CN113215225A - Real-time fluorescent quantitative PCR instrument calibration kit - Google Patents
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Abstract
The invention discloses a real-time fluorescent quantitative PCR instrument calibration kit, which comprises PCR premix solution, PCR enzyme, a standard substance and a primer dry plate, wherein the standard substance is a mixture of two or four kinds of cDNA in cDNA G1, cDNA G2, cDNA G3 and cDNA G4; the target detected by the kit is miRNA, and the target miRNA comprises at least one miRNA with high GC ratio and at least one miRNA with low GC ratio. The invention can comprehensively and accurately evaluate the real performance of the equipment, can observe the marginal effect condition of the equipment, and can effectively avoid the marginal range of obvious influence according to the calibration condition of the kit when a researcher needs to carry out accurate research.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a real-time fluorescent quantitative PCR instrument calibration kit.
Background
With the advance of gene field research, gene detection technology has also been rapidly developed, and the application of real-time fluorescent quantitative PCR instruments is becoming more and more extensive. The real-time fluorescent quantitative PCR detection technology is characterized in that a fluorescent gene is added into a PCR reaction system, the whole PCR process is monitored in real time by utilizing fluorescent signal accumulation, the real-time fluorescent quantitative PCR instrument integrates a plurality of elements, the system is complex, the volume is large, and the calibration of equipment is always a difficult problem. To maintain the accuracy of its detection, the device needs to be calibrated on time each year. At present, the calibration of real-time fluorescence quantitative PCR generally requires a manufacturer or a professional third-party organization to perform calibration, and the manufacturer or the professional third-party organization does not have the required equipment and calibration capability, so the calibration cost is higher, and the calibration mode is relatively extensive, which cannot accurately reflect the actual conditions of the equipment, such as the edge effect of the equipment, but the lack of the calibration mode can show how large the edge effect is probably, and the reaction in what range is relatively stable, so that the low-cost, simple and accurate calibration mode is very important.
At present, there are two main ways for calibrating a fluorescence quantitative PCR instrument: one method is to calibrate the temperature field part of the fluorescence quantitative PCR instrument, and the method cannot calibrate the optical part of the fluorescence quantitative PCR instrument and cannot comprehensively evaluate the performance of equipment; the other method is to use chemical reagents to detect the equipment, generally a biological reagent test disc is used or plasmid DNA with known concentration is adopted, the method is relatively extensive, the performance of the equipment cannot be accurately reacted, the edge effect of the equipment cannot be reflected, the small difference of temperature difference between holes can be exponentially amplified in the gene amplification process, and the experimental result can be influenced for a more precise experiment.
Disclosure of Invention
The invention aims to solve the problem of accurate calibration of a real-time fluorescence quantitative PCR instrument, and provides a calibration kit of the real-time fluorescence quantitative PCR instrument, which can comprehensively and accurately evaluate the real performance of equipment, can observe the marginal effect condition of the equipment, and can effectively avoid the marginal range of obvious influence according to the calibration condition of the kit when a researcher needs to perform accurate research.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a real-time fluorescent quantitative PCR instrument calibration kit comprises PCR premix solution, PCR enzyme, a standard substance and a primer dry plate, wherein the standard substance is a mixture of two or four cDNAs in cDNA G1, cDNA G2, cDNA G3 and cDNA G4, the sequence of the cDNA G1 is shown in SEQ ID No.1, the sequence of the cDNA G2 is shown in SEQ ID No.2, the sequence of the cDNA G3 is shown in SEQ ID No.3, and the sequence of the cDNA G4 is shown in SEQ ID No. 4; the target detected by the kit is miRNA, and the target miRNA comprises at least one miRNA with high GC ratio and at least one miRNA with low GC ratio.
The target miRNAs comprise miRNA G1, miRNA G2, miRNA G3 and miRNA G4, wherein the GC proportion of miRNA G1 and miRNA G4 is 20-40% (low GC proportion), and the GC proportion of miRNA G2 and miRNA G3 is 60-80% (high GC proportion).
The sequence of miRNA G1 is shown in SEQ ID No.5, the sequence of miRNA G2 is shown in SEQ ID No.6, the sequence of miRNA G3 is shown in SEQ ID No.7, and the sequence of miRNA G4 is shown in SEQ ID No. 8.
The cDNA G1 is obtained by reverse transcription by taking miRNA G1 as a template or is directly synthesized according to a sequence; the cDNA G2 is obtained by reverse transcription by taking miRNA G2 as a template or is directly synthesized according to a sequence; the cDNA G3 is obtained by reverse transcription by taking miRNA G3 as a template or is directly synthesized according to a sequence; the cDNA G4 is obtained by reverse transcription with miRNA G4 as template or directly synthesized according to sequence.
The PCR enzyme is DNA polymerase with the concentration of 2U/ul-10U/ul.
The primer dry plate is a 384-pore plate or a 96-pore plate, and positive and negative primer dry powder for detecting target miRNA is contained on the primer dry plate.
The primers for detecting the target miRNA include a primer for detecting miRNA G1:
a forward primer: 5'-GGCATAGGTTATGGCTTTTCA-3' the flow of the air in the air conditioner,
reverse primer: 5'-ACCGCTGGTCACATAGGAATG-3' are provided.
The primers for detecting the target miRNA include a primer for detecting miRNA G2:
a forward primer: 5'-TCATCACTTGAGCGCCT-3' the flow of the air in the air conditioner,
reverse primer: 5'-CTGTCACCTCGGCTCTGTC-3' are provided.
The primers for detecting the target miRNA include a primer for detecting miRNA G3:
a forward primer: 5'-ACCGCACAAAGCCTGCCC-3' the flow of the air in the air conditioner,
reverse primer: 5'-GCCTGGACCCGAGGAGC-3' are provided.
The primers for detecting the target miRNA include a primer for detecting miRNA G4:
a forward primer: 5'-ATCCACCCTCCGTCAAGAGCAA-3' the flow of the air in the air conditioner,
reverse primer: 5'-CAGGCGCGTCACATTTTTCGTT-3' are provided.
The invention has the beneficial effects that:
1. the operation is simple, the corresponding target primer is freeze-dried or dried on the reaction plate in advance, and when the kit is used, the mixed solution is simply prepared and then is directly added into the reaction plate.
2. The GC ratios of the detection targets are different, and the conditions of different genes can be reflected.
3. The reaction is sensitive, the detected target is miRNA, the edge effect of the equipment can be reflected on the experimental result, and the obvious difference region can be avoided when the fine experiment is carried out.
4. The device can be calibrated once, the performance of different aspects of the device can be reflected by different analysis modes of data, and the device can be comprehensively evaluated.
5. The sequence is short, the expenditure of synthetic targets and primers can be reduced, and the cost is low.
Drawings
FIG. 1 is a 384 well plate primer profile.
FIG. 2 is a primer distribution map of a 96-well plate.
Fig. 3 is a marginal effect heatmap.
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples.
In the present invention, the raw materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.
Example (b):
the kit comprises the following components:
| serial number | Components | Specification of | Number of |
| 1 | PCR premix | 3749 ul/ | 2 |
| 2 | PCR enzymes | 120 ul/ | 1 |
| 3 | Standard article | 280 ul/ | 2 |
| 4 | Primer | 1 plate/ | 2 bag |
The PCR enzyme is DNA polymerase with the concentration of 2U/ul-10U/ul.
Wherein the standard substance is a cDNA mixed solution obtained by reverse transcription of 2 or 4 miRNAs, and the cDNA can also be directly synthesized according to a sequence;
wherein the GC contents of target miRNAs are different, the GC proportion of G1/G4 is 20-40%, and the GC proportion of G2/G3 is 60-80%; at a concentration of 102copies/ul-106copies/ul。
Wherein the primer dry plate is a 384-well plate or a 96-well plate, the plate contains positive and negative primer dry powder of the detection target, and the process is drying or freeze-drying.
Specific example 1: production of calibration kit for 384 real-time fluorescent quantitative PCR instrument
The production steps are as follows:
1. and (3) PCR enzyme production: the brands of DNA polymerases selected in this example were: LGC; the goods name is as follows: klear Taq-5000U;
2. diluting DNA polymerase according to the concentration of 5U/ul, subpackaging according to 120 ul/tube, and labeling after subpackaging;
3. and (3) PCR premix liquid production: the PCR Buffer brand selected in this example is: MiRXES; the goods name is as follows: PCR Buffer Premix (10X). Diluting the 10 Xpremixed solution to 2 Xconcentration with non-enzyme and non-nucleic acid water, mixing, subpackaging with 3749 ul/tube, and labeling;
4. production of a standard product: in this example, the cDNA of G1/G2/G3/G4 was synthesized directly, mixed in equal proportion after being prepared into an equal concentration solution according to the concentration report provided by the manufacturer, and diluted to 6 x 104Subpackaging copies/ul at 280 ul/pipe, and labeling after subpackaging;
wherein the cDNA sequence is shown in Table 1:
table 1: cDNA sequence Listing
5. Production of a primer dry plate: preparing the synthesized positive and negative primers G1-G4 into an equal concentration solution according to a concentration report provided by a manufacturer, mixing the solution in equal proportion according to different genes after the preparation is finished, diluting the solution to the concentration of 0.75uM, adding the primers into a 384-pore plate by using a liquid transfer workstation according to the sequence shown in figure 1 after the mixing, transferring the plate into an oven, drying the plate for 80min at the temperature of 55 ℃, sealing the plate by using a sealing film after the drying, filling the plate into an aluminum foil bag, sealing the opening of the aluminum foil bag, and labeling the plate.
Wherein the primer sequences are shown in Table 2:
table 2: primer sequence Listing
6. Assembling: and putting the PCR enzyme, the PCR premixed solution, the standard substance and the primer dry plate into the same reagent box and sealing.
Wherein the miRNA selected from G1/G2/G3/G4 is shown in Table 3, the GC ratio of G1/G4 is low, and the GC ratio of G2/G3 is high;
table 3: MiRNA gene sequence
| Serial number | Name of Gene | Gene sequences | GC％ |
| G1 | hsa-miR-135b-5p | UAUGGCUUUUCAUUCCUAUGUGA(SEQ ID No.5) | 35％ |
| G2 | hsa-miR-339-3p | UGAGCGCCUCGACGACAGAGCCG(SEQ ID No.6) | 70％ |
| G3 | hsa-miR-596 | AAGCCUGCCCGGCUCCUCGGG(SEQ ID No.7) | 76％ |
| G4 | hsa-miR-335-5p | UCAAGAGCAAUAACGAAAAAUGU(SEQ ID No.8) | 30％ |
Specific example 2: production of calibration kit for 96 real-time fluorescent quantitative PCR instrument
The production steps are as follows:
1. and (3) PCR enzyme production: the brands of DNA polymerases selected in this example were: roc; the goods name is as follows: antart Taq DNA Polymerase;
2. diluting DNA polymerase according to the concentration of 5U/ul, subpackaging according to 80 ul/tube, and labeling after subpackaging;
3. and (3) PCR premix liquid production: the PCR Buffer brand selected in this example is: MiRXES; the goods name is as follows: PCR Buffer Premix (10X). Diluting the 10 Xpremixed solution to 2 Xconcentration with non-enzyme and non-nucleic acid water, mixing, subpackaging with 1875 ul/tube, and labeling;
4. production of a standard product: in this example, the cDNA of G1/G2 was synthesized directly, mixed in equal proportion after being prepared into an equal concentration solution according to the concentration report provided by the manufacturer, and diluted to 6 × 104Subpackaging copies/ul according to 280 ul/tube, and labeling after subpackaging, wherein the cDNA sequence is shown in table 1;
5. production of a primer dry plate: preparing synthesized positive and negative primers G1-G2 into an isoconcentration solution according to a concentration report provided by a manufacturer, mixing the solution in an equal proportion according to different genes after the preparation is finished, diluting the solution to the concentration of 1.5uM, adding the primers into a 96-well plate by using a liquid transfer workstation according to the sequence shown in figure 2 after the mixing, adding the primers into each well with 4ul, transferring the mixture into a refrigerator for freezing, performing freeze-drying treatment, sealing the plates by using a sealing plate film after the freeze-drying, and labeling the sealed aluminum foil bags; wherein the corresponding primer sequences are shown in Table 2;
6. assembling: and putting the PCR enzyme, the PCR premixed solution, the standard substance and the primer dry plate into the same reagent box and sealing.
Use of 384 real-time fluorescence quantitative PCR instrument calibration kit
Calibrating the equipment: quantstudio of ABI-384 real-time fluorescence quantitative PCR instrument TM 5
Preparing a premixed solution: taking out 1 tube of PCR premix from the kit, adding 3800ul of nuclease-free water (self-contained), adding 51ul of PCR enzyme, uniformly mixing, adding 7395ul of the premix into a new 15ml centrifuge tube, adding 255ul of standard substance into the centrifuge tube, uniformly mixing, and placing on ice for later use; a96-well deep-well plate was prepared and the premix was transferred to a column of deep-well plates, 900ul per well.
Plate rotating: taking out a primer plate from the kit, transferring the prepared premixed solution to the primer plate by using a discharging gun, adhering a film on the primer plate by using a PCR film after transferring, and centrifuging the primer plate, wherein each hole has 15 ul.
And (3) computer detection: the 384 plate was placed in a PCR machine, programmed, and subjected to PCR reaction.
Setting reaction conditions of a PCR instrument: the baseline was set automatically, the threshold line was set manually to 0.4, and the conditions were set as in Table 4
TABLE 4 setting conditions for real-time fluorescent quantitative PCR instrument
And completing the detection of the second primer plate according to the steps.
Data analysis of 384 real-time fluorescence quantitative PCR instrument calibration kit
The embodiment analyzes the test data in different modes and analyzes the performance of the equipment from different angles.
Marginal effect (heat map) analysis:
and (3) deriving the CT detection result to obtain 2-plate data, averaging the 2-plate data, splitting the data from G1 to G4 after averaging, displaying the data in a color gradation manner after splitting, wherein the obtained result is shown in figure 3, has obvious hot ring effect and shows that the equipment is normal, and can also know the approximate marginal effect range of the equipment according to the result, and if an experiment with higher precision is required, the obviously different area can be avoided.
Uniformity analysis:
the data from plate 1 and plate 2 were analyzed separately and the mean, SD, max, min, max-min values for each gene were calculated and the results are shown in Table 5. Wherein the SD value per gene per plate is required to be 0.125 or less, and the (max-min) value is 0.8 or less, indicating that the uniformity of the apparatus is good.
TABLE 5 data homogeneity analysis
| Mean value of | SD | max | min | max-min | |
| Panel 1-G1 | 23.87 | 0.08 | 24.05 | 23.71 | 0.34 |
| Panel 2-G1 | 23.98 | 0.09 | 24.23 | 23.79 | 0.44 |
| Panel 1-G2 | 21.60 | 0.07 | 21.77 | 21.42 | 0.34 |
| Panel 2-G2 | 21.70 | 0.08 | 21.91 | 21.49 | 0.42 |
| Panel 1-G3 | 21.66 | 0.09 | 21.96 | 21.44 | 0.52 |
| Panel 2-G3 | 21.83 | 0.09 | 22.13 | 21.65 | 0.48 |
| Panel 1-G4 | 21.60 | 0.08 | 21.81 | 21.38 | 0.43 |
| Panel 2-G4 | 21.78 | 0.09 | 21.99 | 21.58 | 0.41 |
And (3) repeatability analysis:
calculating the CT average value of four holes (A1/A2/B1/B2) from G1 to G4, and so on, generating 96 numerical values for each plate, calculating the absolute value of (plate 1-plate 2), counting the number of the numerical values which are more than 0.2, 0.4, 0.6 and 0.8, and according with the number requirement of the table 6, the repeatability of the equipment is good.
TABLE 6 data repeatability analysis
And (3) analyzing the accuracy:
the average CT values of the genes of the plate 1 and the plate 2 are respectively calculated, and the difference value between the genes is calculated, and if the upper limit and the lower limit of the table 7 are met, the equipment accuracy is good.
TABLE 7 data accuracy analysis
| G1-G2 | G1-G3 | G1-G4 | G2-G3 | G2-G4 | G3- | |
| Panel | ||||||
| 1 | 2.267 | 2.285 | 2.361 | 0.018 | 0.094 | 0.076 |
| | 2.300 | 2.323 | 2.325 | 0.023 | 0.025 | 0.002 |
| Upper limit of | 2.668 | 2.459 | 3.027 | 0.367 | 0.431 | 0.969 |
| Lower limit of | 2.068 | 1.935 | 1.691 | -0.666 | -0.389 | -0.747 |
The principle of the kit of the invention is as follows: the performance of the detection equipment is removed through the most intuitive PCR amplification, the selected detection target is miRNA, the sequence is short, the reaction is sensitive, and the slight change of the temperature can influence the detection, so the edge effect of the equipment can be reacted. The genes with different GC ratios have different sensitivity degrees to different systems and temperatures, so that the target genes with high and low GC ratios can represent the amplification conditions of different genes. Meanwhile, the primer dry plate is used, different primers are preset in the reaction plate in advance, experimental operation errors can be reduced, the operation errors are prevented from being substituted into equipment calibration, and a user can use the primer dry plate more conveniently.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
SEQUENCE LISTING
<110> Hangzhou foraging reason Biotechnology Ltd
<120> real-time fluorescent quantitative PCR instrument calibration kit
<130> 2021.5.13
<160> 16
<170> PatentIn version 3.3
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Claims (10)
1. A real-time fluorescent quantitative PCR instrument calibration kit is characterized by comprising a PCR premix solution, a PCR enzyme, a standard substance and a primer dry plate, wherein the standard substance is a mixture of two or four kinds of cDNA in cDNA G1, cDNA G2, cDNA G3 and cDNA G4, the sequence of the cDNA G1 is shown in SEQ ID No.1, the sequence of the cDNA G2 is shown in SEQ ID No.2, the sequence of the cDNA G3 is shown in SEQ ID No.3, and the sequence of the cDNA G4 is shown in SEQ ID No. 4; the target detected by the kit is miRNA, and the target miRNA comprises at least one miRNA with high GC ratio and at least one miRNA with low GC ratio.
2. The real-time fluorescence quantitative PCR instrument calibration kit according to claim 1, wherein the target miRNAs comprise miRNA G1, miRNA G2, miRNA G3 and miRNA G4, wherein the GC proportion of miRNA G1 and miRNA G4 is 20% -40%, and the GC proportion of miRNA G2 and miRNA G3 is 60% -80%.
3. The real-time fluorescent quantitative PCR instrument calibration kit according to claim 2, wherein the sequence of miRNA G1 is shown in SEQ ID No.5, the sequence of miRNA G2 is shown in SEQ ID No.6, the sequence of miRNA G3 is shown in SEQ ID No.7, and the sequence of miRNA G4 is shown in SEQ ID No. 8.
4. The real-time fluorescence quantitative PCR instrument calibration kit according to claim 1 or 2, wherein the cDNA G1 is obtained by reverse transcription using miRNA G1 as a template or directly synthesized according to a sequence; the cDNA G2 is obtained by reverse transcription by taking miRNA G2 as a template or is directly synthesized according to a sequence; the cDNA G3 is obtained by reverse transcription by taking miRNA G3 as a template or is directly synthesized according to a sequence; the cDNA G4 is obtained by reverse transcription with miRNA G4 as template or directly synthesized according to sequence.
5. The real-time fluorescent quantitative PCR instrument calibration kit according to claim 1, wherein the PCR enzyme is DNA polymerase, and the concentration is 2U/ul-10U/ul.
6. The real-time fluorescent quantitative PCR instrument calibration kit according to claim 1 or 2, wherein the primer dry plate is a 384-well plate or a 96-well plate, and positive and negative primer dry powder for detecting target miRNA is contained on the primer dry plate.
7. The real-time fluorescent quantitative PCR instrument calibration kit according to claim 6, wherein the primers for detecting the target miRNA comprise primers for detecting miRNA G1:
a forward primer: 5'-GGCATAGGTTATGGCTTTTCA-3' the flow of the air in the air conditioner,
reverse primer: 5'-ACCGCTGGTCACATAGGAATG-3' are provided.
8. The real-time fluorescent quantitative PCR instrument calibration kit according to claim 6, wherein the primers for detecting the target miRNA comprise primers for detecting miRNA G2:
a forward primer: 5'-TCATCACTTGAGCGCCT-3' the flow of the air in the air conditioner,
reverse primer: 5'-CTGTCACCTCGGCTCTGTC-3' are provided.
9. The real-time fluorescent quantitative PCR instrument calibration kit according to claim 6, wherein the primers for detecting the target miRNA comprise primers for detecting miRNA G3:
a forward primer: 5'-ACCGCACAAAGCCTGCCC-3' the flow of the air in the air conditioner,
reverse primer: 5'-GCCTGGACCCGAGGAGC-3' are provided.
10. The real-time fluorescent quantitative PCR instrument calibration kit according to claim 6, wherein the primers for detecting the target miRNA comprise primers for detecting miRNA G4:
a forward primer: 5'-ATCCACCCTCCGTCAAGAGCAA-3' the flow of the air in the air conditioner,
reverse primer: 5'-CAGGCGCGTCACATTTTTCGTT-3' are provided.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979544A (en) * | 2010-11-02 | 2011-02-23 | 中国计量学院 | Standard sample-based real-time fluorescence PCR relative calibration method |
| CN106399555A (en) * | 2016-11-10 | 2017-02-15 | 三生国健药业（上海）股份有限公司 | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method as well as standard substance and detection kit for real-time fluorescent quantitative PCR detection |
| CN106434924A (en) * | 2016-09-29 | 2017-02-22 | 北京全式金生物技术有限公司 | Kit and method for precise quantification of next-generation sequencing libraries different in GC content by qPCR (quantitative polymerase chain reaction) |
| CN109207566A (en) * | 2018-09-12 | 2019-01-15 | 浙江大学昆山创新中心 | It is a kind of precisely quantitative or concentration correction kit and application to be realized to a variety of DNA standard items plasmid solutions simultaneously |
| CN110484607A (en) * | 2019-03-22 | 2019-11-22 | 中国计量科学研究院 | A kind of real-time fluorescence quantitative PCR instrument calibration standard substance and its application |
| CN112662766A (en) * | 2020-11-19 | 2021-04-16 | 赣南医学院第一附属医院 | Method for detecting familial thoracic aortic aneurysm and interlayer related mutant gene based on high-throughput sequencing technology |
-
2021
- 2021-05-13 CN CN202110523217.0A patent/CN113215225B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979544A (en) * | 2010-11-02 | 2011-02-23 | 中国计量学院 | Standard sample-based real-time fluorescence PCR relative calibration method |
| CN106434924A (en) * | 2016-09-29 | 2017-02-22 | 北京全式金生物技术有限公司 | Kit and method for precise quantification of next-generation sequencing libraries different in GC content by qPCR (quantitative polymerase chain reaction) |
| CN106399555A (en) * | 2016-11-10 | 2017-02-15 | 三生国健药业（上海）股份有限公司 | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method as well as standard substance and detection kit for real-time fluorescent quantitative PCR detection |
| CN109207566A (en) * | 2018-09-12 | 2019-01-15 | 浙江大学昆山创新中心 | It is a kind of precisely quantitative or concentration correction kit and application to be realized to a variety of DNA standard items plasmid solutions simultaneously |
| CN110484607A (en) * | 2019-03-22 | 2019-11-22 | 中国计量科学研究院 | A kind of real-time fluorescence quantitative PCR instrument calibration standard substance and its application |
| CN112662766A (en) * | 2020-11-19 | 2021-04-16 | 赣南医学院第一附属医院 | Method for detecting familial thoracic aortic aneurysm and interlayer related mutant gene based on high-throughput sequencing technology |
Non-Patent Citations (2)
| Title |
|---|
| 吕建新，王培昌主编: "《检验与临床诊断——分子诊断学分册》", 31 May 2010, 人民军医出版社 * |
| 祝天宇等: "荧光定量PCR仪光学校准方法与结果分析", 《计量与测试技术》 * |
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