CN111424094A - Method for detecting hot spot mutation of human TERT gene promoter - Google Patents

Method for detecting hot spot mutation of human TERT gene promoter Download PDF

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CN111424094A
CN111424094A CN202010452615.3A CN202010452615A CN111424094A CN 111424094 A CN111424094 A CN 111424094A CN 202010452615 A CN202010452615 A CN 202010452615A CN 111424094 A CN111424094 A CN 111424094A
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梁国威
张洁
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Aerospace Center Hospital
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Abstract

The invention discloses a method for detecting hot spot mutation of a human TERT gene promoter, which comprises the following steps: a. adopting a DNA enrichment conventional PCR reaction enrichment template aiming at different samples; purifying the PCR product, measuring the concentration by an ultraviolet spectrophotometer, and performing concentration calibration and standard substance preparation; c. TERT-146(C/T) and TERT-124(C/T) mutation rates were quantitatively determined on the calibrated samples using qPCR. The invention adopts a qPCR technical platform developed by clinical routine, and establishes a high-sensitivity TERT hotspot mutation rate quantitative detection method aiming at DNA samples of various sources, wherein the lower limit of the detection mutation rate is up to 0.05%.

Description

Method for detecting hot spot mutation of human TERT gene promoter
Technical Field
The invention relates to the technical field of biology, in particular to a real-time fluorescent quantitative PCR (qPCR) detection method for detecting hot spot mutation-146 (C/T) and-124 (C/T) mutation rates of a human TERT gene promoter.
Background
Telomeres (telomere) refer to a unique structure consisting of DNA and related proteins with a simple repeated 5 '-TTAGGG-3' base series at the end of a chromosome, exist in eukaryotic cells, and have the main functions of maintaining the integrity of the chromosome structure and the stability of a genome, so that the DNA of the telomeres is reduced in each cell division cycle, the length of the telomeres is progressively shortened, and finally, the cell division is stopped, and the cells are forced to age and die. Telomerase is a ribonucleoprotein polymerase, and mainly consists of a telomerase RNA template (hTR), telomerase reverse transcriptase (TERT) and telomerase related protein. Telomerase activation is the major mechanism for maintenance or extension of telomere length, while TERT expression upregulation is thought to be one of the major causes of telomerase activation and activity enhancement.
The scientific journal of 2013 reports that TERT promoter region-124 (C/T or C/A), -146(C/T) high mutation rate (total mutation rate > 70%) is detected in melanoma families and patients for the first time, wherein-124 (C/T) is the main mutation type, and-146 (C/T) mutation and-124 (C/A) mutation are very rare. The mutation can form TTCCGG/ATCCGG sequence which is an Ets/TCF transcription factor binding site and leads to 2-to 4-fold increase of the transcription activity of the TERT gene. The hot spot mutation mainly appears in tumors derived from tissues with low self-renewal rate, mainly comprises 80-90% of glioblastoma multiforme, 60% of hepatocellular carcinoma, 60% of bladder cancer, 70% of basal cell carcinoma, 50% of skin squamous cell carcinoma and up to 30% of thyroid carcinoma, and is related to the invasiveness of thyroid carcinoma, glioblastoma, neuroblastoma and renal cell carcinoma, and the detection of the hot spot mutation has important clinical application value for the diagnosis, curative effect monitoring and prognosis of the tumors. TERT is the earliest somatic genetic change in hepatocellular carcinoma, particularly during the progression of primary hepatocellular carcinoma, with a mutation rate of 6% -19% in poorly cirrhotic nodules, and is the only initiating mutation driver among multiple key signaling pathway gene mutations in hepatocellular carcinoma, representing a critical step in the sequence for transformation to hepatocellular carcinoma as a "gatekeeper", whereas mutations in early hepatocellular carcinoma are significantly increased (61%) and remain stable in both progression and late hepatocellular carcinoma, and therefore, are more likely to be targets for early-stage monitoring of cirrhosis.
Various samples from human bodies can be used for detecting gene somatic mutation, but the concentration difference of DNA obtained from different sample sources is large, and an extremely sensitive detection method is needed for further detecting certain type of somatic mutation in target DNA in a low-concentration sample, and different detection methods are generally needed to detect specific samples. For example, since the concentration of DNA extracted from fresh tissue and peripheral blood is high, and the concentration of free DNA in wax-block-embedded tissue, various body fluids and plasma is extremely low, development of a detection method suitable for various specimen sources is urgently required.
At present, the method for detecting somatic mutation of gene can be divided into methods based on qPCR technology, digital PCR (ddPCR) technology, high-throughput sequencing by second generation (NGS) technology, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MA L DI-TOF MS) technology and other methods according to the difference of main technical principles, and the detection principles, sensitivity and advantages and disadvantages of the methods are different from each other (see Table 1).
TABLE 1 comparison of Gene mutation detection techniques
Figure BDA0002508174550000021
Note that PNA Clamp-PCR (peptide nucleic acids clamped fluorescent PCR), L NA/DNA-PCR (packed nucleic acids/DNA polymerase PCR), locked nucleic acids/DNA chimeric PCR, CO L D-PCR (complete expression at low denaturation temperature multiplex PCR), ARMS (Amplification reaction simulation system), ddPCR (drop Digital PCR), BEAM (beads, expression, Amplification, molecular, Digital PCR-flow Technology), WGS (white-genome-Sequencing, whole genome Sequencing), Digital kallikryzation (Sequencing Digital Amplification), SAFETE (amplified nucleic acid Sequencing, amplified Sequencing.
Since the DNA sequences around TERT promoter region-124 (C/T) and-146 (C/T) mutations are GC-rich (about 80%), and the sequences around these 2 mutation sites are nearly identical to each other, these factors increase the difficulty of designing and screening specific primers and probes, which presents a significant challenge to the establishment of detection methods. At present, for the hot spot mutation rate detection of TERT, the published documents report an NGS method (the lower limit of detection is about 0.1%) and a ddPCR method (the lower limit of detection is about 0.1% -0.05%), and the 2 methods have the problems of complex operation, high detection cost, expensive detection equipment and the like, and are not suitable for clinical routine development.
Disclosure of Invention
The invention provides a method for detecting hot spot mutation of a human TERT gene promoter in order to solve the technical problems.
The invention is realized according to the following technical scheme.
A method for detecting hot spot mutation of a human TERT gene promoter comprises the following steps:
a. adopting a DNA enrichment conventional PCR reaction enrichment template aiming at different samples;
purifying the PCR product, measuring the concentration by an ultraviolet spectrophotometer, and performing concentration calibration and standard substance preparation;
c. TERT-146(C/T) and TERT-124(C/T) mutation rates were quantitatively determined on the calibrated samples using qPCR.
Further, in step a, the specimen comprises tissues containing TERT-124(C/T) and-146 (C/T) mutant sequences, wax-block embedded tissues, whole blood DNA, plasma-free DNA, body fluid specimens or mutant plasmids.
Further, in the step a, the primer sequences of the DNA enrichment conventional PCR reaction are SEQ ID NO.1 and SEQ ID NO. 2.
Further, in the step a, the reaction condition of the DNA enrichment conventional PCR reaction is pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, for 45 cycles.
Further, in the step C, the sequences of a primer, an indicator probe and an L NA inhibition probe for detecting the TERT-146(C/T) mutation rate qPCR amplification reaction are respectively SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, and the sequences of a primer, an indicator probe and a L NA inhibition probe for detecting the TERT-124(C/T) mutation rate qPCR amplification reaction are respectively SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 6.
Further, in the step C, detecting the mutation rate of TERT-146(C/T) and TERT-124(C/T), wherein the condition of qPCR amplification reaction is pre-denaturation at 98 ℃ for 8 min; 15s at 98 ℃, 40s at 58 ℃ and 50 cycles.
Further, in the step b, the DNA enrichment conventional PCR reaction product adopts an ultraviolet spectrophotometer to carry out concentration determination, and the sample is diluted to 10 according to the molecular weight of the amplification product11Copy number/ml concentration was checked.
Further, in the step b, the preparation method of the mutation rate standard substance comprises the following steps: DNA enrichment routine PCR amplification is carried out on a healthy human peripheral blood DNA specimen and a mutant plasmid specimen, and after the product is purified and the concentration is determined, the product is converted into 10 according to the molecular weight11Copy number/ml, at 1011Copy number/ml DNA of healthy human origin as substrate, 10 of the calibrated TERT-146(C/T) or TERT-124(C/T) mutations were added11Copy number/ml calibrator, formulated as 100%, 10%, 1%, 0.5%, 0.1%, 0.05% mutant and 100% wild-type gradient mutation rate standards, respectively.
The present invention obtains the following advantageous effects.
(1) Quantitatively detecting the mutation rate, namely directly reading the mutation rate result of the hot spot of the TERT in the detection sample according to a mutation rate standard curve, wherein the result has significance for clinical disease diagnosis and dynamic treatment monitoring; (2) the accuracy and the specificity are high, and false positive results caused by non-specific PCR amplification of a TaqMan probe, a SYBR dye method and the like can be effectively avoided by adopting the TaqMan-MGB probe as a detection indication probe; (3) the detection sensitivity is high, wherein the detection sensitivity of the mutation rate of-146 (C/T) reaches 0.05 percent, and the detection sensitivity of the mutation rate of-124 (C/T) reaches 0.05 percent; (4) the result interpretation is clear and objective, and the result interpretation is carried out according to a mutation rate standard curve; (5) the specimen has high universality, and the TERT gene promoter-124 (C/T) and-146 (C/T) mutation site sequences are firstly enriched and then detected, so that the original specimen concentration can be detected, and the method is suitable for detecting the TERT hotspot mutation rate in tissues, wax block embedded tissues, whole blood DNA, plasma free DNA and various body fluid specimens; (6) the detection result is widely applied clinically, the TERT hot spot mutation is a molecular marker of various tumors, and has important values for molecular diagnosis, treatment monitoring and prognosis judgment of the mutation-related tumors; (7) the method has strong clinical application and popularization, the qPCR technical platform is a commonly developed technical platform in clinic at present, and the method is a method established on the qPCR technical platform and has wide clinical applicability.
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FIG. 1 is an electrophoretogram of a 163 bpDNA-enriched conventional PCR amplification product of the present invention;
FIG. 2 is a diagram showing the sequencing results of a 163 bpDNA-enriched conventional PCR amplification product of the present invention;
FIG. 3 is a diagram of the qPCR detection principle of mutation rates of-146 (C/T) and-124 (C/T) according to the present invention;
FIG. 4 is a graph of the amplification curve and standard curve of the mutation rate standard of-146 (C/T) according to the present invention;
FIG. 5 is a graph showing the amplification curve and standard curve of the mutation rate standard of the present invention-124 (C/T).
Detailed Description
The invention is further explained below with reference to the drawings and the examples.
The apparatus used in the present invention is as follows: ABI7500 fluorescence quantitative PCR instrument, ABI2700 PCR instrument, AlphaInotech gel imager, Bio-Rad PowerPac electrophoresis apparatus, ultraviolet spectrophotometer, high speed centrifuge, water bath, vortex oscillation instrument, refrigerator, oven, sterilizer, and water for sterilization and injection.
The reagents used in the invention are all from Tiangen Biotechnology (Beijing) Co., Ltd, and comprise 2 × GC-Rich premixed reagent, common DNA product purification reagent and SuperReal fluorescent quantitative premixed reagent (probe method).
The primers, TaqMan probe and L NA inhibition probe used in the invention are all synthesized by Beijing Saiban company, and the TaqMan-MGB probe is synthesized by Shanghai synthesis part of ABI company.
The technical route of the invention comprises 3 steps, (1) DNA enrichment routine PCR reaction enrichment templates are adopted for different samples; (2) purifying the PCR product, measuring the concentration by an ultraviolet spectrophotometer, and performing concentration calibration and standard substance preparation; (3) and (3) carrying out mutation rate quantitative detection on the calibrated sample by adopting qPCR.
1. DNA enrichment routine PCR reaction
(1) Reaction mixture to 2 × GC-Rich premixed reagent were added the following concentrations of each of the substances:
name (R) Dosage (final concentration)
Mastermix 12.5μl(-)
163-F (SEQ ID NO.1) primer 2μl(0.4μM)
163-R (SEQ ID NO.2) primer 2μl(0.4μM)
Sterilized water for injection 6.5μl(-)
DNA template 2μl(100ng～0.05ng)
Total of 25μl
(2) The circulation conditions are as follows: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, for 45 cycles.
(3) The 163-F (SEQ ID NO.1) and 163-R (SEQ ID NO.2) primer sequences are shown in Table 2.
(4) The DNA template: various specimens clinically, including but not limited to the following specimens: tissue, wax-block embedded tissue, whole blood DNA, plasma free DNA, samples of various body fluids such as urine, saliva, etc.
(5) The detection principle is as follows: conventional PCR reaction.
(6) And (3) interpretation of the result: the length of the specific amplified fragment is 163bp, and after 2% agarose gel electrophoresis, the existence of 163bp target amplified fragments and the existence of non-specific amplified fragments are observed, which are shown in attached figures 1 and 2.
Wherein, the lane marked with 1 in FIG. 1 and FIGS. 2A and 2B: electrophoresis images and sequencing result images of DNA amplification products from apparent healthy human peripheral blood; lane numbered 2 in fig. 1 and fig. 2C: electrophoresis images and sequencing result images of amplification products of the standard plasmid derived from the-124 (C/T) mutant plasmid; lanes marked with 3 in FIG. 1 and FIG. 2D electrophoresis and sequencing results of amplification products from the-146 (C/T) mutant plasmid standard.
2. Concentration calibration and mutation rate standard formulation
(1) After the DNA enrichment conventional PCR reaction product is purified by a DNA purification reagent, an ultraviolet spectrophotometer is adopted to carry out concentration determination, and a sample is diluted to 10 according to the molecular weight of an amplification product11Copy number/ml concentration was checked.
(2) The mutation rate standard is prepared by the following method: DNA enrichment routine PCR amplification is carried out on a healthy human peripheral blood DNA sample, a-146 (C/T) mutant plasmid and a-124 (C/T) mutant plasmid, and the product is converted into 10 according to the molecular weight after being purified and the concentration is measured11Copy number/ml, at 1011Copy number/ml keyHuman-origin DNA as a substrate, to which 10 of the calibrated-146 (C/T) or-124 (C/T) mutation was added11Copy number/ml calibrator, formulated as 100%, 10%, 1%, 0.5%, 0.1%, 0.05% mutant and 100% wild-type gradient mutation rate standards, respectively.
3. TERT-146(C/T) mutation rate detection reaction
(1) Reaction mixture liquid: to the SuperReal fluorescent quantitation premix reagent (probe method) were added the following concentrations of the various substances:
name (R) Dosage (final concentration)
Mastermix 10.0μl(-)
Primer 146-F (SEQ ID NO.3) 1μl(0.25μM)
146-R (SEQ ID NO.4) primer 1μl(0.25μM)
146-L NA (SEQ ID NO.5) inhibition probe 0.2μl(0.20μM)
146-P (SEQ ID NO.6) Probe 1μl(0.25μM)
ROX 0.2μl(-)
Sterilized water for injection 4.6μl(-)
DNA template (1 × 10)11Number of copies/ml) 2μl(1×1010Number of copies/ml)
Total of 20μl
(2) The cycle condition of-146 (C/T): pre-denaturation at 98 ℃ for 8 min; denaturation at 98 ℃ for 15s, fluorescence at 58 ℃ for 40s (fluorescence values collected), 50 cycles.
(3) The 146-F (SEQ ID NO.3), 146-R (SEQ ID NO.4) primers, 146-L NA (SEQ ID NO.5) inhibition probe and 146-P (SEQ ID NO.6) probe sequences for the-146 (C/T) mutation are shown in Table 2.
(4) The DNA template to be detected (1 × 10)11Copy number/ml) uv spectrophotometer calibration product from PCR enriched product.
(5) The detection principle is that on a qPCR technology platform, an Amplification Retardation Mutation System (ARMS) is combined with L NA inhibition probes to inhibit wild type amplification to achieve the principle of selectively amplifying mutant allele, the ARMS principle is that when mismatch occurs at the 3 ' end of a primer, the amplification efficiency of the primer is obviously reduced, the specificity of the primer can be obviously increased by introducing artificial mismatched bases at the 3 ' end-2 to-4 positions of the primer, in the 146-F (SEQ ID NO.6) primer, mismatched base G is introduced at the 3 ' end-3 positions to increase the specificity, for L NA inhibition probes, the primers have extremely strong base recognition capability (particularly for T-C mismatch) and the annealing temperature higher than that of the primers, can be preferentially combined with wild type allele in competition during PCR annealing, occupy primer combination sites, so that more primers are combined with mutant allele, and finally achieve the purpose of selectively amplifying mutant allele.
(6) The result is interpreted according to the concentration of 1 × 1011A qPCR amplification standard curve of 100% -0.05% mutation of copy number/ml and a 100% wild gradient mutation rate standard substance is read, the mutation rate of a sample to be detected is read, and the result shows that the lower limit of the mutation rate detection is 0.05% referring to an attached figure 4; the linear range of the mutation rate is 100-0.05% (Slope ═ 3.97, R)20.999), M: mutation; w: and (5) wild.
4. TERT-124(C/T) mutation rate detection reaction
(1) Reaction mixture liquid: to the SuperReal fluorescent quantitation premix reagent (probe method) were added the following concentrations of the various substances:
name (R) Dosage (final concentration)
Mastermix 10.0μl(-)
124-F (SEQ ID NO.7) primer 1μl(0.25μM)
124-R (SEQ ID NO.8) primer 1μl(0.25μM)
124-L NA (SEQ ID NO.9) inhibition probe 0.25μl(0.25μM)
146-P (SEQ ID NO.6) Probe 1μl(0.25μM)
ROX 0.2μl(-)
Sterilized water for injection 4.55μl(-)
DNA template (1 × 10)11Number of copies/ml) 2μl(1×1010Number of copies/ml)
Total of 20μl
(2) Cycling conditions of said-124 (C/T): pre-denaturation at 98 ℃ for 8 min; 98 ℃ for 15s,58 ℃ for 40s (fluorescence values collected), 50 cycles.
(3) The 124-F (SEQ ID NO.7), 124-R (SEQ ID NO.8) primers, 124-L NA (SEQ ID NO.9) inhibition probe and 146-P (SEQ ID NO.6) probe sequences for the-124 (C/T) mutation are shown in Table 2.
(4) The DNA template (1 × 10)11Copy number/ml) calibration product from a uv spectrophotometer of PCR enriched product.
(5) The detection principle is as follows: see FIG. 3B for the same-146 (C/T) detection principle.
(6) The result is interpreted according to the concentration of 1 × 1011A qPCR amplification standard curve of 100% -0.05% mutation of copy number/ml and a 100% wild gradient mutation rate standard substance is read, the mutation rate of a sample to be detected is read, and the result shows that the lower limit of the mutation rate detection is 0.05% referring to an attached figure 5; the linear range of the mutation rate is 100-0.05% (Slope ═ 3.76, R)20.999), M: mutation; w: and (5) wild.
TABLE 2 enrichment, detection primers and probes for TERT-146(C/T) and-124 (C/T) mutations
Figure BDA0002508174550000081
Note that the underlined bases in the 146-F and 124-R primer sequences are mismatched bases for increased specificity of allele discrimination, and the underlined bases in the 146-L NA and 124-L NA sequences are locked nucleic acid bases.

Claims (8)

1. A method for detecting hot spot mutation of a human TERT gene promoter is characterized by comprising the following steps: the method comprises the following steps:
a. adopting a DNA enrichment conventional PCR reaction enrichment template aiming at different samples;
purifying the PCR product, measuring the concentration by an ultraviolet spectrophotometer, and performing concentration calibration and standard substance preparation;
c. TERT-146(C/T) and TERT-124(C/T) mutation rates were quantitatively determined on the calibrated samples using qPCR.
2. The method for detecting hot spot mutation of the human TERT gene promoter according to claim 1, characterized in that: in step a, the specimen comprises tissues containing TERT-124(C/T) and-146 (C/T) mutant sequences, wax block embedded tissues, whole blood DNA, plasma free DNA, body fluid specimens or mutant plasmids.
3. The method for detecting hot spot mutation of the human TERT gene promoter according to claim 1, characterized in that: in step a, the primer sequences of the DNA enrichment conventional PCR reaction are SEQ ID NO.1 and SEQ ID NO. 2.
4. The method of claim 3, wherein the hot spot mutation of the human TERT gene promoter is detected by: in the step a, the reaction condition of the DNA enrichment conventional PCR reaction is pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, for 45 cycles.
5. The method for detecting hot spot mutation of the human TERT gene promoter as claimed in claim 1 or 3, wherein in step C, the primer, indicator probe and L NA inhibition probe sequences for detecting the TERT-146(C/T) mutation rate qPCR amplification reaction are respectively SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, and the primer, indicator probe and L NA inhibition probe sequences for detecting the TERT-124(C/T) mutation rate qPCR amplification reaction are respectively SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 6.
6. The method of claim 5, wherein the hot spot mutation in the human TERT gene promoter is detected by: in the step C, detecting the mutation rate of TERT-146(C/T) and TERT-124(C/T), wherein the condition of qPCR amplification reaction is pre-denaturation at 98 ℃ for 8 min; 15s at 98 ℃, 40s at 58 ℃ and 50 cycles.
7. The method for detecting hot spot mutation of the human TERT gene promoter according to claim 1, characterized in that: in the step b, the concentration of the DNA enrichment conventional PCR reaction product is measured by adopting an ultraviolet spectrophotometer, and a sample is diluted to 10 according to the molecular weight of an amplification product11Copy number/ml concentration was checked.
8. The method for detecting hot spot mutation of the human TERT gene promoter according to claim 1, characterized in that: in the step b, the preparation method of the mutation rate standard substance comprises the following steps: DNA enrichment routine PCR amplification is carried out on a healthy human peripheral blood DNA specimen and a mutant plasmid specimen, and after the product is purified and the concentration is determined, the product is converted into 10 according to the molecular weight11Copy number/ml, at 1011Copy number/ml DNA of healthy human origin as substrate, 10 of the calibrated TERT-146(C/T) or TERT-124(C/T) mutations were added11Copy number/ml calibrator, formulated as 100%, 10%, 1%, 0.5%, 0.1%, 0.05% mutant and 100% wild-type gradient mutation rate standards, respectively.
CN202010452615.3A 2019-10-18 2020-05-26 Method for detecting hot spot mutation of human TERT gene promoter Pending CN111424094A (en)

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CN106795563A (en) * 2014-08-25 2017-05-31 杜克大学 For method that is quick and delicately detecting hot spot mutation
CN104805206A (en) * 2015-04-29 2015-07-29 苏州工业园区为真生物医药科技有限公司 Kit for detecting TERT (telomerase reverse transcriptase) gene promoter mutation, and detection method of kit
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