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

Abstract

The invention discloses a method for detecting hot spot mutation of human TERT gene promoter, comprising the following steps: a. using DNA enrichment conventional PCR reaction to enrich templates for different samples; b. PCR products are purified and the concentration is determined by ultraviolet spectrophotometer , carry out concentration calibration and standard preparation; c. Quantitatively detect the mutation rate of TERT-146 (C/T) and TERT-124 (C/T) in the calibrated samples by qPCR. The invention adopts the qPCR technology platform carried out in clinical routine to establish a high-sensitivity quantitative detection method of TERT hot spot mutation rate for DNA samples from various sources, and the lower limit of detection mutation rate reaches 0.05%. The method of the invention has the advantages of high detection sensitivity and specificity. It has the characteristics of strong, accurate and quantitative detection of mutation rate, low cost and suitable for clinical routine development.

Description

A method for detecting hot spot mutation of human TERT gene promoter

technical field

The invention relates to the field of biotechnology, in particular to a real-time fluorescence quantitative PCR (qPCR) detection method for detecting the mutation rates of human TERT gene promoter hot spot mutations -146 (C/T) and -124 (C/T).

Background technique

Telomere refers to a unique structure composed of DNA and related proteins containing simple repeating 5'-TTAGGG-3' base series at the end of chromosomes. It exists in eukaryotic cells and its main function is to maintain the integrity of chromosome structure and genome. Stability. With each cell division cycle, telomere DNA is reduced, and telomere length is progressively shortened, eventually leading to the cessation of cell division, forcing cells to senesce and die. Telomerase is a ribonucleoprotein polymerase mainly composed of telomerase RNA template (hTR), telomerase reverse transcriptase (TERT) and telomerase-related proteins. The activation of telomerase is the main mechanism for the maintenance or extension of telomere length, and the up-regulation of TERT expression is considered to be one of the main reasons for the activation and activity of telomerase.

In 2013, Science Journal first reported the detection of high mutation rates (total mutation rate>70%) in TERT promoter regions -124 (C/T or C/A), -146 (C/T) in melanoma pedigrees and patients , among which -124(C/T) is the main mutation type, followed by -146(C/T) mutation, -124(C/A) mutation is extremely rare. Mutations can form the TTCCGG/ATCCGG sequence, which is the binding site for the Ets/TCF transcription factor, resulting in a 2- to 4-fold increase in TERT gene transcriptional activity. This hotspot mutation occurs mainly in tumors of tissue origin with low self-renewal rate, mainly including glioblastoma multiforme 80-90%, hepatocellular carcinoma 60%, bladder cancer 60%, basal cell carcinoma 70%, skin Squamous cell carcinoma 50% and up to 30% of thyroid cancer, and is associated with aggressiveness of thyroid cancer, glioblastoma, neuroblastoma and renal cell carcinoma, detection of this hot spot mutation for the diagnosis, efficacy of the above tumors Monitoring and prognosis judgment have important clinical application value. Especially in the progression of primary hepatocellular carcinoma, this hot spot mutation of TERT is the earliest somatic genetic alteration in hepatocellular carcinoma, and its mutation rate is 6%-19% in poor nodules of liver cirrhosis, and it is a hepatic The only initiating mutation driver gene among multiple key signaling pathway gene mutations in cell carcinoma, which acts as a "gatekeeper" key step in the transformation sequence to hepatocellular carcinoma, and the mutation is significantly increased in early hepatocellular carcinoma ( 61%) and remained stable in advanced and advanced hepatocellular carcinoma, therefore, this mutation is more likely to be a target for early carcinogenesis surveillance in cirrhosis.

Various samples from human body can be used for gene somatic mutation detection, but the DNA concentration that can be obtained from different sample sources is quite different. Further detection of a certain type of somatic mutation in target DNA in low-concentration samples requires extremely sensitive detection. Methods, usually different detection methods need to be tested for specific specimens. For example, the concentration of DNA extracted from DNA in fresh tissue and peripheral blood is high, while the concentration of free DNA in paraffin-embedded tissue, various body fluids, and plasma is extremely low. Therefore, it is urgent to develop detection methods suitable for various specimen sources.

At present, the methods of gene somatic mutation detection can be divided into: based on qPCR technology, digital PCR (ddPCR) technology, high-throughput next-generation sequencing (NGS) technology, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) MALDI-TOF MS) method and other methods, their detection principle, sensitivity, advantages and disadvantages are different from each other (see Table 1).

Table 1 Comparison of gene mutation detection techniques

Note: PNA Clamp-PCR (peptide nucleic acids clamp PCR, peptide nucleic acid-clamp fluorescence PCR); LNA/DNA-PCR (locked nucleic acids/DNA chimera PCR, locked nucleic acids/DNA chimera PCR); COLD-PCR (complete enrichment PCR) coamplification at lower denaturation temperature PCR, low denaturation temperature complex PCR); ARMS (amplification refractory mutation system, mutation amplification block system); ddPCR (droplet digital PCR, droplet digital PCR); BEAMing (beads, emulsion, amplification, magnetics, digital PCR-flow technology); WGS (whole-genome sequencing, whole genome sequencing); Digital karyotyping (digital karyotyping analysis); PARE (personalized analysis of rearranged ends, individualized analysis of rearranged ends); Targeted Sequencing (targeted Sequencing); TAm-Seq (tagged-amplicon deep sequencing, tagged amplification deep sequencing); SAFE-SeqS (safe-sequencing system, safe sequencing system); CAPP-Seq (Cancer Personalized Profiling by DeepSequencing, deep sequencing tumor individualized documentation method); iDES (integrated digital errorsuppression, integrated digital error suppression); WES (whole-exome sequencing, whole exome sequencing); cSMART (CirculatingSingle-Molecule Amplification and Resequencing Technology, circular single-molecule amplification and resequencing technology) .

Since the DNA sequences around the -124 (C/T) and -146 (C/T) mutations in the TERT promoter region are rich in GC (about 80%), and the sequences around these 2 mutation sites are almost identical to each other, these factors It increases the difficulty of designing and screening specific primers and probes, and brings great challenges to the establishment of detection methods. At present, for the detection of the hot spot mutation rate of TERT, the NGS method (the lower limit of detection is about 0.1%) and the ddPCR method (the lower limit of detection is about 0.1%-0.05%) have been reported in the public literature. High cost and expensive testing equipment are not suitable for clinical routine development.

SUMMARY OF THE INVENTION

In order to solve the above technical problem, the present invention provides a method for detecting hot spot mutation of human TERT gene promoter.

The present invention is realized according to the following technical solutions.

A method for detecting hot spot mutation of human TERT gene promoter, comprising the following steps:

a. Use DNA enrichment conventional PCR reaction enrichment template for different specimens;

b. The PCR product is purified and the concentration is determined by UV spectrophotometer, and concentration calibration and standard preparation are carried out;

c. Quantitative detection of TERT-146(C/T) and TERT-124(C/T) mutation rates on the calibrated samples by qPCR.

Further, in step a, the specimen includes tissue containing TERT-124 (C/T) and -146 (C/T) mutation sequences, paraffin-embedded tissue, whole blood DNA, plasma cell-free DNA, body fluid samples or Mutant plasmid.

Further, in step a, the primer sequences of the DNA enrichment conventional PCR reaction are SEQ ID NO.1 and SEQ ID NO.2.

Further, in step a, the reaction conditions of the conventional PCR reaction for DNA enrichment are 98°C for 3 min of pre-denaturation; 45 cycles of 98°C denaturation for 30s, 60°C annealing for 30s, and 72°C extension for 30s.

Further, in step c, the sequences of primers, indicator probes and LNA inhibition probes for TERT-146 (C/T) mutation rate detection qPCR amplification reaction are respectively SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6; TERT-124 (C/T) mutation rate detection primers, indicator probes and LNA inhibition probe sequences for qPCR amplification reaction are SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO. ID NO.9 and SEQ ID NO.6.

Further, in step c, the mutation rates of TERT-146 (C/T) and TERT-124 (C/T) were detected, and the qPCR amplification reaction conditions were pre-denaturation at 98°C for 8min; 98°C for 15s, 58°C for 40s, 50 cycle.

Further, in step b, the concentration of the DNA-enriched conventional PCR reaction product is measured by an ultraviolet spectrophotometer, and according to the molecular weight of the amplified product, the sample is diluted to a concentration of 10 ¹¹ copies/ml to be tested.

Further, in step b, the mutation rate standard preparation method is as follows: DNA samples from healthy human peripheral blood and mutant plasmid samples are amplified by DNA enrichment conventional PCR, the products are purified and the concentration is determined, and then converted into 10 ¹¹ copies according to the molecular weight. /ml, using 10 ¹¹ copies/ml of healthy human DNA as the matrix, adding 10 ¹¹ copies/ml calibrators of the TERT-146 (C/T) or TERT-124 (C/T) mutation after calibration, respectively prepared A gradient mutation rate standard of 100%, 10%, 1%, 0.5%, 0.1%, 0.05% mutant and 100% wild was used.

The present invention obtains the following beneficial effects.

(1) Quantitative detection of mutation rate, according to the mutation rate standard curve, the hot spot mutation rate result of TERT in the test specimen is directly read, and the result is meaningful for clinical disease diagnosis and dynamic treatment monitoring; (2) High accuracy and specificity, using TaqMan-MGB probe as detection indicator probe can effectively avoid false positive results caused by non-specific PCR amplification such as TaqMan probe and SYBR dye method; (3) The detection sensitivity is high, among which the -146(C/T) mutation rate is detected The sensitivity reached 0.05%, and the detection sensitivity of -124(C/T) mutation rate reached 0.05%; (4) The interpretation of the results was clear and objective, and the results were interpreted according to the mutation rate standard curve; (5) The universality of the specimen was high, because the first rich The TERT gene promoter -124(C/T) and -146(C/T) mutation site sequences are collected and then detected, and the original sample concentration can be detected. It is suitable for tissue, wax block embedded tissue, whole tissue Detection of TERT hot spot mutation rate in blood DNA, plasma cell-free DNA, and various body fluid samples; (6) The detection results are widely used in clinical practice. TERT hot spot mutation is a molecular marker of various tumors, which is useful for molecular diagnosis and treatment of tumors related to this mutation. Monitoring and prognosis judgment are of great value; (7) The clinical application is highly popularized, the qPCR technology platform is a technology platform commonly developed in clinical practice, and the present invention is a method based on the qPCR technology platform, and has wide clinical applicability.

Description of drawings

Fig. 1 is the electrophoresis diagram of 163bp DNA enrichment conventional PCR amplification product of the present invention;

Fig. 2 is the sequencing result diagram of 163bp DNA enrichment conventional PCR amplification product of the present invention;

Figure 3 is a schematic diagram of the present invention -146 (C/T) and -124 (C/T) mutation rate qPCR detection;

Fig. 4 is the amplification curve and standard curve diagram of the present invention-146 (C/T) mutation rate standard substance;

Figure 5 is an amplification curve and a standard curve diagram of the -124 (C/T) mutation rate standard of the present invention.

Detailed ways

The present invention will be further described below with reference to the accompanying drawings and embodiments.

The instruments used in the present invention are as follows: ABI7500 fluorescence quantitative PCR instrument, ABI2700 PCR instrument, AlphaInnotech gel imager, Bio-Rad company PowerPac electrophoresis instrument, UV spectrophotometer, high-speed centrifuge, water bath, vortex shaker, refrigerator , oven, autoclave, sterile water for injection.

Reagents used in the present invention: The following reagents are all from Tiangen Biochemical Technology (Beijing) Co., Ltd., including 2×GC-Rich premix reagent, common DNA product purification reagent, and SuperReal fluorescence quantitative premix reagent (probe method).

The primers, TaqMan probes and LNA inhibitory probes used in the present invention are all synthesized by Beijing Saibaisheng Company; TaqMan-MGB probes are synthesized by Shanghai Synthesis Department of ABI Company.

The technical route of the present invention includes 3 steps, (1) first, for different samples, DNA enrichment is used to enrich the template by conventional PCR reaction; (2) the PCR product is purified and the concentration is determined by an ultraviolet spectrophotometer, and concentration calibration and standard preparation are performed; (3) Quantitative detection of mutation rate of the calibrated samples by qPCR.

1. DNA enrichment conventional PCR reaction

(1) Reaction mixture: Add the following concentrations of various substances to the 2×GC-Rich premix reagent:

name 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) Sterile water for injection 6.5μl(-) DNA template 2μl (100ng～0.05ng) total 25μl

(2) The cycle conditions: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 30s, annealing at 60°C for 30s, extension at 72°C for 30s, 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 clinical specimens, including but not limited to the following specimens: tissue, paraffin-embedded tissue, whole blood DNA, plasma free DNA, various body fluids such as urine, saliva and other specimens.

(5) The detection principle: conventional PCR reaction.

(6) Interpretation of the results: the length of the specific amplified fragment is 163 bp. After electrophoresis on a 2% agarose gel, it is observed whether there is a 163 bp target amplified fragment, and whether there is a non-specific amplified fragment, see Figure 1, 2.

Among them, the swimming lane numbered 1 in Figure 1 and Figures 2A and 2B: derived from the electrophoresis and sequencing results of peripheral blood DNA amplification products of apparently healthy people; the swimming lane numbered 2 in Figure 1 and Figure 2C: derived from - 124(C/T) mutant plasmid standard amplification product electropherogram and sequencing result map; lane numbered 3 in Fig. 1 and Fig. 2D: Electropherogram of amplified product derived from -146(C/T) mutant plasmid standard and sequencing results.

2. Concentration calibration and mutation rate standard preparation

(1) After the DNA enrichment conventional PCR reaction product is purified by DNA purification reagent, the concentration is determined by using an ultraviolet spectrophotometer. According to the molecular weight of the amplified product, the sample is diluted to a concentration of 10 ¹¹ copies/ml to be tested.

(2) The mutation rate standard was prepared as follows: DNA samples from healthy human peripheral blood, -146(C/T) mutant plasmid and -124(C/T) mutant plasmid were amplified by DNA enrichment conventional PCR, and the product was purified , After the concentration is determined, it is converted into 10 ¹¹ copies/ml according to the molecular weight, and 10 ¹¹ copies/ml of healthy human-derived DNA is used as the matrix. After calibration, -146 (C/T) or -124 (C/T) mutation 10 ¹¹ copies/ml calibrators, formulated as gradient mutation rate standards of 100%, 10%, 1%, 0.5%, 0.1%, 0.05% mutant and 100% wild, respectively.

3. TERT-146 (C/T) mutation rate detection reaction

(1) Reaction mixture: Add the following concentrations of various substances to the SuperReal fluorescence quantitative premix reagent (probe method):

name Dosage (final concentration) Mastermix 10.0μl(-) 146-F (SEQ ID NO.3) primer 1μl (0.25μM) 146-R (SEQ ID NO.4) primer 1μl (0.25μM) 146-LNA (SEQ ID NO. 5) inhibition probe 0.2μl (0.20μM) 146-P (SEQ ID NO. 6) probe 1μl (0.25μM) ROX 0.2μl(-) Sterile water for injection 4.6μl(-) DNA template (1×10¹¹ copies/ml) 2μl (1×10¹⁰copy number/ml) total 20μl

(2) Cyclic conditions of the -146 (C/T): pre-denaturation at 98°C for 8 min; denaturation at 98°C for 15s, 58°C for 40s (collecting fluorescence values), 50 cycles.

(3) The 146-F (SEQ ID NO. 3), 146-R (SEQ ID NO. 4) primers for the -146 (C/T) mutation, and the 146-LNA (SEQ ID NO. 5) inhibition probe The needle and 146-P (SEQ ID NO. 6) probe sequences are shown in Table 2.

(4) The DNA template to be tested (1×10 ¹¹ copy number/ml) is from the UV spectrophotometer calibration product of the PCR enriched product.

(5) Detection principle: On the qPCR technology platform, the amplification retardation mutation system (ARMS) combined with the LNA inhibitory probe is used to inhibit the wild-type amplification to achieve the principle of selective amplification of mutant alleles. The principle of ARMS is based on the fact that when the mismatch occurs at the 3' end of the primer, the amplification efficiency of the primer will be significantly reduced, and the primer specificity can be significantly increased by introducing artificial mismatch bases at the -2--4 positions of the 3' end of the primer. The 146-F (SEQ ID NO. 6) primer introduced a mismatched base G at the 3'-end-3 position to increase specificity. For LNA inhibitory probes, it has a strong base recognition ability (especially for T-C mismatches) and a higher annealing temperature than the primers. During PCR annealing, it can competitively bind the wild-type allele preferentially, occupying the primer binding site, so that the primers bind more mutant alleles, and finally achieve the purpose of selectively amplifying mutant alleles. In addition, TaqMan-MBG probe was used for amplification signal collection, which has strong sequence recognition ability, avoids false positive results caused by non-specific products, and significantly increases the accuracy of detection results, see FIG. 3A .

(6) Interpretation of results: Read the mutation rate of the sample to be tested according to the qPCR amplification standard curve of the 100%-0.05% mutant and 100% wild-type gradient mutation rate standards at a concentration of 1×10 ¹¹ copies/ml, see Figure 4, the results show that the detection limit of mutation rate is 0.05%; the linear range of mutation rate is 100%-0.05% (Slope= ^-3.97 , R2=0.999), M: mutant; W: wild.

4. TERT-124 (C/T) mutation rate detection reaction

(1) Reaction mixture: Add the following concentrations of various substances to the SuperReal fluorescence quantitative premix reagent (probe method):

name 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-LNA (SEQ ID NO. 9) inhibition probe 0.25μl (0.25μM) 146-P (SEQ ID NO. 6) probe 1μl (0.25μM) ROX 0.2μl(-) Sterile water for injection 4.55μl(-) DNA template (1×10¹¹ copies/ml) 2μl (1×10¹⁰copy number/ml) total 20μl

(2) Cyclic conditions of -124 (C/T): pre-denaturation at 98°C for 8 min; 98°C for 15s, 58°C for 40s (collecting fluorescence values), 50 cycles.

(3) The 124-F (SEQ ID NO. 7), 124-R (SEQ ID NO. 8) primers for the -124 (C/T) mutation, and the 124-LNA (SEQ ID NO. 9) inhibition probe The needle and 146-P (SEQ ID NO. 6) probe sequences are shown in Table 2.

(4) The DNA template (1×10 ¹¹ copies/ml) is from the calibration product of the UV spectrophotometer of PCR enriched products.

(5) Detection principle: the same as -146 (C/T) detection principle, see Figure 3B.

(6) Interpretation of results: Read the mutation rate of the sample to be tested according to the qPCR amplification standard curve of the 100%-0.05% mutant and 100% wild-type gradient mutation rate standards at a concentration of 1×10 ¹¹ copies/ml, see Figure 5, the results show that the detection limit of mutation rate is 0.05%; the linear range of mutation rate is 100%-0.05% (Slope= ^-3.76 , R2=0.999), M: mutant; W: wild.

Table 2 Enrichment, detection primers and probes for TERT-146 (C/T) and -124 (C/T) mutations

Note: The underlined bases in the 146-F and 124-R primer sequences are mismatched bases, which are used to increase the specificity of allele discrimination; the underlined bases in the 146-LNA and 124-LNA sequences are locked nucleic acid bases.

Claims (8)

1. a method for detecting human TERT gene promoter hot spot mutation is characterized in that: comprise the following steps: a. Use DNA enrichment conventional PCR reaction enrichment template for different specimens; b. The PCR product is purified and the concentration is determined by UV spectrophotometer, and concentration calibration and standard preparation are carried out; c. Quantitative detection of TERT-146 (C/T) and TERT-124 (C/T) mutation rates on the calibrated specimens by qPCR. 2. A method for detecting hot spot mutation of human TERT gene promoter according to claim 1, characterized in that: in step a, the specimen comprises TERT-124 (C/T) and -146 (C/T) ) mutated sequences of tissue, paraffin-embedded tissue, whole blood DNA, plasma cell-free DNA, body fluid samples or mutant plasmids. 3. a kind of method of detecting human TERT gene promoter hot spot mutation according to claim 1, is characterized in that: in step a, the primer sequence of DNA enrichment conventional PCR reaction is SEQ ID NO.1 and SEQ ID NO. 2. 4. The method for detecting hot spot mutation of human TERT gene promoter according to claim 3, characterized in that: in step a, the reaction conditions of DNA enrichment conventional PCR reaction are 98 ℃ of pre-denaturation for 3min; 98 ℃ of denaturation for 30s , annealing at 60°C for 30s, and extending at 72°C for 30s, for 45 cycles. 5. A method for detecting hot spot mutation of human TERT gene promoter according to claim 1 or 3, characterized in that: in step c, the TERT-146 (C/T) mutation rate detects the primers of the qPCR amplification reaction, The sequences of indicator probe and LNA inhibitor probe are SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively; TERT-124(C/T) mutation rate detection qPCR amplification reaction The sequences of primers, indicator probes and LNA inhibition probes are SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.6, respectively. 6. The method for detecting hot spot mutation of human TERT gene promoter according to claim 5, characterized in that: in step c, TERT-146 (C/T) and TERT-124 (C/T) mutation rates are detected , qPCR amplification reaction conditions were pre-denaturation at 98°C for 8 min; 98°C for 15s, 58°C for 40s, 50 cycles. 7. a kind of method for detecting human TERT gene promoter hot spot mutation according to claim 1, is characterized in that: in step b, described DNA enrichment conventional PCR reaction product adopts ultraviolet spectrophotometer to carry out concentration measurement, according to amplification. To increase the molecular weight of the product, the sample was diluted to a concentration of 10 ¹¹ copies/ml for testing. 8. A method for detecting hot spot mutation of human TERT gene promoter according to claim 1, characterized in that: in step b, the method for preparing a mutation rate standard product is: using a healthy human peripheral blood DNA sample and a mutant plasmid sample through DNA enrichment routine PCR amplification, product purification and concentration determination, converted to 10 ¹¹ copies/ml according to molecular weight, using 10 ¹¹ copies/ml of healthy human DNA as the matrix, adding calibrated TERT-146 (C/T ) or TERT-124(C/T) mutated 10 ¹¹ copies/ml calibrators formulated to gradient mutation rates of 100%, 10%, 1%, 0.5%, 0.1%, 0.05% mutant and 100% wild, respectively Standard.

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