CN110724742A - Kit for detecting hot spot mutation of human TERT gene promoter - Google Patents

Abstract

The present invention discloses a kit for detecting hot spot mutation of human TERT gene promoter, which is characterized by comprising: DNA enrichment detection kit for TERT gene promoter -124 (C/T) and -146 (C/T) mutation sites Collection of conventional PCR master mixes, ‑124 (C/T) and ‑146 (C/T) mutant plasmids and human peripheral blood wild-type DNA standards and ‑124 (C/T) and ‑146 (C/T) mutations Rate detection qPCR master mix. The kit of the invention adopts the fluorescent quantitative PCR technology platform carried out in clinical routine to detect DNA samples from various sources. The kit has the advantages of high detection sensitivity, strong specificity, accurate and quantitative detection of mutation rate, low cost, and is suitable for clinical routine development. Features.

Description

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

technical field

The invention relates to the field of biotechnology, in particular to a sample enrichment, standard preparation and sample calibration, and mutation rate detection for detecting human TERT gene promoter hot spot mutations -146 (C/T) and -124 (C/T) real-time quantitative PCR (qPCR) detection kit.

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 kits suitable for various specimen sources.

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 kit for detecting hot spot mutation of human TERT gene promoter.

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

A kit for detecting hot spot mutation of human TERT gene promoter, including DNA enrichment conventional PCR premix reagent for detection of TERT gene promoter -124 (C/T) and -146 (C/T) mutation sites, - 124(C/T) and -146(C/T) mutant plasmids and human peripheral blood wild-type DNA standards and -124(C/T) and -146(C/T) mutation rate detection qPCR master mix reagents.

Further, the conventional PCR premix reagent for DNA enrichment includes primers, and the primer sequences are SEQ ID NO.1 and SEQ ID NO.2.

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

Further, the reaction conditions of DNA enrichment conventional PCR reaction were 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.

Further, to detect the mutation rate of TERT-146(C/T) and TERT-124(C/T), the qPCR amplification reaction conditions were pre-denaturation at 98°C for 8 min; 98°C for 15s, 58°C for 40s, 50 cycles.

Further, the preparation methods of -124(C/T) and -146(C/T) mutant plasmids and human peripheral blood wild-type DNA standards are: using healthy human peripheral blood DNA samples and -124(C/T) and -124(C/T) and The -146(C/T) mutant plasmid sample was amplified by DNA enrichment conventional PCR, the product was purified and the concentration was determined, converted into 10 ¹¹ copies/ml according to the molecular weight, and 10 ¹¹ copies/ml of healthy human-derived DNA was used as the matrix , adding 10 ¹¹ copies/ml calibrator of TERT-146(C/T) or TERT-124(C/T) mutation after calibration, respectively, to prepare 100%, 10%, 1%, 0.5%, 0.1%, Gradient mutation rate standards of 0.05% mutant and 100% wild.

Further, the kit is suitable for tissues containing TERT-124(C/T) and -146(C/T) mutant sequences, paraffin-embedded tissues, whole blood DNA, plasma cell-free DNA, body fluid samples or mutant plasmids detection of samples.

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 result interpretation is clear and objective, and the result is interpreted according to the mutation rate standard curve; (5) The universality of the specimen is high, because the TERT gene promoters -124 (C/T) and -146 (C/T) are enriched first. The mutation site sequence is then detected, and the original sample concentration will not affect the detection results. The kit is suitable for the detection of TERT hotspot mutation rate in tissue, paraffin-embedded tissue, whole blood DNA, plasma cell-free DNA, and various body fluid samples; ( 6) The kit has a wide range of clinical applications, and the hot spot mutation of TERT is a molecular marker of various tumors, which is of great value for the molecular diagnosis, treatment monitoring and prognosis judgment of the mutation-related tumors; (7) The clinical application is highly popularized, and qPCR technology The platform is a technical platform commonly carried out in clinical practice at present, and the kit of the present invention is a method established 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;

Fig. 3 is the amplification curve and standard curve diagram of the present invention-146 (C/T) mutation rate standard substance;

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

Fig. 5 is the amplification curve and standard curve diagram of the present invention-124 (C/T) mutation rate standard substance;

Fig. 6 is a graph showing the amplification curve and the standard curve for the detection of the mutation rate of apparently healthy human -124 (C/T) according to the present invention.

Detailed ways

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

The self-provided instruments required by the kit of the present invention are as follows: various fluorescence quantitative PCR instruments, various conventional PCR thermal cyclers, ultraviolet spectrophotometers, high-speed centrifuges, vortex shakers, and sterile water for injection.

Self-provided reagents required by the kit of the present invention: DNA extraction kit, common DNA product purification reagent.

1. DNA enrichment conventional PCR reaction

(1) PCR reaction mixed reagents: Add various substances with the following concentrations to the premixed reagents:

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 1.

(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: ordinary 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 and -146(C/T), -124(C/T) mutant plasmid samples were amplified by DNA enrichment conventional PCR, and the products were purified, After the concentration was determined, it was converted into 10 ¹¹ copies/ml according to the molecular weight, and 10 ¹¹ copies/ml of healthy human-derived DNA was used as the matrix, and 10 of the calibrated -146 (C/T) or -124 (C/T) mutations were added. 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) qPCR reaction mixture reagent: add various substances in the following concentrations to the premix reagent:

name Dosage (final concentration) Master mix 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) Cycling conditions of the -146 (C/T): pre-denaturation at 98°C for 8 min; denaturation at 98°C for 15s, annealing at 58°C for 40s, 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 1.

(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) 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 3, 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, R ² =0.999); M: mutant; W: wild. According to the measurement results of apparently healthy people, the lower limit of detection of the -146(C/T) mutation rate detected by the kit is 0.05%, see Figure 4, the results show that the mutation rates of apparently healthy people are all less than 0.05%; M: mutation ; W: wild.

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

(1) qPCR reaction mixture reagent: add various substances in the following concentrations to the premix reagent:

name Dosage (final concentration) Master mix 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

The cycle conditions of -124 (C/T) described in a(2): pre-denaturation at 98°C for 8 min; 98°C for 15s, 58°C for 40s (collecting the fluorescence value), 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 1.

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

(5) 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, R ² =0.999) M: mutant; W: wild. According to the measurement results of apparently healthy people, the lower limit of detection of the -124(C/T) mutation rate detected by the kit is 0.05%, see Figure 6, the results show that the mutation rates of apparently healthy people are all less than 0.05%; M: mutation ; W: wild.

Table 1 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 (7)

1. A kit for detecting hot spot mutation of a human TERT gene promoter is characterized in that: comprises DNA enrichment conventional PCR premixed reagent for detecting TERT gene promoter-124 (C/T) and-146 (C/T) mutation sites, 124(C/T) and-146 (C/T) mutation plasmids, human peripheral blood wild type DNA standard and-124 (C/T) and-146 (C/T) mutation rate detection qPCR premixed reagent.

2. The kit for detecting hot spot mutation of the human TERT gene promoter according to claim 1, wherein the kit comprises: the DNA enrichment conventional PCR premixed reagent comprises primers, wherein the sequences of the primers are SEQ ID NO.1 and SEQ ID NO. 2.

3. The kit for detecting hot spot mutation of the human TERT gene promoter according to claim 1 or 2, wherein the kit comprises: the-124 (C/T) and-146 (C/T) mutation rate detection qPCR premixed reagent comprises a primer, an indicator probe and an LNA (low noise amplifier) inhibition probe, and the sequences of the primer, the indicator probe and the LNA inhibition probe for the 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; the primer, the indicator probe and the LNA inhibitory probe for detecting qPCR amplification reaction with TERT-124(C/T) mutation rate have the sequences of SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.6 respectively.

4. The kit for detecting hot spot mutation of the human TERT gene promoter according to claim 1, wherein the kit comprises: 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 kit for detecting hot spot mutation of the human TERT gene promoter according to claim 1, wherein the kit comprises: detecting mutation rates 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.

6. The kit for detecting hot spot mutation of the human TERT gene promoter according to claim 1, wherein the kit comprises: the preparation method of the-124 (C/T) and-146 (C/T) mutant plasmids and the human peripheral blood wild type DNA standard comprises the following steps: DNA enrichment routine PCR amplification is carried out on a healthy human peripheral blood DNA sample and-124 (C/T) and-146 (C/T) mutant plasmid samples, and the product is converted into 10 according to the molecular weight after purification and concentration determination¹¹Copy 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.

7. The kit for detecting hot spot mutation of the human TERT gene promoter according to claim 1, wherein the kit comprises: the kit is suitable for detecting tissues containing TERT-124(C/T) and-146 (C/T) mutant sequences, wax block embedded tissues, whole blood DNA, plasma free DNA, body fluid samples or samples of mutant plasmids.

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