CN106065414A - Noninvasive cancer of pancreas polygenes detection method and kit based on blood plasma cfDNA detection technique - Google Patents

Abstract

The invention belongs to the field of medical biology, and relates to a non-invasive pancreatic cancer multi-gene detection method and a kit based on plasma cfDNA detection technology. The invention discloses a non-invasive pancreatic cancer multi-gene detection method and a kit based on plasma cfDNA detection technology, including methods for collecting blood, separating plasma, and purifying cfDNA; designing a panel for a hot spot mutation region characteristic of pancreatic cancer; PCR primer amplification, and library construction , a pipeline method for high-throughput next-generation sequencing and data analysis. The present invention can detect the dynamic changes of the characteristic mutation type and mutation frequency of pancreatic cancer in plasma cfDNA according to the progress of treatment and the development of the course of the disease for each individual, so as to provide information for the monitoring of the course of pancreatic cancer and the emergence of drug resistance.

Description

Non-invasive multi-gene detection method and test for pancreatic cancer based on plasma cfDNA detection technology Kit

technical field

The invention belongs to the field of medical biology, and relates to a non-invasive pancreatic cancer multi-gene detection method and a kit based on plasma cfDNA detection technology.

Background technique

Pancreatic cancer is one of the common malignant tumors of the digestive system. It usually refers to pancreatic tubular adenocarcinoma. Its characteristics of insidious onset, difficulty in early diagnosis, rapid progression, and easy recurrence and metastasis lead to poor clinical treatment effect, and it is currently the malignant tumor with the worst prognosis. One, the median survival period is about 6 months, and the overall 5-year survival rate is 3 to 5%, which seriously threatens human health and is called "the king of cancer".

Studies have shown that human cancer is caused by the accumulation of mutations in oncogenes and tumor suppressor genes. The vast majority of gene mutations are non-functional or non-pathogenic mutations, called "Passenger Mutations"; a small number of gene mutations have carcinogenic functions, The mutation common to most tumors is called "Driver Mutation". This type of mutation is the main cause of tumorigenesis, and it is also an important molecular marker for early diagnosis and early warning.

With the continuous development and advancement of genomic technology, some high-frequency mutations have been found in pancreatic cancer genome research. For example, the somatic mutation rate of KRAS in ductal adenocarcinoma is greater than 90%, which is the oncogene with the highest mutation rate in pancreatic cancer. KRAS mutations are mainly concentrated at specific codons (most commonly found at codon 12), which have been shown to be critical for the development of pancreatic cancer.

Circulating DNA, also known as cell-free DNA (cfDNA), is a kind of extracellular DNA in a cell-free state that exists in animal, plant and human body fluids. At present, studies have shown that cell-free DNA in plasma of tumor patients has molecular genetic changes consistent with DNA in tumor tissue (such as gene mutation, hypermethylation of tumor suppressor gene promoters, microsatellite instability, and loss of heterozygosity, etc.). In the early diagnosis and early warning monitoring of pancreatic cancer, collecting peripheral blood is more convenient, non-invasive and widely applicable than other samples such as pancreatic juice and tumor. Therefore, detection of tumor cfDNA mutations in plasma has become one of the important directions for early diagnosis and early warning research of pancreatic cancer.

With the continuous improvement of trace DNA detection technology and the rapid development of next-generation sequencing technology, more convenient, economical and efficient capture sequencing technology has been developed. Low yield) was subverted, and the goal of "one-time sampling, all screening" was realized. The advantage of gene capture sequencing technology is that it takes full advantage of the advantages of modern second-generation high-throughput sequencing to capture and detect tens to thousands of genes and millions of sites at one time. gene fragments were detected. Furthermore, the technique is highly sensitive. For example, there are hundreds of copies of mitochondrial DNA in each human cell, and the detection limit of traditional Sanger sequencing technology is 10-15%, so many low-abundance mitochondrial heterogeneities cannot be detected by traditional techniques. The heterogeneity of >2% of all sites in mitochondrial DNA can be accurately detected by capture sequencing technology.

In summary, the present invention provides a method for multigene detection of pancreatic cancer plasma cfDNA based on trace DNA detection technology and next-generation sequencing technology, and according to the self-designed pancreatic cancer panel (which basically covers the high-frequency mutations of pancreatic cancer site) developed into a kit. Finally, according to the detection of the dynamic changes of pancreatic cancer characteristic mutation type and mutation frequency in plasma cfDNA, it can be used to provide information for the monitoring of pancreatic cancer disease course and the emergence of drug resistance.

Contents of the invention

The invention provides a non-invasive multi-gene detection method for pancreatic cancer based on plasma cfDNA detection technology.

A non-invasive pancreatic cancer multigene detection method based on plasma cfDNA detection technology, comprising the following steps:

(1) Separation of plasma;

(2) purify cfDNA;

(3) Mix 10 ng of DNA from step (2) with the pancreatic cancer characteristic hotspot mutation panel, and perform a single-tube multiplex PCR reaction to amplify the target gene; the pancreatic cancer characteristic hotspot mutation panel includes specific primers for 37 genes shown in Table 1.

(4) library construction;

(5) High-throughput sequencing;

(6) Data analysis.

in,

In step (1): add whole blood into the EDTA tube containing anticoagulant; transfer the EDTA tube to a refrigerated centrifuge at 4°C within 2 hours and centrifuge at 1900g (3000rpm) for 10min, the lower layer is blood cells, and the upper layer is plasma, transfer them separately Transfer to EP tubes, store at -80°C, and save.

Step (2) cfDNA purification adopts Gnomegen's purification kit (Circulating DNA Purification Kit), and applies magnetic bead purification technology to enrich DNA fragments below 300 bp from plasma.

Step (4) The library was constructed using Gnomegen’s library preparation kit (DNA Seq NGS Library Preparation Kit For Amplicon Sequencing).

Step (5) Ion torrent sequencing platform is used for sequencing, Proton, the read length is 150bp, and the sequencing depth is 10000x.

Step (6) The bioinformatics analysis process for detecting low-frequency mutations in plasma cell-free DNA is as follows

1. Raw data quality inspection

2. filter

a) remove the adapter sequence in read;

b) remove the primer sequence in the read;

c) remove reads containing N bases greater than 10% of the entire reads;

d) Remove reads with a read length <50bp;

e) Remove low-quality reads: the number of bases with Q>20 is less than 70% of the total length of the reads;

3. Compare

Use the BWA tool to compare the clean reads back to the reference genome. BWA compresses the genome sequence through Burrows Wheeler transformation and builds an index, and then locates the reads through search and backtracking. When searching, base substitution can be used to achieve allowable mismatches;

4. Somatic Mutation Detection

Filter out variant sites detected in both plasma cfDNA samples and blood cell samples with similar frequencies

When the frequency is <10%, the frequency difference is <2%

When the frequency is >10%, the frequency difference is <10%.

In addition, the present invention also provides a primer combination (combination of upstream primers and/or combination of downstream primers as shown in Table 1) for detecting pancreatic cancer polygenes in plasma cfDNA and the primer combination in the preparation of non-invasive pancreatic cancer polygenes. Application in genetic testing reagents or kits.

In addition, a detection reagent and a detection box for detecting pancreatic cancer polygenes in plasma cfDNA are also provided.

A detection reagent for detecting multiple genes of pancreatic cancer in plasma cfDNA, comprising a primer combination (combination of upstream primers and/or combination of downstream primers shown in Table 1) for detecting multiple genes of pancreatic cancer in plasma cfDNA.

A detection kit for detecting multiple genes of pancreatic cancer in plasma cfDNA, comprising a primer combination (combination of upstream primers and/or combination of downstream primers shown in Table 1) for detecting multiple genes of pancreatic cancer in plasma cfDNA.

The present invention will be further described below, the steps of the non-invasive pancreatic cancer multi-gene detection method based on plasma cfDNA detection technology:

(1) Collect blood and separate plasma

Add whole blood to EDTA tubes containing anticoagulant. Transfer the EDTA tube to a refrigerated centrifuge at 4°C within 2 hours and centrifuge at 1900g (3000rpm) for 10min. The lower layer is blood cells and the upper layer is plasma, which are transferred to EP tubes and stored at -80°C for preservation.

(2) Purification of cfDNA

The cfDNA purification adopts the Circulating DNA Purification Kit of Gnomegen Company, and the magnetic bead purification technology is used to concentrate and enrich the DNA fragments below 300bp from the plasma, which can more effectively remove the contamination of large fragments of genomic DNA, especially suitable for free DNA from the plasma of tumor patients Research on the detection of tumor-associated gene mutation information. The specific process was carried out according to the kit instructions.

(3) Pancreatic cancer characteristic hotspot mutation panel, PCR primer amplification

The panel covers the entire exon regions of 5 genes and hotspot mutation regions of 32 genes (see Table 1). And each sample (about 10ng DNA) is mixed with self-customized primers, and a single-tube multiplex PCR reaction is performed to amplify the target gene.

(4) Library construction

DNA Seq NGS Library Preparation Kit For AmpliconSequencing from Gnomegen was used. The specific process was carried out according to the kit instructions.

(5) High-throughput sequencing

The Ion torrent sequencing platform was used for sequencing, Proton, with a read length of 150bp and a sequencing depth of 10000x.

(6) Data analysis

According to Figure 1, the bioinformatics analysis process for detecting low-frequency mutations in plasma cell-free DNA is used to ensure the accuracy and sensitivity of detection.

The invention discloses a non-invasive pancreatic cancer multi-gene detection method based on plasma cfDNA detection technology, a reagent and a kit, including methods for collecting blood, separating plasma, and purifying cfDNA; designing a panel for a hot spot mutation region characteristic of pancreatic cancer; PCR primer amplification, Workflow methods for library construction, high-throughput next-generation sequencing, and data analysis. The present invention can detect the dynamic changes of the characteristic mutation type and mutation frequency of pancreatic cancer in plasma cfDNA according to the blood of each individual along with the progress of treatment and the development of the disease, so as to provide information for the monitoring of the course of pancreatic cancer and the emergence of drug resistance.

The invention discloses a non-invasive pancreatic cancer multi-gene detection method and a kit based on plasma cfDNA detection technology, including methods for collecting blood, separating plasma, and purifying cfDNA; designing a panel for a hot spot mutation region characteristic of pancreatic cancer; PCR primer amplification, and library construction , a pipeline method for high-throughput next-generation sequencing and data analysis. The present invention can detect the dynamic changes of the characteristic mutation type and mutation frequency of pancreatic cancer in plasma cfDNA according to the progress of treatment and the development of disease course for each individual, so as to provide information for monitoring the course of pancreatic cancer and the emergence of drug resistance.

Description of drawings

Fig. 1 is the distribution map of mutations in patients No. 1-3 of the present invention.

detailed description

The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the protection scope of the present invention.

The materials and reagents required in the examples of the present invention can be purchased in the market unless otherwise specified.

Example 1

The whole blood (provided by the First Affiliated Hospital of Zhejiang University) from 3 patients with pancreatic cancer from No. 1 to No. 3 was subjected to multi-gene detection test of cfDNA in plasma of pancreatic cancer and multi-gene detection of DNA in tumor tissue, and the consistency was compared, and the judgment was made. Whether the mutations detected in plasma cfDNA can be used as individual monitoring markers for patients.

1. Take materials, separate

1.1 5ml of whole blood from 3 patients with pancreatic cancer No. 1-3 were added into the EDTA tube containing anticoagulant. Transfer the EDTA tube to a refrigerated centrifuge at 4°C within 2 hours and centrifuge at 1900g (3000rpm) for 10min. The lower layer is blood cells and the upper layer is plasma, which are transferred to EP tubes and stored at -80°C for preservation.

1.2 Take 30 mg of each tumor surgical tissue, put them directly into EP tubes and store them immediately at -80°C or in liquid nitrogen.

2 DNA purification and quantification

2.1 The Gnomegen Circulating DNA Purification Kit was used for the purification of plasma cfDNA, and the Qiamp DNA Blood Mini Kit was used for the purification of blood cell genomic DNA. The specific process was carried out according to the kit standard instructions. The purified plasma free DNA was quantified by Qubit to detect the concentration of free DNA. The purified blood cell genomic DNA was quantified by Qubit to detect the DNA concentration.

2.2 Qiagen DNA Mini Kit was used for the purification of tumor tissue genomic DNA, and the specific process was carried out in accordance with the standard instructions of the kit. The purified tumor tissue genomic DNA was quantified by Qubit to detect the DNA concentration.

3. Target gene PCR amplification

The pancreatic cancer multi-gene detection kit contains specific primers for 37 genes of the pancreatic cancer characteristic hotspot mutation panel, which cover the entire exon region of 5 genes and 32 gene hotspot mutation regions (see Table 1). And each sample (about 10ng DNA) is mixed with self-customized primers, and a single-tube multiplex PCR reaction is performed to amplify the target gene.

Table 1:

4. Library construction and sequencing

The library was constructed using DNA Seq NGS Library Preparation Kit For Amplicon Sequencing from Gnomegen. The Ion torrent sequencing platform was used for sequencing, Proton, with a read length of 150bp and a sequencing depth of 10000x.

5. Bioinformatics analysis process

5.1 Raw data quality inspection

5.2 Filtration

f) remove the adapter sequence in the read;

g) remove the primer sequence in the read;

h) remove reads containing N bases greater than 10% of the entire reads;

i) Remove reads with a read length <50bp;

j) Remove low-quality reads (the number of bases with Q>20 is less than 70% of the total length of the reads).

5.3 Comparison

Clean reads were aligned back to the reference genome using the BWA tool. BWA compresses and indexes the genome sequence through the Burrows Wheeler transformation, and then locates the reads through search and backtracking. During the search, base substitution can be used to achieve allowable mismatches.

5.4 Somatic mutation detection

When comparing somatic mutations detected in tumor tissue samples with those detected in plasma cfDNA samples, the following filtering was done:

a). Filter out mutation sites that are detected in both tumor tissue and blood cell samples and have similar frequencies.

When the frequency is <10%, the frequency difference is <2%.

When the frequency is >10%, the frequency difference is <10%.

2. Filter out the variant sites detected in both plasma cfDNA samples and blood cell samples with similar frequencies.

When the frequency is <10%, the frequency difference is <2%.

When the frequency is >10%, the frequency difference is <10%.

6. Data Interpretation and Results

Somatic mutation detection report

Conclusion: It is found that the detection effect of plasma cfDNA is consistent with that of tumor tissue, and has the effect of replacing biopsy tissue.

The mutation distribution map is shown in Figure 1.

Clinical significance (mutation frequency greater than 5% in cfDNA):

Patient 1's individual high-frequency mutations are FBXW7-chr4:153249329A>G and MAP2K4-chr17:12044664T>G, which have the value of prognostic monitoring.

The individual high-frequency mutations of patient 2 are BRCA2-chr13:32911371A>G and ARID1A-chr1:27059176C>T, which have the value of prognostic monitoring.

The individualized high-frequency mutation of patient 3 is KMT2C-chr7:151879447T>A, which has the value of prognostic monitoring.

Research value: It is found that the chr19:1226599T>C mutation site on the STK11 gene has a change affecting its protein S419P, and it is shared by three patients, which has research value.

Claims (10)

1. A noninvasive pancreatic cancer polygene detection method based on a plasma cfDNA detection technology is characterized by comprising the following steps:

(1) separating the plasma;

(2) purifying the cfDNA;

(3) mixing the DNA10ng obtained in the step (2) with the pancreatic cancer characteristic hot spot mutation panel, and carrying out single-tube multiplex PCR reaction to amplify a target gene; pancreatic cancer-characteristic hotspot mutation panel included primers specific for the 37 genes shown in table 1.

Table 1:

(4) constructing a library;

(5) high-throughput sequencing;

(6) and (6) analyzing the data.

2. The noninvasive pancreatic cancer polygene detection method based on the plasma cfDNA detection technology as claimed in claim 1, wherein in step (1): adding whole blood into an EDTA tube containing an anticoagulant; transferring EDTA tube into 4 deg.C refrigerated centrifuge for 10min at 1900g (3000rpm) for 2h, transferring the lower layer to blood cell and the upper layer to blood plasma, respectively transferring to EP tube, storing at-80 deg.C, and storing.

3. The noninvasive pancreatic cancer polygene detection method based on plasma cfDNA detection technology as claimed in claim 1, wherein in step (2), the cfDNA is purified by using a purification Kit (Circulating DNAPurification Kit) from Gnomegen company, and the DNA fragments with the size of less than 300bp are concentrated and enriched from plasma by using magnetic bead purification technology.

4. The noninvasive pancreatic cancer polygene detection method based on the plasma cfDNA detection technology of claim 1, wherein the library construction in the step (4) adopts a library construction Kit (DNA Seq NGS library preparation Kit For amplification Sequencing) of Gnomegen company.

5. The noninvasive pancreatic cancer polygene detection method based on the plasma cfDNA detection technology of claim 1, wherein the sequencing in step (5) is performed by using an Ion torrent sequencing platform, Proton, read length is 150bp, and 10000X sequencing depth.

6. The noninvasive pancreatic cancer polygene detection method based on plasma cfDNA detection technology as claimed in claim 1, wherein the bioinformatics analysis procedure of step (6) for detecting low frequency mutation in plasma free DNA is as follows

1. Raw data quality inspection

2. Filtration

a) Removing an adapter sequence in the read;

b) removing primer sequences in the read;

c) removing reads containing N bases which are more than 10 percent of the whole reads;

d) removing reads with the read length less than 50 bp;

e) removing low-quality reads: the number of bases with Q >20 is less than 70% of the total length of reads;

3. comparison of

Comparing clean reads back to a reference genome by using a BWA tool, compressing and indexing the genome sequence by using BWA through Burrows Wheeler conversion, positioning reads through searching and backtracking, and realizing allowable mismatch through base substitution during searching;

4. somatic mutation detection

Filtering out variant sites with similar frequency detected in both plasma cfDNA samples and blood cell samples

When the frequency is less than 10%, the frequency difference is less than 2%

When the frequency is > 10%, the frequency difference is < 10%.

7. A primer combination for detecting multiple genes of pancreatic cancer in plasma cfDNA, characterized by an upstream primer and a downstream primer as shown in table 1.

8. The application of the primer combination of claim 7 in the preparation of noninvasive pancreatic cancer polygene detection reagents or kits.

9. A detection reagent for detecting multiple genes in pancreatic cancer in plasma cfDNA, comprising the primer combination of claim 7.

10. A detection kit for detecting multiple genes of pancreatic cancer in plasma cfDNA, comprising the primer combination of claim 7.