DNA sample.
Optionally, in step (1), the DNA sample of the sample to be detected is amplified and fluorescence detected by multiplex fluorescent quantitative PCR based on the nucleic acid reagent or the kit described in any one of the above.
Optionally, the reaction system for amplification and fluorescence detection comprises:
4-5 mu L of PCR amplification premix;
10 mu M of primer shown in SEQ ID NO.1 is 0.06-0.08 mu L;
10 mu M of primer shown in SEQ ID NO.2 is 0.06-0.08 mu L;
10 mu M of primer shown in SEQ ID NO.3 is 0.10-0.15 mu L;
10 mu M of primer shown in SEQ ID NO.4 is 0.10-0.15 mu L;
0.04-0.05 mu L of reference dye;
1-2 mu L of DNA sample;
the enzyme-free water was supplemented to 10. Mu.L.
Optionally, the reaction process of amplification and fluorescence detection comprises:
95℃15min；
15s at 95℃for 38 cycles;
58℃20s；
collecting Ct value corresponding to human mitochondrial DNA at 72 ℃ for 30 s;
and collecting Ct value corresponding to the reference gene at 85 ℃ for 30 s.
Optionally, the standard curve is obtained based on at least 5 concentration gradients of a reference standard solution;
optionally, the concentration of any one of the reference standard solutions in the at least 5 concentration gradients is 0.0625-10 times that of the DNA sample solution;
optionally, in the reference standard solution with at least 5 concentration gradients, the lowest concentration of the reference standard solution is not higher than the concentration of the DNA sample solution, and the highest concentration of the reference standard solution is not lower than the concentration of the DNA sample solution;
optionally, in the reference standard solution with at least 5 concentration gradients, the lowest two concentrations of the reference standard solution are smaller than the concentration of the DNA sample solution, and the highest two concentrations of the reference standard solution are larger than the concentration of the DNA sample solution;
alternatively, the concentration of the DNA sample solution is 0.625-40 ng/. Mu.L.
The beneficial effects of the present disclosure are:
1. the nucleic acid reagent, the kit and the detection method provided by the disclosure take the albumin gene with higher stability as the reference gene, remarkably improve the fluorescence collection effect of MMQPCR, are used for detecting the copy number of human mitochondrial DNA, have the advantages of simple operation, small systematic error, high accuracy and high sensitivity, and are suitable for the rapid detection of large-sample-number and large-age-span people.
2. The nucleic acid reagent, the kit and the detection method provided by the disclosure have stable peaks of human mitochondrial DNA and internal reference genes, smooth dissolution curves and no obvious nonspecific amplification products; the primer sequence capable of specifically amplifying the albumin gene is increased by a sequence rich in GC-clamp compared with a conventional primer sequence, so that the Tm value of the reference gene in a single-color multiple fluorescent quantitative PCR detection result can be remarkably improved, and when the single-color multiple fluorescent quantitative PCR detection is carried out on a sample, the condition of fluorescence collection overlapping between the mitochondrial gene and the reference gene does not exist.
3. The nucleic acid reagent, the kit and the detection method provided by the disclosure optimize the proportion of the primers, and obviously reduce the peak value difference between the mitochondrial DNA peak and the internal reference gene peak in the PCR melting curve. The primer concentration and the primer PCR sample loading amount are optimized, so that the primer finally reaches a specific ratio in a reaction system. Experimental results show that on one hand, under the specific primer proportioning condition of the disclosure, the PCR reaction can keep the amplification efficiency at 90.00% -110.00% and obtain more ideal (> 0.990) standard curve linear R 2 The method comprises the steps of carrying out a first treatment on the surface of the On the other hand, the use of the specific primer-pair of the present disclosure can significantly reduce the peak difference between the two peaks of the human mitochondrial DNA peak and the internal reference gene peak in the PCR melting curve, and by observing the melting curves of the two genes, the specific amplification of the two genes is clearly observed.
4. The nucleic acid reagent, the kit and the detection method provided by the disclosure use specific amplification and fluorescence detection reaction processes, are beneficial to effectively collecting signals of two genes and reduce mutual interference. In the earlier cycle of the PCR reaction program, when the PCR temperature is 72 ℃, the Ct value of the human mitochondrial DNA is collected, the signal of the reference gene is still at a base line at the moment, and when the PCR temperature is 85 ℃, the Ct value of the reference gene is collected, and the mitochondrial DNA product is completely melted, the signal is not collected, so that the signal interference is reduced.
Additional features and advantages of the present disclosure will be set forth in the detailed description which follows.
Drawings
In order to more clearly illustrate the embodiments of the present disclosure or the prior art, the drawings that are required in the detailed description or the prior art will be briefly described, it will be apparent that the drawings in the following description are some embodiments of the present disclosure, and other drawings may be obtained according to the drawings without inventive effort for a person of ordinary skill in the art.
FIG. 1 is a graph of amplification of a single copy gene (albumin) in a standard series of standards in the assay of example 2 of the present disclosure;
FIG. 2 is a graph showing amplification of mitochondrial genes in a standard series of standards in the assay of example 2 of the disclosure;
FIG. 3 is a graph showing the melting curve of mitochondrial genes in a standard series of standards in the detection method of example 2 of the present disclosure, wherein the left peak in the graph is mitochondrial gene signal and the right peak is internal reference gene signal;
FIG. 4 is an electrophoresis chart of PCR products of mitochondrial genes in a standard series of the standard in the detection method of example 2 of the present disclosure, wherein the lower band is mitochondrial genes and the upper band is internal reference genes;
FIG. 5 shows amplification efficiencies of mitochondrial gene (a) and reference gene (b) in a standard series of standards in the detection method of example 2 of the present disclosure.
Detailed Description
The following examples are provided for a better understanding of the present disclosure and are not limited to the preferred embodiments described, but are not intended to limit the disclosure and the scope of protection, and any product that is the same or similar to the present disclosure, either in light of the present disclosure or by combining the present disclosure with other prior art features, falls within the scope of protection of the present disclosure.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
Example 1
The embodiment provides a kit for detecting human mitochondrial DNA copy number, which comprises 8 products in total, and specifically comprises:
1. reference standard 1:
the concentration of human whole blood DNA in the reference standard is 90 ng/. Mu.L, and a sample is obtained from 3000 human whole blood DNA mixed samples (the ages of 20-60 years);
2. primer 4:
respectively are provided withComprises specific amplification of human mitochondrial gene upstream and downstream primers (adding ddH with corresponding volume according to the tube body prompt) 2 Concentration 10. Mu.M after O) and specific amplified single copy gene (albumin) upstream and downstream primers (adding ddH in corresponding volumes as suggested by tube length 2 post-O concentration was 10 μm). The specific primer sequences are as follows:
3. SYBR qPCR mix 1 branch:
contains reaction buffer solution,Green I、dNTPs、Mg 2+ 2X premix of rTaq DNA polymerase and anti-DNA polymerase antibody;
4. ROX reference dye 1 branches;
5、ddH 2 o1 branch.
Example 2
A method for detecting the copy number of human mitochondrial DNA for non-diagnostic purposes using the kit of example 1, comprising the steps of:
1. extracting genome DNA to be detected and preparing a sample to be detected with the DNA concentration of 5 ng/. Mu.L.
2. And preparing a reference standard substance solution with standard series concentration gradients according to the concentration of DNA in the sample solution to be detected by taking a standard substance in the kit, so that the DNA concentration of the sample solution to be detected is contained in the standard series concentration gradients, wherein the standard series concentration gradients in the embodiment are 40 ng/mu l, 20 ng/mu l, 10 ng/mu l, 5 ng/mu l, 2.5 ng/mu l, 1.25 ng/mu l and 0.625 ng/mu l.
3. Detecting the sample solution to be detected and the series of reference standard solutions by using a single-color multiplex fluorescence quantitative PCR (MMQPCR) method respectively, taking an Albumin (ALB) gene as an internal reference gene, and respectively measuring a Ct value Ct1 corresponding to human mitochondrial DNA and a Ct value Ct corresponding to the internal reference gene in the sample solution to be detected 2 And Ct value Ct corresponding to human mitochondrial DNA in each reference standard solution 1 ' and reference Gene pairsCt value Ct of the application 2 ′。
The reaction system of the single-color multiplex fluorescent quantitative PCR (MMQPCR) method is shown in Table 1. The sample adding method specifically comprises the step of adding each reagent in a sample solution to be detected/a reference standard solution and a kit into the same reaction hole according to the sample adding amount in the following table 1 for reaction.
TABLE 1 reaction System for Monochromatic multiplex fluorescence quantitative PCR (MMQPCR) method
The reaction procedure for the single-color multiplex fluorescent quantitative PCR (MMQPCR) method was set as follows:
Stage1(hold stage)：1cycle：95℃15min；
stage2 (PCR Stage): 38cycle:95 ℃ for 15s;58 ℃ for 20s; collecting Ct value corresponding to human mitochondrial DNA at 72 ℃ for 30 s; ct values corresponding to the reference genes were collected at 85℃for 30 s.
4. Detecting Ct value Ct corresponding to human mitochondrial DNA in each obtained reference standard solution, and Ct value Ct corresponding to' and internal reference genes 2 ' as shown in table 2.
TABLE 2 Ct of reference standard solutions 1 ' value and Ct 2 ' value
Based on the data in Table 2, the Ct values Ct corresponding to human mitochondrial DNA in each reference standard solution were calculated 1 ' Ct value Ct corresponding to reference Gene 2 ' is the ordinate, respectively takes the log corresponding to each reference standard solution 10 (Mt)、log 10 And (S) drawing a first standard curve and a second standard curve by taking the abscissa as the (S), wherein Mt is the content of human mitochondrial DNA, and S is the content of internal reference gene DNA. Wherein the first isThe standard curve is shown in fig. 5a, and the expression is: ct is (Ct) 1 ′＝-3.48×log 10 (Mt)+23.465，R 2 =0.999; the second standard curve is shown in fig. 5b, and the expression is: ct is (Ct) 2 ′＝-3.39×log 10 (S)+30.936，R 2 ＝0.999。
As can be seen from FIGS. 5a and 5b, in the range of the DNA content of the reference standard solutions of 0.625 ng/. Mu.l to 40 ng/. Mu.l, the Ct value corresponding to the human mitochondrial DNA and the Ct value corresponding to the internal reference gene in each reference standard solution correspond to the log of each reference standard solution, respectively 10 (Mt)、log 10 (S) is in a linear relationship, which shows that the method has better sensitivity.
5. Detecting average Ct of Ct values corresponding to human mitochondrial DNA in the obtained sample solution to be detected 1 = 20.968; average Ct of Ct values corresponding to reference genes 2 = 29.045; and carrying out calculation by taking the first standard curve and the second standard curve to obtain the relative copy number Mt/S ratio of the human mitochondrial DNA in the sample solution to be detected, wherein the relative copy number Mt/S ratio is 5.22/3.61=1.45.
6. Detection effect
Example 2 the results of the assay show that the single copy gene (albumin) and the standard system of human mitochondrial gene in the reference standard obtained using the kit of example 1 using the assay method of example 2 show stable peaks (see fig. 1, fig. 2), smooth melting curves with little bimodal difference (see fig. 3), no significant non-specific amplification product, and no significant non-specific amplification product in the electropherogram (see fig. 4).
As can be seen from FIG. 4, in the agarose gel electrophoresis analysis result of the PCR product, the size of the target gene amplification product band is the same as the design size, the band is clear, the size of the internal reference gene amplification product band is the same as the design size, the band is clear, and no nonspecific amplification product band exists in the product, which indicates that the specificity of the method is better.
The preferred embodiments of the present disclosure have been described in detail above, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.
In addition, the specific features described in the foregoing embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present disclosure does not further describe various possible combinations.
Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.
Claims (10)
1. A nucleic acid reagent for human mitochondrial DNA copy number detection, wherein the nucleic acid reagent comprises a primer capable of specifically amplifying human mitochondrial DNA and a primer capable of specifically amplifying an albumin gene.
2. The nucleic acid reagent of claim 1, wherein the primer capable of specifically amplifying human mitochondrial DNA comprises the primer set forth in SEQ ID No. 1-2;
and/or the primer capable of specifically amplifying the albumin gene comprises a primer shown as SEQ ID NO. 3-4.
3. The nucleic acid reagent according to claim 2, wherein the molar ratio of the primer shown in SEQ ID NO.1, the primer shown in SEQ ID NO.2, the primer shown in SEQ ID NO.3 and the primer shown in SEQ ID NO.4 is (0.06-0.08): 0.10-0.15.
4. A kit for human mitochondrial DNA copy number detection, comprising the nucleic acid reagent of any one of claims 1 to 3.
5. The kit of claim 4, further comprising a PCR amplification premix, a reference standard, a reference dye, and enzyme-free water;
optionally, the PCR amplification premix comprises a reaction buffer,Green I、dNTPs、Mg 2+ 2 x premix of rTaq DNA polymerase and anti-DNA polymerase antibody;
optionally, the reference standard comprises a human whole blood DNA extract;
alternatively, the human whole blood DNA extract is obtained by mixing whole blood DNA of not less than 3000 persons;
optionally, the reference dye comprises ROX reference dye;
alternatively, the PCR amplification pre-mix is used in an amount of more than 100 times, preferably 500 times, the amount of the reference dye by volume.
6. A multiplex fluorescent quantitative PCR method for detecting the copy number of human mitochondrial DNA for non-diagnostic purposes, comprising the steps of:
(1) Amplifying and fluorescence detecting a DNA sample of a sample to be detected by adopting a multiplex fluorescence quantitative PCR method, and determining a Ct value Ct corresponding to human mitochondrial DNA in the DNA sample 1 And Ct value Ct corresponding to the reference gene 2 Wherein the reference gene is an albumin gene;
(2) Ct value Ct based on the corresponding Ct value of the human mitochondrial DNA in the DNA sample 1 Determining the relative content Mt of the human mitochondrial DNA in the DNA sample; ct value Ct based on the corresponding Ct value of the internal reference gene in the DNA sample 2 Determining the relative content S of the reference gene in the DNA sample according to a second preset standard curve;
wherein the first preset standard curve indicates a Ct value Ct corresponding to human mitochondrial DNA in the reference standard 1 ' correlation with the relative content of human mitochondrial DNA in the reference standard; the second preset standard curve indicates the Ct value Ct corresponding to the reference gene in the reference standard substance 2 ' correlation with the relative content of reference genes in the reference standardA relationship;
(3) Determining the relative copy number Mt/S of the human mitochondrial DNA and the reference gene in the DNA sample based on the relative content Mt of the human mitochondrial DNA and the relative content S of the reference gene in the DNA sample.
7. The method according to claim 6, wherein in the step (1), the amplification and fluorescence detection of the DNA sample of the sample to be detected are performed by using a multiplex fluorescent quantitative PCR method based on the nucleic acid reagent according to any one of claims 1 to 3 or the kit according to claim 4 or 5.
8. The method of claim 7, wherein the reaction system for amplification and fluorescence detection comprises:
4-5 mu L of PCR amplification premix;
10 mu M of primer shown in SEQ ID NO.1 is 0.06-0.08 mu L;
10 mu M of primer shown in SEQ ID NO.2 is 0.06-0.08 mu L;
10 mu M of primer shown in SEQ ID NO.3 is 0.10-0.15 mu L;
10 mu M of primer shown in SEQ ID NO.4 is 0.10-0.15 mu L;
0.04-0.05 mu L of reference dye;
1-2 mu L of DNA sample;
the enzyme-free water was supplemented to 10. Mu.L.
9. The method according to claim 7 or 8, wherein the reaction process of amplification and fluorescence detection comprises:
95℃15min；
15s at 95℃for 38 cycles;
58℃20s；
collecting Ct value corresponding to human mitochondrial DNA at 72 ℃ for 30 s;
and collecting Ct value corresponding to the reference gene at 85 ℃ for 30 s.
10. The detection method according to any one of claims 6 to 9, wherein the standard curve is obtained based on a reference standard solution of at least 5 concentration gradients;
optionally, the concentration of any one of the reference standard solutions in the at least 5 concentration gradients is 0.0625-10 times that of the DNA sample solution;
optionally, in the reference standard solution with at least 5 concentration gradients, the lowest concentration of the reference standard solution is not higher than the concentration of the DNA sample solution, and the highest concentration of the reference standard solution is not lower than the concentration of the DNA sample solution;
optionally, in the reference standard solution with at least 5 concentration gradients, the lowest two concentrations of the reference standard solution are smaller than the concentration of the DNA sample solution, and the highest two concentrations of the reference standard solution are larger than the concentration of the DNA sample solution;
alternatively, the concentration of the DNA sample solution is 0.625-40 ng/. Mu.L.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102439171A (en) * | 2008-12-22 | 2012-05-02 | 犹他大学研究基金会 | Monochrome multiplex quantitative pcr |
| CN104372002A (en) * | 2014-10-20 | 2015-02-25 | 江苏亿康基因科技有限公司 | Internal reference system for human individual cell genome or mitochondrion DNA (deoxyribonucleic acid) PCR (polymerase chain reaction) amplification |
| WO2017065839A1 (en) * | 2015-10-16 | 2017-04-20 | Coopersurgical, Inc. | Quantification of mitochondrial dna and methods for determining the quality of an embryo |
| CN110387410A (en) * | 2018-04-19 | 2019-10-29 | 深圳华大生命科学研究院 | A kind of detection method of mitochondria copy number and sequence variations |
| CN113862343A (en) * | 2021-10-11 | 2021-12-31 | 中国疾病预防控制中心职业卫生与中毒控制所 | Human telomere length detection kit and detection method for non-diagnosis purpose |
| CN114317693A (en) * | 2021-12-31 | 2022-04-12 | 中国疾病预防控制中心职业卫生与中毒控制所 | Human 5S ribosome DNA copy number detection kit and detection method |
-
2023
- 2023-02-22 CN CN202310148732.4A patent/CN116479105A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102439171A (en) * | 2008-12-22 | 2012-05-02 | 犹他大学研究基金会 | Monochrome multiplex quantitative pcr |
| CN104372002A (en) * | 2014-10-20 | 2015-02-25 | 江苏亿康基因科技有限公司 | Internal reference system for human individual cell genome or mitochondrion DNA (deoxyribonucleic acid) PCR (polymerase chain reaction) amplification |
| WO2017065839A1 (en) * | 2015-10-16 | 2017-04-20 | Coopersurgical, Inc. | Quantification of mitochondrial dna and methods for determining the quality of an embryo |
| CN110387410A (en) * | 2018-04-19 | 2019-10-29 | 深圳华大生命科学研究院 | A kind of detection method of mitochondria copy number and sequence variations |
| CN113862343A (en) * | 2021-10-11 | 2021-12-31 | 中国疾病预防控制中心职业卫生与中毒控制所 | Human telomere length detection kit and detection method for non-diagnosis purpose |
| CN114317693A (en) * | 2021-12-31 | 2022-04-12 | 中国疾病预防控制中心职业卫生与中毒控制所 | Human 5S ribosome DNA copy number detection kit and detection method |
Non-Patent Citations (4)
| Title |
|---|
| 何静;夏虹;张昌明;刘志红;施少林;: "线粒体DNA拷贝数检测方法的建立及在肾脏疾病诊断中的应用", 肾脏病与透析肾移植杂志, no. 04, pages 342 - 348 * |
| 杨麒巍;杜珍武;于杉;高素洁;赵冠杰;张琳;卢佳;任明;张桂珍;: "孕妇血浆DNA中内参基因拷贝数稳定性的评价", 吉林大学学报(医学版), no. 03, pages 523 - 539 * |
| 邹小波 等: "现代食品检测技术", 中国轻工业出版社, pages: 334 * |
| 高玉晓;吴永红;叶巧;李志慧;张成岗;: "利用单线虫两重PCR技术对秀丽隐杆线虫线粒体DNA拷贝数进行相对定量的方法", 军事医学, vol. 37, no. 07, pages 544 * |
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