CN116254346B - Digital PCR kit for thyroid cancer detection - Google Patents

Digital PCR kit for thyroid cancer detection Download PDF

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CN116254346B
CN116254346B CN202310534174.5A CN202310534174A CN116254346B CN 116254346 B CN116254346 B CN 116254346B CN 202310534174 A CN202310534174 A CN 202310534174A CN 116254346 B CN116254346 B CN 116254346B
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祝令香
程寻
杨文军
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Beijing Targeting One Biotechnology Co ltd
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Abstract

The invention provides a digital PCR kit for thyroid cancer detection, which is used for simultaneously detecting TERT-C250T, TERT-C228T and BRAF V600E mutation, wherein a buffer system in the kit comprises Tris-HCl, glycerol and dNTPs, KCl, mgCl 2 The kit comprises Taq DNA polymerase, UNG enzyme, betaine and formamide, wherein the final concentration of the betaine is 0.4-0.6M, the volume concentration of the formamide is 0.5% -2% and the final concentration of the Taq DNA polymerase is more than 3U. The invention realizes that the detection sensitivity of the target sequence under the background of complex genome can reach 0.2 percent by optimizing the final concentration of each component in the detection system.

Description

Digital PCR kit for thyroid cancer detection
Technical Field
The invention relates to the field of digital PCR detection, in particular to a digital PCR kit for thyroid cancer detection.
Background
At present, the most common examination mode for identifying benign and malignant thyroid nodule is thyroid B ultrasonic, but the examination mode is greatly influenced by subjective factors such as experience, level and the like of B ultrasonic doctors, and the positive rate is 30-80% different. If the B-ultrasonic examination indicates that the nodule is possibly malignant, a puncture biopsy is needed, and the benign and malignant nodule is judged according to whether tumor cells are observed or not.
Puncture pathology is a gold standard for diagnosing benign and malignant thyroid nodules, but diagnosis of puncture pathology is highly required for pathologists due to the limited number of nodule puncture cells and the influence of smears. Even the internationally best thyroid pathology diagnosis laboratory has 20-40% of successfully punctured nodules which cannot judge benign and malignant.
With the development of molecular biology, specific molecular markers of thyroid cancer are continuously discovered, so that the diagnosis rate of malignant nodules can be remarkably increased through cytopathology combined with molecular diagnosis.
BRAF and TERT genes are two important genes in the aspects of thyroid cancer diagnosis and prognosis judgment, wherein the BRAF gene is an oncogene, codes for a serine/threonine specific kinase and is involved in regulating various physiological functions in cells, such as cell growth, differentiation, apoptosis and the like. The mutation rate of the BRAF gene in thyroid cancer of Asian population is very high and is about 80%. In the aspect of diagnosis, in patients of III and IV types with undefined cell meaning of puncture, the BRAF gene detection can lead the accuracy of the pre-operation diagnosis of thyroid cancer to reach more than 95 percent. Therefore, the BRAF gene provides a very good index of molecular auxiliary diagnosis for thyroid cancer diagnosis.
TERT gene is an index for determining prognosis and recurrence. The main mutation sites are C228T and C250T mutation of the TERT gene promoter. In the diagnosis of thyroid cancer, if mutation of TERT gene is found, it is predicted that the prognosis of the patient will be poor, especially some of the hypothyroid papillary cancers. If there are both TERT mutations and BRAF mutations, i.e., double mutant patients, this suggests that the tumor may be more aggressive and have a higher probability of recurrence and metastasis than patients without genetic mutation, so that a wider range of surgical protocols are favored in the selection of surgical protocols to ensure surgical thoroughness, and the prognosis is worse for close follow-up observations after surgery. TERT and BRAF gene detection provide a molecular basis for preoperatively judging benign malignancy and prognosis of thyroid cancer patients. The method has very common detection significance in a wide range of thyroid tumor patients, and the detection of the two genes can be widely carried out in various areas and hospitals in the future.
Disclosure of Invention
In order to solve the problems, the invention provides a digital PCR kit for thyroid cancer detection, which is used for simultaneously detecting TERT-C250T, TERT-C228T and BRAF V600E mutation, wherein a buffer system in the kit comprises Tris-HCl, glycerol, dNTPs, KCl, mgCl2, taq DNA polymerase, UNG enzyme, betaine and formamide, the final concentration of the betaine is 0.4-0.6M, the final volume concentration of the formamide is 0.5% -2% and the final concentration of the Taq DNA polymerase is more than 3U.
In one embodiment, the final concentration of Taq DNA polymerase is 3-4U.
In one embodiment, the final Tris-HCl concentration is 0.04-0.05M.
In one embodiment, the final glycerol volume concentration is 4-8%.
In one embodiment, the dATP to dUTP, dCTP to dUTP, and dGTP to dUTP mass ratios of dNTPs are all between 1:2 and 1:3.
In one embodiment, the final KCl concentration is 20-50mM.
In one embodiment, the final UNG enzyme concentration is 0.2U or more.
In one embodiment, the final concentration of UNG enzyme is between 0.2 and 0.4U.
In one embodiment, the primer concentration is not less than 400nM and the probe concentration is not less than 200nM.
In one embodiment, the MgCl 2 The final concentration was 2-6mM.
TERT gene mutations C228T and C250T are present in the promoter region, which has a higher GC content (80% or more). When the conventional PCR reaction solution is used for amplification, no amplification exists or the amplification effect is poor. The GC content of the area where the BRAF gene mutation is located is 39-40%, and the GC content is lower, if the two genes are subjected to double detection, the PCR amplification buffer solution is required to be optimized to meet the requirement of simultaneously amplifying and detecting the two genes, so that the invention optimizes the PCR amplification buffer solution, and realizes the purpose of simultaneously detecting the TERT gene promoter mutation and the BRAF gene V600E mutation in a one-tube system.
In the invention, when dual amplification establishment and optimization are carried out on the TERT gene and the BRAF gene, the evaluation standard is that the two genes are amplified simultaneously; the amplified copy numbers of the two genes are basically equivalent; and the theoretical copy number (for example, about 3000 copies of 10ng genome DNA) is met, and the system amplification efficiency is higher, so that higher detection sensitivity can be achieved. If the copy number of one gene amplification is high and the copy number of the other gene amplification is low, the sensitivity of the two genes is different and the sensitivity of the lower copy number gene is lower in the double detection.
In addition, since micro-droplet digital PCR is used as a detection platform, the droplet stability of a detection system needs to be considered during evaluation. When the liquid drops are generated, the surfactant gathers on the surfaces of the liquid drops in the water phase, the surface tension of the water-oil interface is reduced through molecular assembly, spherical liquid drops with the lowest interface energy are formed, and the stability of the water-in-oil micro liquid drops is maintained. Only if the liquid drop is generated stably and the liquid drop detection is stable (the liquid drop number meets a certain standard), the detection result is accurate and reliable. The digital PCR platform of New Yi manufacturing technology (Beijing) limited company and matched consumables (a sample preparation instrument, a chip analyzer, a matched micro-droplet generation chip, a generation oil, a micro-droplet detection chip and a detection oil) are used for generating micro-droplets with the volume of about 0.5nL, the theoretical micro-droplet number generated by a 30 mu L reaction system is about 6 ten thousand, when the micro-droplets are detected and analyzed, the more the detected droplet number is (but the droplet number should not exceed the theoretical generated droplet number), the higher the sensitivity and the stability of detection are, particularly when a low-copy target (lower than 50 copies) is detected, if the target-containing droplet is lost, the lower the quantitative result is caused, when the target copy number is higher (higher than 50 copies), the influence on the quantitative result is not great by a poisson distribution formula, but at least 50% of generated droplets are usually required to be detected, the detection result is stable and reliable, namely the detected droplet number reaches more than 3 ten thousand, and the detection result meets the standard.
Specifically, the concentration of each component in the buffer solution is regulated based on the conventional buffer solution, betaine, formamide and different combinations are added at the same time, and the amplification efficiency, the signal to noise ratio and the droplet stability of simultaneous amplification of two genes are comprehensively considered. When a conventional buffer solution is used, only the BRAF gene can be amplified, further, after betaine is added into the buffer solution, the TERT gene and the BRAF can be amplified simultaneously, but the copy number of the BRAF is obviously higher than that of the TERT gene by more than 2 times, the final concentration of the betaine is adjusted to be increased from 0.25M to 0.5M, the amplified copy number of the TERT gene is increased, but still lower than that of the BRAF gene, and the concentration of the betaine is continuously increased to 0.75M, so that the generated liquid drop number is obviously reduced, and the addition of betaine with higher concentration can influence the liquid drop stability; further optimize betaine concentration at 0.4-0.6M, TERT copy number is basically equivalent to BRAF copy number, slightly lower than BRAF, and the drop number is more stable, basically more than 3 ten thousand.
In the invention, in order to further improve the BRAF copy number and the TERT copy number, the detection sensitivity is increased, other PCR enhancers are further supplemented into the buffer solution, and after formamide is added, the TERT gene copy number is further increased, basically consistent with the BRAF copy number, and the liquid drop is stable, so that the detection sensitivity is improved.
In the invention, the components and enzyme amount in the buffer are regulated, when the enzyme amount of the DNA polymerase is increased to 3U when the concentration of the Tris is increased from 0.02M to 0.05mM, the detected signal to noise ratio (the ratio of the median of the positive signal intensity to the median of the negative signal intensity) is obviously increased, so that the optimal final concentration of the Taq enzyme is 3-4U, and the optimal concentration of the Tris is 0.04-0.05mM.
In the invention, the glycerol concentration in the buffer solution is regulated, no amplification is carried out when no glycerol is added in the system, the number of liquid drops and the detection copy number are lower when the final glycerol concentration is increased to 2%, the number of liquid drops and the copy number are both more in accordance with the expected standard when the glycerol concentration is increased to 4% -8%, and the number of liquid drops exceeds the expected theoretical value when the glycerol concentration is further increased to 10%, so that the glycerol concentration is 4% -8%.
In the invention, the proportion of 3 dNTPs (dATP, dCTP, dGTP) and dUTP in the system is regulated, and the single dNTP final concentration is 200 mu M as a reference, when the proportion of the dNTPs to the dNTPs is 1:1 and 1:4, the amplified copy number and the signal-to-noise ratio are lower, and when the proportion of the dNTPs to the dNTPs is 1:2 and 1:3, the amplified copy number and the signal-to-noise ratio are higher, so that the expected result is met. Thus, the ratio of 3 dNTPs (dATP, dCTP, dGTP) to dUTP is 1:2 to 1:3 (i.e., dATP, dCTP, dGTP is 200. Mu.M and dUTP is 400-600. Mu.M).
In the invention, the concentration of KCl in the buffer solution is regulated, no KCl is added, no amplification is carried out, and when the final concentration of KCl is increased to 20-50mM, the copy number and the signal-to-noise ratio are higher, so that the method meets the expectations; further increasing the final KCl concentration to 100mM decreases the copy number and signal to noise ratio, and thus the final KCl concentration is 20-50mM.
In the present invention, mgCl in the conditioning buffer 2 Concentration without MgCl 2 No amplification, when MgCl is added 2 The copy number and the signal to noise ratio are higher when the final concentration is 2-6mM, which accords with the expectations; further increase of MgCl 2 At a final concentration of 8mM, the copy number is reduced, and MgCl 2 The final concentration was 2-6mM.
In the invention, the concentration of UNG is regulated to achieve the purpose of pollution prevention, and when UNG is increased to more than 0.2U, 1 ten thousand copies of PCR products containing U can be degraded to achieve the effect of pollution prevention.
In the invention, the concentration of the primer probe is regulated, when the concentration of the primer probe is 400nM and 200nM and above respectively, the amplified copy number and the signal-to-noise ratio are higher, which meet the expectations, so that the final concentration of the primer probe is 400nM and 200nM and above respectively.
In the invention, the amplification procedure is optimized, the copy number and the signal-to-noise ratio are higher when the annealing temperature is 56-60 ℃, and the copy number and the signal-to-noise ratio are higher when the cycle number is 40 cycles or more, so that the annealing temperature in the amplification procedure is 56-60 ℃ and the amplification cycle number is 40 cycles or more.
In the invention, the template pretreatment is further optimized to further improve the detection sensitivity, and the detection copy number can be improved by 60% -80% when the denaturation temperature is 85 ℃ -98 ℃ and the denaturation time is more than 30 seconds.
The invention provides a high-sensitivity TERT and BRAF gene dual-detection kit with simple operation based on a digital PCR method and a multiplex PCR technology. The detection sensitivity of the target sequence under the complex genome background can reach 0.2% by optimizing the final concentration of betaine, the final concentration of formamide, the final concentration of Tris and the final concentration of Taq DNA polymerase. The invention has the advantages that 3 mutation sites of two genes can be detected by one tube, the required sample size is small, and 1-3 FFPE (paraffin embedded tissue slice) samples or 0.1 FNA (fine needle puncture) samples can be detected, thereby being applicable to thyroid nodule auxiliary diagnosis.
Drawings
Fig. 1 is a graph of the linear correlation results of the detection of different mutation rates of BRAF V600E of the present invention.
FIG. 2 is a graph showing the results of the linear correlation of the detection of different mutation rates of TERT C228T according to the invention.
FIG. 3 is a graph showing the results of the linear correlation of the detection of different mutation rates of TERT C250T according to the invention.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present application, the present invention will be further described with reference to examples, and it is apparent that the described examples are only some of the examples of the present application, not all the examples. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, shall fall within the scope of the present application.
Example 1 betaine concentration optimization experiment
1. Human wild-type genomic DNA was used as a template. The basic system is prepared by optimizing the betaine concentration in the system on the basis, and preparing the betaine concentration of 0M, 0.25M, 0.5M and 0.75M respectively to prepare the systems 1-4. Evaluation indexes include amplification efficiency (detection of copy number) and droplet stability (total droplet number). Primer probe sequences are shown in Table 1 below, and primer and probe sequences of the invention are shown in Table 2 below.
TABLE 1
。
TABLE 2
。
Remarks + represents LNA
Using System 1-4, 10ng of wild-type genomic DNA was added for detection.
2. Digital PCR workflow
2.1. And (3) preparing micro-droplets, namely adding a 30 [ mu ] L PCR reaction system into a sample hole of a droplet generation chip (New manufacturing technology (Beijing) limited company) and a sample preparation instrument (New manufacturing technology (Beijing) limited company), adding 160 [ mu ] L droplet generation oil into an oil hole, placing the chip and the 8-row tube into the preparation instrument, covering a rubber mat, and preparing the micro-droplets.
2.2. PCR amplification 8 rows of tubes containing microdroplets were placed on a PCR apparatus for amplification with the basic amplification procedure set forth in Table 3.
TABLE 3 Table 3
。
2.3. After PCR, placing 8 rows of pipes and a liquid drop detection chip (New Youyi manufacturing technology (Beijing) Co., ltd.) into a fixture, respectively adding 430 mu L and 500 mu L detection oil into the oil holes, covering a rubber pad, and placing the chip into a chip analyzer (New Youyi manufacturing technology (Beijing) Co., ltd.) for liquid drop detection.
2.4. And (3) data analysis, namely detecting each micro droplet through a chip analyzer after PCR amplification, recording the fluorescence signal intensity of the micro droplet signal, and detecting the corresponding fluorescence signal of the droplet containing the target gene to be detected. The fluorescence intensity in the micro-droplets is digitized through a fluorescence classification threshold value, the micro-droplets with stronger fluorescence are judged to be 1 (positive), the micro-droplets with weaker fluorescence are judged to be 0 (negative), the numbers of 1 and 0 are counted, and the total copy number of the target genes in the template can be calculated through the correction of a poisson distribution formula.
The test results are shown in Table 4 below, in the basic system (without betaine), the TERT gene is not amplified, the BRAF gene is amplified normally, the copy number is in the expected range, after betaine is increased, the TERT gene starts to amplify, the TERT amplified copy number is increased along with the increase of the betaine concentration, the TERT copy number is about 1/3 of the copy number of the BRAF gene when the final betaine concentration is 0.25M, the TERT copy number is slightly lower than the BRAF copy number when the betaine concentration is 0.5M, but when the betaine concentration is continuously increased to 0.75M, the drop stability is reduced, a plurality of drops are fused, the total drop number is obviously reduced, the drop number is reduced to be about 2.4 Mo Zuo from more than 4 ten thousand on average, and the standard that the total drop number is greater than 3 ten thousand is not met, so the final betaine concentration is set between 0.25 and 0.5M, and the drop number is required to be lower than 0.75M.
TABLE 4 Table 4
。
Based on the system 1, betaine concentration was further optimized, systems 5 to 8 were prepared, betaine final concentrations were 0.3, 0.4, 0.5, and 0.6M, respectively, and 10ng of genomic DNA was used as a template, and the detection procedure was as above, and the detection results were as shown in Table 5 below. The betaine concentration is 0.4-0.6M, the TERT copy number is slightly lower than BRAF, and the drop number is more stable and basically more than 3.5 ten thousand.
TABLE 5
。
EXAMPLE 2 formamide concentration optimization experiments
The formamide is used as a polar solvent, has great advantages in the aspects of water-in-oil droplet polymerization and stability, can promote the annealing of a primer probe and a template, reduces the denaturation temperature of DNA with a secondary structure, and is a common PCR enhancer. In the invention, formamide is further introduced into the system, and the concentration is optimized, so that the TERT amplification copy number is further improved, the purposes of stable amplification and detection of TERT and BRAF double genes are achieved, and the detection sensitivity is improved. Human wild-type genomic DNA was used as a template. Based on the system 6 (the final concentration of betaine is 0.4M), the systems 9 to 14 are prepared so that the final volume concentration of formamide in the system is 0.1 percent, 0.5 percent, 1 percent, 2 percent, 4 percent and 8 percent respectively, and the formamide concentration of the system is optimized, and evaluation indexes comprise amplified copy number and total liquid drop number. The primer probe sequences are shown in Table 2, and the experimental operation steps are the same. The results are shown in Table 6 below, which shows that after the final 0.1% formamide concentration is added, the TERT copy number is slightly increased, and when the final 0.5% -2% formamide concentration is added, the TERT copy number is further increased, which is basically equivalent to BRAF, and is consistent with the expected theoretical value (about 3000 copies of 10ng DNA), the droplet number is basically more than 4 ten thousand (6 ten thousand theoretical values of generated droplet number), and the droplets are stable and the droplet detection rate is high. If the final volume concentration of the formamide is further increased to 4% and 8%, the copy numbers detected by BRAF and TERT are reduced, and the detected droplet number is further increased and is higher than the theoretical generated droplet number (6 ten thousand), at the moment, the formamide serving as a surfactant influences the droplet volume, reduces the droplet volume, and accordingly the detected droplet number is increased, and at the moment, the copy number of a target is calculated according to the poisson distribution formula according to the generated droplet number preset by an instrument, and the quantification is lower. Therefore, the formamide volume concentration is more proper at 0.5-2%, and the droplet size is not influenced, so that the copy numbers of the BRAF gene and the TERT gene can be stably detected, the detected droplet number is stable, and the result is reliable.
TABLE 6
。
Example 3 optimization experiments of enzyme amount and TRIS concentration
Because the digital PCR detection is end point detection, the invention improves the detection signal to noise ratio (the ratio of the median of the positive signal to the median of the negative signal) by further optimizing the enzyme amount of the system and the TRIS concentration in the buffer solution. Based on system 10 (final betaine concentration of 0.4M and final formamide volume concentration of 0.5%), systems 15-20 were prepared, and the enzyme amount and TRIS final concentration were adjusted, respectively. Then 10ng of human wild-type genomic DNA was added for detection, as described above. The evaluation indexes are mainly copy number and signal to noise ratio.
The detection results are shown in Table 7 below. When the enzyme amount is increased from 2U to 3U, the BRAF and TERT detection copy numbers are basically equivalent to the theoretical copy numbers, the signal-to-noise ratio (the ratio of positive signals to negative signals) of the two genes is increased from 1.5-2 to 3.5-4, the enzyme amount is further increased to 4U, and no further increase is caused to the signal-to-noise ratio, so that the enzyme amount can be 3-4U. When the TRIS concentration is further increased from 0.02M to 0.04-0.05M under the condition of the enzyme amount being fixed to 3U, the signal to noise ratio can be further improved to about 4-5. When the TRIS concentration is increased to 0.06M, the signal to noise ratio is lowered, so that the TRIS concentration is 0.04-0.05M.
TABLE 7
。
Example 4 optimization of Glycerol concentration in System
Based on system 20 (final betaine concentration of 0.4M, final formamide volume concentration of 0.5%, taq DNA polymerase of 3U, tris of 0.06M), other component concentrations such as system 1, glycerol concentration was changed only, and systems 21-26 were prepared, wherein glycerol final concentrations were 0, 2%, 4%, 6%, 8%,10%, respectively, using 10ng of genomic DNA as a template, the detection procedure was as above, and the detection results were as shown in Table 8 below. When no glycerol (0%) is added into the system, the liquid drops are fused, when the glycerol concentration in the system is increased to 2%, the liquid drop number is about 2.5-3 ten thousand, the liquid drop number is lower than an evaluation standard, when the glycerol concentration is increased to 4-8%, the liquid drop number is stabilized to 4-5.5 ten thousand, the BRAF and TERT copy numbers basically accord with the expected result (about 3000 copies), and when the glycerol reaches 10%, the liquid drop number is higher than the expected 6 ten thousand, so that the glycerol concentration is 4-8%.
TABLE 8
。
Example 5 optimization of dNTP concentration in System
dUTP and UNG are added into the system, so that anti-pollution measures can be effectively established, and the PCR product containing U is degraded. dTTP is replaced by dUTP, and the concentration proportion of each dNTP needs to be optimized to achieve optimal amplification efficiency. Based on system 20, only the ratio of 3 dNTPs (dATP, dCTP, dGTP) to dUTP concentrations was changed to 1:1, 1:2, 1:3, 1:4 (the base concentrations were 200. Mu.M each for 3 dNTPs (dATP, dCTP, dGTP) in the system, and dUTP was 200. Mu.M, 400. Mu.M, 600. Mu.M, 800. Mu.M, respectively), and systems 27-30 were prepared using 10ng of genomic DNA as a template, and the detection procedures were as described above, with the detection results shown in Table 9 below. When the ratio of other 3 dNTPs (dATP, dCTP, dGTP) to dUTP in the system is 1:2 and 1:3, the amplified copy number accords with the expectation, and the signal-to-noise ratio of the detection signal is higher; when the ratio is 1:1, the amplification efficiency is poor, the copy number is reduced, and the signal-to-noise ratio of the detection signals is low and is basically indistinguishable; when the concentration ratio is 1:4, the signal to noise ratio of the detection signal is slightly reduced. Thus, the ratio of the other 3 dNTPs (dATP, dCTP, dGTP) to dUTP is 1:2 to 1:3 (i.e., dATP/dCTP/dGTP of 200. Mu.M each and dUTP of 400. Mu.M to 600. Mu.M).
TABLE 9
。
Example 6 optimization of KCl concentration in System
Based on system 20, KC1 concentration was varied to prepare systems 31-34 with final KCl concentrations of 0, 20, 50, 100mM, respectively, using 10ng of genomic DNA as template, the assay procedure was as above, and the assay results were as follows. When KC1 is not added into the system, no amplification is carried out, when the concentration of KC1 in the system is increased to 20-50mM, the copy numbers of BRAF and TERT basically accord with the expected result, the signal to noise ratio of the detection signal is high, when KC1 reaches 100mM, the copy numbers of BRAF and TERT are reduced, and the signal to noise ratio is also reduced, so that the concentration of KCl is 20-50mM.
Table 10
。
Example 7 optimization of UNG concentration in System
Based on system 20, the UNG concentration was varied to prepare systems 35-39, and final UNG concentrations were 0, 0.1, 0.2, 0.3, and 0.4U, respectively, using prepared U-containing PCR products (i.e., PCR products amplified using system 20) at different concentrations (1E 4, 1E3, and 100 copies) as templates, and the detection steps were as above, and the detection results were as shown in Table 11 below. When UNG is not added into the system, no pollution prevention effect exists, and the products containing U are not degraded; when UNG is increased to 0.1U, the U-containing product with the copy number of 1000 can be degraded, and when UNG is increased to more than 0.2U, the U-containing product with the copy number of 1E4 can be degraded, so that the purpose of preventing PCR product pollution can be achieved. Therefore, the UNG concentration is 0.2U or more, preferably 0.2 to 0.4U.
TABLE 11
。
Example 8 optimization of primer probe concentration in System
Based on the system 20, changing the concentration of primer probes to prepare a system 40-43, wherein the final concentration of the primers is 200nM, 400nM, 600 nM and 800nM respectively; the corresponding probe concentrations were 100, 200, 300, 400nM, using 10ng of genomic DNA as template, and the detection procedure was as above, with the following table 12. When the concentration of the primer probe in the system is 200-100nM, the signal-to-noise ratio of the detection signal is low, and the amplification efficiency is low; when the primer probe concentration in the system is 400-200nM, 600-300nM and 800-400nM, the signal-to-noise ratio is high, thus meeting the detection requirement. Thus, the primer concentration in the system is at least 400nM and above, and the probe concentration is at least 200nM and above.
Table 12
。
EXAMPLE 9 MgCl in System 2 Optimization of concentration
Based on system 20, mgCl was changed 2 Concentration, formulation System 44-48, mgCl 2 Final concentrations were 0, 2, 4, 6, 8mM, respectively, using 10ng genomic DNA as template, the detection procedure was as above, and the detection results were as shown in table 13 below. When MgCl is not added into the system 2 In the absence of amplification, mgCl in the system 2 When the concentration is increased to 2mM, the BRAF and TERT copy numbers basically accord with the expected result, the signal to noise ratio of the detection signal is higher, and when MgCl is used 2 The copy number was expected to be further increased at 4-6mM and the signal to noise ratio was further increased, and BRAF and TERT copy numbers were decreased at 8mM MgCl2, thus MgCl 2 The concentration is 2-6mM.
TABLE 13
。
Example 10 optimization of amplification procedure
The annealing temperature and the number of amplification cycles, which mainly affect the amplification efficiency, were optimized based on the system 45 of Table 14 below, and the annealing temperature of the primer was 56 to 63℃and the annealing temperature of the probe was 67 to 72℃as shown in Table 2, so that the annealing temperatures were set to 56, 58, 60, 62℃based on the basic amplification procedure in example 1 above (cycle number: 40), and the cycle numbers were set to 35, 45, 50 based on the basic amplification procedure in example 1 above (annealing 60 ℃); using 10ng of genomic DNA as a template, the detection procedure was as above, and the detection results were as shown in Table 15 below. When the annealing temperature is 56-60 ℃, the signal to noise ratio of the detection signal is higher, and when the annealing temperature is 62 ℃, BRAF is not amplified, so the annealing temperature of the system is 56-60 ℃. When the cycle is 35, the signal-to-noise ratio of the detection signal is low, the TERT is basically free of positive signals, and when the cycle number is more than 40, the signal-to-noise ratio of the detection signal is higher, so that the amplification cycle number of the system is more than 40, when the amplification cycle number is increased to 50 cycles, the signal-to-noise ratio is not increased, and the cycle number of the system is 40-45 cycles in consideration of the influence of the detection duration.
TABLE 14
。
TABLE 15
。
EXAMPLE 11 template nucleic acid pretreatment optimization
The pretreatment of the template nucleic acid is optimized based on the system 45, and the template nucleic acid is subjected to the thermal denaturation pretreatment by dispersing the template into a micro-reaction unit for reaction, so that the template nucleic acid can be converted into single strands and then enter the micro-reaction unit, the detection copy number is further improved, the detection sensitivity is further improved, the nucleic acid is subjected to the treatment at 75, 80, 85, 90, 95 and 98 ℃ for 5 minutes, and the denaturation treatment time is set to 30 seconds, 1, 3, 5 and 10 minutes under the condition of fixing the denaturation temperature at 95 ℃; using 10ng of genomic DNA as a template, the detection procedure was as above, and the detection results were as shown in Table 16 below. When the thermal denaturation temperature is 75-80 ℃, the detection copy number is increased by about 30%, and when the denaturation temperature is 85-98 ℃, the detection copy number is increased by about 60-80%, so that the denaturation temperature is 85-98 ℃ and the denaturation time is from 30 seconds to 10 minutes, the detection copy number can be increased, and the denaturation time is more than 30 seconds.
Table 16
。
Example 12 test sensitivity test
Using clinical samples of BRAF V600E positive and TERT C250T and C228T positive, FFPE samples were taken in 1-2 pieces with a cell number of FNA samples of 0.1, and then nucleic acid was extracted from the samples. The extracted nucleic acid was subjected to gradient dilution with wild-type genomic DNA to different mutation rates (Ratio), with mutation rates=mutant copy number/(mutant copy number+wild-type copy number) set to 50%,20%,5%,2.5%,1%,0.2%, respectively. Each concentration sample was repeated 3 times using the detection system described above for system 45. The results of the measurements are shown in the following table, with the linear curves shown in FIGS. 1-3. The result shows that the BRAF and TERT double amplification system of the invention can accurately detect mutant type, has high linear correlation and R 2 All are larger than 0.99, and 0.2% of mutation can be accurately detected, so that clinical requirements are met. The BRAF V600E different mutation rate detection results are shown in Table 17, the TERT C228T different mutation rate detection results are shown in Table 18, and the TERT C250T different mutation rate detection results are shown in Table 19.
TABLE 17
。
TABLE 18
。
TABLE 19
。
It is to be understood that this invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also encompassed by the appended claims.

Claims (4)

1. A digital PCR kit for thyroid cancer detection is characterized by comprising a kit buffer system, a primer and a probe, wherein the kit buffer system comprises Tris-HCl, glycerol and dNTPs, KCl, mgCl 2 The preparation method comprises the steps of (1) preparing Taq DNA polymerase, UNG enzyme, betaine and formamide, wherein the final concentration of the betaine is 0.4-0.6M, the final volume concentration of the formamide is 0.5% -2%, and the use amount of the Taq DNA polymerase is more than 3U in every 30 microliters of a reaction system;
TERT gene upstream primer SEQ ID No.1:5'-CCTGCCCCTTCACCTTCCAG-3', TERT gene downstream primer SEQ ID No.2:5'-AGCGCTGCCTGAAACTCG-3', TERT gene wild type probe SEQ ID No.3:5'-CAGCC+CCTT+CCGGG-3', TERT-C228T mutant probe SEQ ID No.4:5'-GACCC+CT+TC+CGGGTC-3', TERT-C250T mutant probe SEQ ID No.5:5 '-CGACCCCT+CCCGGGG-3', BRAF gene upstream primer SEQ ID No.6:5'-CATGAAGACCTCACAGTAAAAATAGG-3', BRAF gene downstream primer SEQ ID No.7:5'-CAACTGTTCAAACTGATGGGACC-3', BRAF V600E mutant probe SEQ ID No.8:5'-AG+CTA+ CAGAGAAAT +CTCGATG-3', BRAF wild type probe SEQ ID No.9:5 'AG+CTA+ CAGTGAAAT +CTCGATG-3';
the final concentration of Tris-HCl is 0.04-0.05M; the final volume concentration of the glycerol is 4-8%; the final concentration of KCl is 20-50mM; the MgCl 2 The final concentration is 2-6mM; the UNG usage amount is more than 0.2U per 30 microliters of final usage amount in the reaction system; dATP: dUTP, dCTP: dUTP and dGTP: dUTP mass ratio in dNTPs is 1:2 to 1:3.
2. The digital PCR kit according to claim 1, wherein the Taq DNA polymerase is used in an amount of 3 to 4u per 30 microliters of the reaction system.
3. The digital PCR kit of claim 1, wherein the UNG enzyme is used in an amount of 0.2 to 0.4U per 30 microliters of the reaction system.
4. The digital PCR kit of claim 1, wherein the primer concentration is not less than 400nM and the probe concentration is not less than 200nM.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111748628A (en) * 2020-07-14 2020-10-09 润安医学科技(苏州)有限公司 Primer and kit for detecting thyroid cancer prognosis related gene variation
WO2022144432A1 (en) * 2020-12-31 2022-07-07 Ingenierie Et Analyse En Genetique Environnementale Pathogen detection in liquid matrix

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111748628A (en) * 2020-07-14 2020-10-09 润安医学科技(苏州)有限公司 Primer and kit for detecting thyroid cancer prognosis related gene variation
WO2022144432A1 (en) * 2020-12-31 2022-07-07 Ingenierie Et Analyse En Genetique Environnementale Pathogen detection in liquid matrix

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Microfluidic Droplet Digital PCR Is a Powerful Tool for Detection of BARF and TERT Mutations in Papilary Thyroid Carcinomas;Ylli,D等;《Cancers》;第11卷(第12期);第1-14页 *

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