CN108893536B - Method and kit for detecting copy number variation of c-MET gene from human peripheral blood CTC - Google Patents

Abstract

The invention discloses a method for detecting copy number variation of a c-MET gene from human peripheral blood CTC, a kit for detecting copy number variation of the c-MET gene from human peripheral blood CTC by using the method and application of the kit in treatment and detection of tumor patients at different stages. The method and the kit are beneficial to the guiding individual administration of the patient, the detection result can be used for clinical individual treatment, the effectiveness of the treatment is predicted, the clinical administration selection is convenient, the treatment effect is obviously improved, and the patient is benefited to the maximum extent; meanwhile, the problem that tissue samples are difficult to obtain can be avoided, and monitoring can be carried out in the treatment period of a patient.

Description

Method and kit for detecting copy number variation of c-MET gene from human peripheral blood CTC

Technical Field

The invention relates to the field of tumor cell detection, in particular to a method and a kit for detecting copy number variation of a c-MET gene from human peripheral blood CTC.

Background

With the progress of biological analysis technology and the development of information technology, as well as the deep understanding based on genetic Medicine, the concept of Precision Medicine (Precision Medicine) is abstracted from the concepts of system Medicine, transformation Medicine, 4P Medicine, and the like, and becomes a highly comprehensive and detailed new medical model, and the level of disease prevention and disease treatment is expected to be improved. Liquid biopsy was first proposed by Sorrells in 1974 and is a non-invasive method of pathological detection of the systemic circulation of a patient, which monitors CTCs, ctDNA fragments or exosomes released into the blood by tumors or metastases; the method can be used for quickly and atraumatically diagnosing, treating and monitoring tumor patients. The liquid biopsy technology is gradually applied to the diagnosis and treatment of tumors. ctDNA is a DNA fragment released into blood by tumor cells, and is suitable for early screening, personalized medication guidance, drug resistance monitoring and the like; CTC is a tumor cell released into peripheral circulating blood from a solid tumor or a metastatic lesion, and is suitable for prognosis, detection of a therapeutic effect, and the like. For individuals, whether CTC, ctDNA or exosome, there is a large fluctuation at the patient individual level; particularly, in the case where the concentrations of CTC and ctDNA are relatively low in the early detection, the detection rate of ctDNA is significantly reduced.

The CTC detection detects trace CTCs in peripheral blood by capturing and detecting the CTCs, and monitors the trend of the change of the types and the quantity of the CTCs so as to monitor the tumor dynamics in real time, evaluate the treatment effect and realize real-time individual treatment. As the CTC technology matured, the physician was able to promptly follow a new treatment regimen by measuring the change in the number of CTCs, indicating that the chemotherapeutic drug was effective if the number of CTCs decreased significantly, and ineffective if the number of CTCs increased. The recurrence and metastasis of the tumor are closely related to CTC, and the monitoring of CTC can predict the recurrence risk of the tumor earlier than the conventional imaging examination means. However, the content of CTCs in peripheral blood is extremely low, and the enrichment and detection of CTCs are the biggest challenges facing clinical application. Thus, there is a pressing need for a detection technique and method that enables qualitative liquid biopsy of CTCs.

The c-MET protooncogene is present in the long arm of human chromosome 7, the protein product is the c-MET tyrosine kinase receptor, and the c-MET gene is expressed in both embryonic and adult stages. The ligand of c-MET is Hepatocyte Growth Factor (HGF), secreted by mesenchymal cells, and the binding of HGF and c-MET promotes proliferation, migration, differentiation and morphological changes of cells. The HGF/c-MET signaling pathway is subject to complex and high regulation and plays an important role in cell proliferation, differentiation and movement, while c-MET gene overexpression in tumor tissues is a main cause of drug resistance of EGFR-TKI causing non-small cell lung cancer. Therefore, there is an urgent need and great significance to develop a method for detecting copy number variation of c-MET gene from human peripheral blood CTC, in order to guide individual administration to patients by the method, so that the method is suitable for different stages of tumor patients, including but not limited to early stage, middle stage, late stage, pre-and post-operation, treatment detection and recurrence detection.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: a method for detecting copy number variation of c-MET gene from human peripheral blood CTC is provided. The advantages of high sensitivity, high specificity and easy detection of CTC and the advantages of high flux, high sensitivity, time saving and the like of high-flux sequencing are utilized; the method of hybrid capture is used for carrying out target high-throughput sequencing on the hotspot mutation region of the related gene in the enriched CTC, so that the patient can be subjected to guided individual administration, and the method is suitable for different stages of tumor patients, including but not limited to early stage, middle stage, late stage, before and after operation, treatment detection, relapse detection and the like.

In order to solve the technical problems, the invention provides a method for detecting copy number variation of a c-MET gene from human peripheral blood CTC, which specifically comprises the following steps:

(a) enriching CTC from human peripheral blood by subtraction method;

(b) obtaining a c-MET gene copy number variation primer and a specific taqman probe;

(c) obtaining an internal reference gene specific primer and a specific taqman probe;

(d) establishing a detection method of c-MET gene copy number variation, and detecting the c-MET gene copy number from human peripheral blood CTC by adopting a 3D digital PCR platform;

the 5 'end of the specific taqman probe for detecting copy number variation of the c-MET gene is marked with FAM fluorescent group, and the 3' end is marked with TAMRA fluorescent group.

The base sequence of the taqman probe corresponding to the c-MET gene is shown as SEQ ID NO: 1 is shown in the specification;

specific upstream and downstream primers for detecting c-MET gene subtype in human peripheral blood CTC have base sequences shown in SEQ ID NO: 2 (upstream primer) and SEQ ID NO: 3 (downstream primer);

the base sequence of the taqman probe corresponding to the internal reference gene is shown as SEQ ID NO: 4 is shown in the specification;

the base sequences of the specific upstream and downstream primers of the internal reference gene subtype are shown as SEQ ID NO: 5 (upstream primer) and SEQ ID NO: 6 (downstream primer).

The invention also provides a kit for detecting the copy number variation of the c-MET gene from human peripheral blood CTC by using the method and application of the kit in tumor patient detection.

The kit comprises:

the base sequence of the specific taqman probe for detecting copy number variation of c-MET gene is shown as SEQ ID NO: 1 is shown in the specification;

the c-MET gene copy number variation primer has a base sequence shown as SEQ ID NO: 2 and SEQ ID NO: 3 is shown in the specification;

the base sequence of the specific taqman probe corresponding to the internal reference gene is shown as SEQ ID NO: 4 is shown in the specification;

the base sequence of the internal reference gene primer is shown as SEQ ID NO: 5 and SEQ ID NO: and 6.

The invention has the beneficial effects that:

(1) the method can be used for detecting the copy number variation of the c-MET gene in human peripheral blood CTC, and utilizes the advantages of high sensitivity, high specificity and easy detection of CTC and the advantages of high flux, high sensitivity, time saving and the like of high-throughput sequencing; performing target high-throughput sequencing on a hot spot mutation region of a related gene in the enriched CTCs by using a hybridization capture method; the detection result can be used for clinical individualized treatment, the effectiveness of treatment is predicted, clinical medication selection is facilitated, the treatment effect is obviously improved, and patients are benefited to the greatest extent; meanwhile, the problem that tissue samples are difficult to obtain can be avoided, and monitoring can be carried out in the treatment period of a patient.

(2) The kit disclosed by the invention is simple in design, simple and convenient to operate, rapid and high in flux; the kit is applicable to different stages of tumor patients, including but not limited to early stage, middle stage, late stage, before and after operation, treatment detection and recurrence detection.

Drawings

FIG. 1 is a process of detecting copy number variation of c-MET gene from human peripheral blood CTC according to the present invention.

FIG. 2 is a quantitative curve of fluorescent quantitative PCR detection system to 10-fold serial dilution of c-MET gene standard.

FIG. 3 is a quantitative curve of a fluorescent quantitative PCR detection system for a 10-fold serial dilution of an internal reference gene standard.

FIG. 4 shows the result of detecting the copy number variation of c-MET gene in CTC cells.

Detailed Description

The technical solution of the present invention is described in detail below with reference to the accompanying drawings and specific embodiments, but the scope of the present invention is not limited to the embodiments.

Example 1: enrichment of CTC from human peripheral blood using subtraction

The instrument comprises the following steps: balance, centrifuge, pipettor, vibration mixer.

Reagent: 10 × CRC wash (Tanzhou Saite organisms), Cytelligen cell separation (Tanzhou Saite organisms), and immunomagnetic bead binding buffer (from IP kit, Shanghai Changchini organisms).

The method comprises the following steps:

(1) collecting peripheral blood of a patient: to avoid epithelial cell contamination, 6.0mL of blood was collected using the blood collection tube provided after discarding the first 2mL of blood, and the tube was immediately mixed 5-10 times end-to-end.

(2) Balancing the blood collecting tube with a balance, reversing the head and the tail, uniformly mixing, centrifuging at room temperature, and rotating at 2000rpm for 20 min. Discarding the supernatant to 5mm above the red-brown precipitate, adding 10 × CRC cleaning solution until the yellow line of the blood collection tube label is full of blood, covering with a yellow tube cap, and reversing the head and the tail to mix evenly for 10 times.

(3) Adding 3mL of Cytelligen cell separation solution into the centrifuge tube A, turning the head and the tail of the blood collection tube gently and evenly, slowly adding the blood cells in the blood collection tube to the top layer of the separation solution along the liquid level of the tube wall of the centrifuge tube A by using a manual pipettor, and avoiding adding the blood cells into the separation medium. After the superposition, a clear interface can be seen, and a small amount of red blood cells slowly precipitate to the bottom of the tube. After trimming, centrifuge at room temperature, 450rpm for 7 min.

(4) After centrifugation, three layers of liquid can be seen, the upper Layer is yellow liquid (Layer 1), the middle Layer is transparent liquid (Layer 2), and the bottom Layer is brownish red precipitated red blood cells (Layer 3). However, some erythrocytes were present in the middle layer of the individual patients due to treatment, etc., and were not centrifuged to the bottom of the tube, and the supernatant was aspirated as follows. All the liquid on the bottom layer red blood cells in centrifuge tube A was transferred to a 50mL centrifuge tube B, and the liquid was first aspirated from the liquid surface boundary between the upper layer and the middle layer using a 1mL sample applicator, and the supernatant was aspirated from the liquid surface when 2mL remained, and the bottom layer red blood cells were never aspirated. The liquid was then shaken up vertically.

(5) And (3) fully and uniformly mixing the washed immunomagnetic beads with the buffer solution, slowly adding the magnetic beads into the centrifuge tube B according to the proportion of 400 mu L per tube, and shaking the centrifuge tube while adding. The centrifuge tubes were fixed on a shaker at an angle of 35-40 ℃ and shaken at room temperature for 20min at 125rpm (the maximum of the liquid level shaking was between 30-35mL of the scale).

(6) Placing centrifuge tube B on magnetic frame, stretching 5mL gun head into the center of tube bottom to gently blow 2min later, transferring liquid to 50mL centrifuge tube C along the center of centrifuge tube B carefully after 2min, and taking care not to absorb adsorbed magnetic beads.

(7) Add 10 × CRC wash to 45mL to centrifuge tube C, trim, reverse mix, centrifuge at 700rpm for 4min at room temperature. Discard the supernatant to 500. mu.L, mix the precipitated cells by gentle shaking using a shaking mixer, transfer to 15mL centrifuge tube D (without discarding the tip), wash centrifuge tube C with 1mL 10 × CRC wash, transfer the wash to 15mL centrifuge tube D with the same non-discarded tip, add 10 × CRC wash to 14mL centrifuge tube D, balance, reverse mix, centrifuge at 950rpm for 4min at room temperature. Obtaining the enriched human peripheral blood Circulating Tumor Cells (CTC).

Example 2: establishment of c-MET gene copy number variation detection method

This example optimizes and confirms the detection system of c-MET gene and reference gene.

Primers and probes: purchased from Shanghai Producers.

The base sequence of the taqman probe corresponding to the c-MET gene is shown as SEQ ID NO: 1, and the following components:

attagcctct gtctcggtgg caggttcc；

specific upstream and downstream primers for detecting c-MET gene subtype in human peripheral blood CTC have base sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3, showing:

an upstream primer: tcattggttc caatcacagc t, respectively;

a downstream primer: gccaccgaga cagaggctaa t are provided.

The base sequence of the taqman probe corresponding to the internal reference gene is shown as SEQ ID NO: 4, and (2) is as follows:

agcctcccct cctcatgcct tcttgcct；

the base sequences of the specific upstream and downstream primers of the internal reference gene subtype are shown as SEQ ID NO: 5 and SEQ ID NO: 6, showing:

an upstream primer: ggctgctcac atattctg, respectively;

a downstream primer: cattgatggc aacaatatcc are provided.

The c-MET gene is used as a positive standard substance, and the base sequence of the c-MET gene is shown as SEQ ID NO: 7 is shown in the specification; GAPDH is used as an internal reference standard, and the base sequence of the GAPDH is shown as SEQ ID NO: shown in fig. 8.

The initial concentrations of the positive standard and the internal reference standard are both 6 × 10⁶copies/. mu.L, diluted in 10-fold gradient with water (no DNA water) as a blank, high fidelity enzyme from TaKaRa, and direct amplification Buffer: 20mM Tris-HCl (pH 8.8, 25 ℃), 10mM KCl, 10mM (NH)₄)₂SO₄，2mM MgSO₄0.1% Triton X-100, 100. mu.g/ml Bovine Serum Albumin (BSA), 1.0mol/L betaine; the quantitative curve is determined and the standard curve is drawn by adopting a fluorescent quantitative PCR method (the fluorescent quantitative PCR reaction system is shown in table 1) and utilizing a Roche fluorescent quantitative PCR instrument for detection and analysis.

TABLE 1 fluorescent quantitative PCR reaction System

Reagent	Volume (μ L)
		2×Buffer	12.5
dNTP	0.5
		Upstream primer	0.1
Downstream primer	0.1
		Probe Probe	0.2
UNG	0.1
		High fidelity enzyme	0.5
H2O	3.1
		Standard article	2
Total up to	20

Reaction conditions are as follows: the program is set as one-step Real-Time PCR: pre-denaturation at 95 deg.C for 1 min; then, denaturation is carried out at 95 ℃ for 5s in each step; the annealing was extended at 60 ℃ for 20s for 40 cycles, and the absorbance was read at each time during the extension phase. As the PCR system measures the absorbance after the end of each cycle, a quantitative curve (see FIG. 2, FIG. 3) with the cycle number as the abscissa and the absorbance as the ordinate and a standard curve with the logarithm of the standard concentration as the abscissa and the Ct value as the ordinate are obtained. The Ct value refers to the number of cycles that the fluorescence signal in each reaction system has undergone to reach a set threshold. The Ct value of each template has a linear relation with the logarithm of the initial copy number of the template, and the more the initial copy number is, the smaller the Ct value is; the lower the initial copy number, the greater the Ct value.

The detection results of the c-MET standard are shown in Table 2, and the detection results of the internal reference standard are shown in Table 3.

TABLE 2 detection results of c-MET standards

TABLE 3 test results of reference standard

Example 3: detection of c-MET Gene copy number variation in CTC cells

The composition of a detection kit for detecting copy number variation of the c-MET gene by 3D PCR.

The c-MET gene copy number variation detection kit comprises 1 × 3D Digital PCR Master Mixv2, upstream and downstream primers 0.1. mu. mol/L respectively, and probes 0.2. mu. mol/L respectively. The kit components are shown in table 4.

Primers and probes: purchased from Shanghai Producers.

The base sequence of the taqman probe corresponding to the c-MET gene is shown as SEQ ID NO: 1 is shown.

Specific upstream and downstream primers for detecting c-MET gene subtype in human peripheral blood CTC have base sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

The base sequence of the taqman probe corresponding to the internal reference gene is shown as SEQ ID NO: 4, respectively.

The base sequences of the specific upstream and downstream primers of the internal reference gene subtype are shown as SEQ ID NO: 5 and SEQ ID NO: and 6.

TABLE 43D PCR detection of c-MET gene copy number variation detection kit components

The instrument comprises the following steps: QuantStaudio 3D Digital PCR ChipLoader instrument, flat plate PCR instrument.

The total volume of the 3D-dPCR system is 15 mu L, and the system comprises the following components: 1X 3D Digital PCR Master Mixv2, specific exogenous sequence upstream and downstream primers and probes, 5. mu.L of CTC sample.

The configured system is automatically loaded into each micropore on the Chip through a QuantStaudio (TM) 3D Digital PCR (polymerase chain reaction) Chip loader instrument, and 3D Digital PCR Chip immersion liquid is used for covering the surface of the Chip and sealing the Chip after the system is loaded.

Placing the chip on a flat PCR instrument for amplification, wherein the reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 40s for a total of 40 cycles.

And finally, scanning and detecting on a 3D PCR instrument, and analyzing the result. As shown in FIG. 4, the dotted region a is a c-Met gene positive reaction well, the dotted region b is a c-Met gene and GAPDH gene double positive reaction well, and the dotted region c is a GAPDH gene positive reaction well. And (3) calculating the ratio of the number of positive reaction holes of the c-MET gene to the number of positive reaction holes of the internal reference gene GAPDH, and when the ratio is more than 1.2, indicating that the copy number of the c-MET gene is increased.

The gene amplification is a gene mutation form of c-Met, the c-Met mutation may cause EGFR-TKI drug resistance, and the detection of the c-Met mutation of a tumor patient is favorable for assisting in guiding the administration of a patient with non-small cell lung cancer, so that the disease-free survival period of the patient is prolonged.

As noted above, while the present invention has been shown and described with reference to certain preferred embodiments, it is not to be construed as limited thereto. Various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> Jiangsu province national hospital (the first subsidiary hospital of Nanjing medical university)

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aaaggttcat tggttccaat cacagctcat aggtagagca aagaaagggt ggatggattg 180

aaaagattag cctctgtctc ggtggcaggt tcccacctcg caagcaattg gaaacaaaac 240

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ctacatggtg agccccaaag ctggtgtggg aggagccacc tggctgatgg gcagcccctt 300

Claims (1)

1. A kit for detecting copy number variation of the c-MET gene from human peripheral blood CTCs, the kit comprising:

3D Digital PCR Master Mixv2, a specific upstream primer of c-MET gene subtype, a specific downstream primer of c-MET gene subtype, a specific taqman probe for detecting copy number variation of c-MET gene, a specific upstream primer of an internal reference gene subtype, a specific downstream primer of the internal reference gene subtype and a specific taqman probe corresponding to the internal reference gene;

wherein, the base sequence of the specific taqman probe for detecting the copy number variation of the c-MET gene is shown as SEQ ID NO: 1, and the following components:

attagcctct gtctcggtgg caggttcc；

the specific upstream and downstream primers of the c-MET gene subtype have base sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3, showing:

an upstream primer: tcattggttc caatcacagc t, respectively;

a downstream primer: gccaccgaga cagaggctaa t, respectively;

the base sequence of the specific taqman probe corresponding to the internal reference gene is shown as SEQ ID NO: 4, and (2) is as follows:

agcctcccct cctcatgcct tcttgcct；

the base sequences of the specific upstream and downstream primers of the internal reference gene subtype are shown as SEQ ID NO: 5 and SEQ ID NO: 6, showing:

an upstream primer: ggctgctcac atattctg, respectively;

a downstream primer: cattgatggc aacaatatcc, respectively;

the 5 'end of the specific taqman probe for detecting copy number variation of the c-MET gene is marked with FAM fluorescent group, and the 3' end is marked with TAMRA fluorescent group.

Images