阅读说明：本技术 用于检测尿液外泌体cnv的试剂在制备泌尿系统肿瘤检测试剂盒中的应用 (Application of reagent for detecting urine exosome CNV in preparation of urinary system tumor detection kit ) 是由 王晓楠 钱祺 缪家顺 张亚楠 于 2021-07-19 设计创作，主要内容包括：本发明公开了用于检测尿液外泌体CNV的试剂在制备泌尿系统肿瘤检测试剂盒中的应用。所述检测尿液外泌体CNV的试剂包括尿液外泌体提取试剂、外泌体DNA提取试剂和外泌体DNA建库测序试剂,进一步通过软件对外泌体DNA的测序数据进行分析处理,获得尿液外泌体CNV数据。本发明通过实验发现：尿液外泌体CNV的检测可以用来区分膀胱癌患者与膀胱良性病变患者,并且相对于尿脱落细胞CNV检测,尿液外泌体CNV检测方法对于泌尿系统肿瘤的诊断具有更高的准确性。(The invention discloses an application of a reagent for detecting urine exosome CNV in preparation of a urinary system tumor detection kit. The reagent for detecting the urine exosome CNV comprises a urine exosome extraction reagent, an exosome DNA extraction reagent and an exosome DNA library-building sequencing reagent, and further analyzes and processes sequencing data of exosome DNA through software to obtain urine exosome CNV data. The invention discovers that: the detection of the urine exosome CNV can be used for distinguishing bladder cancer patients from bladder benign lesion patients, and compared with the detection of the urine exfoliated cell CNV, the urine exosome CNV detection method has higher accuracy for the diagnosis of urinary system tumors.)

1. Application of a reagent for detecting urine exosome CNV in preparation of a urinary system tumor detection kit.

2. The use of claim 1, wherein: the kit judges whether the tumor is the urinary system tumor or not by detecting the abnormality of the urine exosome CNV.

3. Use according to claim 2, characterized in that: the urinary system tumor comprises prostatic cancer, bladder cancer, renal cancer, urothelial cancer and testicular cancer.

4. The use of claim 1, wherein: the reagent for detecting the urine exosome CNV comprises a urine exosome extraction reagent, an exosome DNA extraction reagent and an exosome DNA library construction sequencing reagent.

5. The use of claim 4, wherein: the exosome extraction reagent is selected from a reagent used for extracting exosomes by an ultra-high speed centrifugation method, a reagent used for extracting exosomes by an ultrafiltration method, a reagent used for extracting exosomes by a PEG precipitation method, a reagent used for extracting exosomes by a magnetic bead method, or a commercially available urine exosome preparation kit.

6. The use of claim 4, wherein: the exosome DNA extraction reagent is selected from a reagent used for extracting exosome DNA by an alkaline lysis method, a reagent used for extracting exosome DNA by a phenol extraction method, a reagent used for extracting exosome DNA by a silica gel membrane centrifugal column method, a reagent used for extracting exosome DNA by a magnetic bead method, or a commercially available exosome DNA extraction kit.

7. The use of claim 4, wherein: the exosome DNA Library-establishing sequencing reagent is selected from Truseq Nano DNA Sample Prep Kit of Illumina, NEBNext Ultra II DNA Library Prep Kit of NEB for Illumina, Kapa Hyper Prep Kit of Kapa, and NovonzaUniversal Pro DNA Library Prep Kit for Illumina。

8. The use of claim 4, wherein: the method for detecting urine exosomes CNV includes the following steps,

1) using a fastp software to truncate and remove a low-quality sequence and a joint sequence from original sequencing data of a fastq-format sequence, and evaluating the sequencing quality, wherein a key parameter is-q 25;

2) comparing the filtered sequencing data to a reference genome hg19 by using bwa software, sequencing the BAM comparison files according to the comparison positions by using SAMtools, further removing repeated sequences from the BAM files, and reducing the interference of optical repeat and PCR (polymerase chain reaction) repeated sequences;

3) using R packet QDNAseq to calculate and identify log2 ratio in a fixed bin interval of the BAM file obtained in the step 2, wherein the size of the bin interval is respectively selected from 500kb and 1 Mb; the log2 ratio of bin segments was obtained and then segmentation was performed using the log2-transformation method to identify CNV.

Technical Field

The invention belongs to the technical field of biomedical diagnosis, and particularly relates to an application of a reagent for detecting urine exosome CNV in preparation of a urinary system tumor detection kit.

Background

Cancer is a well-known genomic disease, and the genomic aberrations of interest in cancer are mostly somatic aberrations, since tumors arise from normal cells with acquired aberrations in their genome. Copy Number Variation (CNV) is one of the most important somatic aberrations in cancer, because activation of oncogenes is often attributed to chromosomal copy number amplification, while inactivation of oncogenes is often caused by mutation-related heterozygous or homozygous deletions, including proliferation and deletion 1 of specific DNA fragments. A number of studies have now found a link between CNV and cancer.

Exosomes, also known as exosomes, 30-150 nm in diameter, are vesicles encapsulated by lipid bilayer membranes released into the extracellular environment after fusion of intracellular multivesicular bodies (MVBs) with the cell membrane, contain proteins, lipids and nucleic acids, and serve as important mediators of cell-cell communication in various physiological and pathological pathways. Current research shows that: exosomes have high abundance in body fluid (blood, saliva, urine, ascites, amniotic fluid, spinal fluid and the like), and have wide clinical application prospect when being used for detecting the contents.

The copy number variation of exosomes is used for detecting CNV of nucleic acids in exosomes, and the CNV is used as a new detection means and has an important effect on the auxiliary diagnosis of tumors.

Although there are many methods for detecting tumor cells or exosomes, for example, CNV is detected using urine-shed cells, and proteins and RNA are detected in exosomes. However, no report is available in the prior art about the differentiation of tumor cells from healthy cells by detecting the copy number variation of exosomes.

Disclosure of Invention

The invention aims to provide application of a reagent for detecting urine exosome CNV in preparation of a urinary system tumor detection kit, and provides a novel method for detecting urinary system tumors.

The technical scheme adopted by the invention for realizing the purpose is as follows:

application of a reagent for detecting urine exosome CNV in preparation of a urinary system tumor detection kit. The kit judges whether the tumor is the urinary system tumor or not by detecting the abnormality of the urine exosome CNV.

As a preferred embodiment, the urological tumours comprise prostate cancer, bladder cancer, kidney cancer, urothelial cancer, testicular cancer.

In a preferred embodiment, the reagent for detecting urine exosome CNV comprises a urine exosome extraction reagent, an exosome DNA extraction reagent and an exosome DNA library-building sequencing reagent.

In a preferred embodiment, the exosome extraction reagent is selected from a reagent used for extracting exosomes by an ultra-high-speed centrifugation method, a reagent used for extracting exosomes by an ultrafiltration method, a reagent used for extracting exosomes by a PEG precipitation method, a reagent used for extracting exosomes by a magnetic bead method, or a commercially available urine exosome preparation kit.

In a preferred embodiment, the exosome DNA extraction reagent is selected from a reagent used for extracting exosome DNA by an alkaline lysis method, a reagent used for extracting exosome DNA by a phenol extraction method, a reagent used for extracting exosome DNA by a silica gel membrane centrifugal column method, a reagent used for extracting exosome DNA by a magnetic bead method, or a commercially available exosome DNA extraction kit.

As a preferred embodiment, the exosome DNA Library-building sequencing reagent is selected from Truseq Nano DNA Sample Prep Kit of Illumina, NEBNext Ultra II DNA Library Prep Kit of NEB for Illumina, Kapa HyperPrep Kit of Kapa, NovonzaUniversal Pro DNA Library Prep Kit for Illumina。

As a preferred embodiment, the method for detecting urinary exosomes CNV comprises the steps of:

1) using a fastp software to truncate and remove a low-quality sequence and a joint sequence from original sequencing data of a fastq-format sequence, and evaluating the sequencing quality, wherein a key parameter is-q 25;

2) comparing the filtered sequencing data to a reference genome hg19 by using bwa software, sequencing the BAM comparison files according to the comparison positions by using SAMtools, further removing repeated sequences from the BAM files, and reducing the interference of optical repeat and PCR (polymerase chain reaction) repeated sequences;

3) using R packet QDNAseq to calculate and identify log2 ratio in a fixed bin interval of the BAM file obtained in the step 2, wherein the size of the bin interval is respectively selected from 500kb and 1 Mb; the log2 ratio of bin segments was obtained and then segmentation was performed using the log2-transformation method to identify CNV.

Compared with the prior art, the invention has the following beneficial effects:

the invention establishes a new method for detecting the tumor of the urinary system, and finds out through experiments that: the detection of the urine exosome CNV can be used for distinguishing bladder cancer patients from bladder benign lesion patients, and compared with the detection of urine exfoliated cell CNV, the urine exosome CNV detection method has higher accuracy for the diagnosis of urinary system tumors.

Drawings

FIG. 1 shows the CNV detection results of the DNA of the tumor cell line 22RV1 in example 1 of the present invention.

FIG. 2 shows the CNV detection result of the exosome DNA of the tumor cell line 22RV1 cell culture supernatant in example 1 of the present invention.

FIG. 3 shows the CNV detection results of the DNA of the tumor cell line H3122 in example 1 of the present invention.

FIG. 4 shows the CNV detection result of the exosome DNA of the tumor cell line H3122 cell culture supernatant in example 1 of the present invention.

FIG. 5 shows the CNV detection result of the exosome DNA of the urine sample of the healthy volunteer in example 2 of the present invention.

FIG. 6 shows the CNV test results of urine exosomes of bladder cancer patient in example 3 of the present invention.

FIG. 7 shows the CNV test results of urine exosomes of the benign bladder lesion patient in example 3 of the present invention.

FIG. 8 shows the CNV measurement of exfoliated urine cells of a patient with bladder cancer in example 4 of the present invention.

Detailed Description

The technical solution of the present invention will be described in detail with reference to examples. The reagents and biomaterials used below were all commercial products unless otherwise specified.

Example 1: tumor cell DNA CNV detection and tumor cell exosome DNA CNV detection

Tumor cell and cell DNA extraction:

preparation of 22RV1 cells and H3122 cells at 5X 10 cells each⁶The DNA was extracted using QIAamp DNA Mini Kit (50) (cat # 51304) from QIAGEN, the detailed procedure is described in the Kit instructions, and the obtained DNA was eluted using 30uL of eluent.

Collecting exosomes of tumor cell culture supernatant and extracting exosome DNA:

the 22RV1 serum-free cell culture supernatant and the H3122 serum-free cell culture supernatant were prepared at 50mL and 500g each, centrifuged for 10min to remove cell debris, the supernatant was 120000g, centrifuged at 4 ℃ for 2H, the supernatant was removed, the pellet was resuspended in 100uL of PBS buffer, and then extracted using QIAamp DNA Mini Kit (50) (cat # 51304) from QIAGEN, the detailed procedure was as described in the Kit instructions, and the obtained DNA was eluted with 30uL of eluent.

DNA sample quantification: the obtained DNA samples were quantified using Nanodrop, and 50ng of each quantified sample was used for the subsequent construction of a DNA library.

DNA library construction and sequencing:

the obtained cell line DNA and cell line culture supernatant exosome DNA were usedConstructing a DNA Library by using the Universal Pro DNA Library Prep Kit for Illumina (the product number is ND608), wherein the detailed operation process is shown in the specification of the Kit, the prepared Library is sent to a plain code organism for sequencing, the both ends are 150, and the required amount of each sample data is 3G.

Sequencing data CNV analysis:

1) truncation removal of low quality sequences and linker sequences on raw sequencing data (fastq format sequences) using fastp software, assessment of sequencing quality, key parameters: -q 25.

2) The filtered sequencing data were aligned to the reference genome using bwa software (hg19), the BAM alignment files were sorted by alignment position using SAMtools, and further BAM files were subjected to duplicate removal, reducing interference of optical and PCR repeats.

3) And (3) carrying out calculation identification on the log2 ratio in a fixed bin interval on the BAM file obtained in the step 2 by using R package QDNAseq, wherein the size of the bin interval is 500 kb. The log2 ratio of bin segments was obtained and then segmentation was performed, using log2-transformation, to analyze CNV. The analytical results were as follows: fig. 1 shows the results of CNV analysis of tumor cell line 22RV1, fig. 2 shows the results of CNV analysis of tumor cell line 22RV1, fig. 3 shows the results of CNV analysis of tumor cell line H3122, and fig. 4 shows the results of CNV analysis of tumor cell line H3122.

As can be seen from fig. 1 and 2: the CNV detection results of the genomic DNA of cell line 22RV1 were consistent with those of the corresponding exosome DNA. As can be seen from fig. 3 and 4: the CNV detection results of the genomic DNA of cell line H3122 were consistent with those of the corresponding exosome DNA. CNV abnormalities that indicate the genomic level of the cell can also be detected by CNV abnormalities in exosome DNA.

Example 2: detection of urine exosomes CNV of healthy people

Preparing urine exosomes and exosome DNA of healthy people: urine samples of 6 healthy volunteers (numbered U1-U6) were collected, 30mL of each urine sample was centrifuged at 1600g for 10min, and the supernatant was collected, then 1/2 volumes of an aqueous solution of PEG8000 (30% by mass/volume) were added, mixed, and left overnight at 4 ℃. After that, 3000g was centrifuged for 10min, and resuspended in 100uL PBS to obtain urine exosome suspension. The obtained exosomes were extracted using the QIAamp DNA Mini Kit (50) (cat # 51304) from QIAGEN, and the detailed procedure is described in the Kit instructions. The obtained DNA was eluted with 30uL of eluent and quantified using Nanodrop, and 50ng of each quantified sample was used for the subsequent construction of DNA library.

The specific construction, sequencing and CNV analysis process of the urine exosome DNA library of the healthy human is the same as that of the example 1. The results of CNV analysis of urine exosome DNA are shown in figure 5. And (3) displaying a detection result: the DNA of the urine exosomes can be used for analyzing and detecting the CNV, and the CNV detection results of the urine exosomes DNA of healthy volunteers are normal.

Example 3: detection of urine exosomes CNV (CNV) of patients with bladder cancer and patients with benign bladder diseases

30mL of each of 5 urine samples (Nos. T1-T5) of bladder cancer patients and 30mL of each of 5 urine samples (Nos. N1-N5) of bladder benign lesion patients were taken, the preparation process of exosomes and exosome DNAs is shown in example 2, and the construction, sequencing and analysis of exosome sample DNA libraries are shown in example 1.

The results of the CNV test on the urine exosomes of the patients with bladder cancer are shown in FIG. 6, and the CNV test on the urine exosomes of the patients with benign bladder diseases is shown in FIG. 7. And (3) displaying a detection result: obvious CNV abnormality can be detected in urine samples of patients with bladder cancer, and CNV of urine exosomes of patients with benign bladder diseases is basically normal. The detection of the urine exosome CNV can be used for distinguishing bladder cancer patients from bladder benign lesion patients and can be used as an auxiliary diagnosis of bladder cancer.

Example 4: the patent of the invention is compared with the CNV detection of the DNA of urine cast-off cells

A cell pellet (numbered as S1-S5 in sequence) obtained by centrifuging 1600g of a urine sample of a bladder cancer patient in example 3 for 10min is added with 100uL of PBS for resuspension, so as to obtain a resuspension solution of urine exfoliated cells. The obtained urine exfoliated cell suspension was extracted using the QIAamp DNA Mini Kit (50) (cat No. 51304) from QIAGEN, and the detailed procedure is described in the Kit instructions. The obtained DNA was eluted with 30uL of eluent and quantified using Nanodrop, and 50ng of each quantified sample was used for the subsequent construction of DNA library. Construction of a DNA library of a sample of urine exfoliated cells, sequencing and analysis of CNV are shown in example 1. CNV measurements of urine exfoliated cells from patients with bladder cancer are shown in fig. 8. And (3) displaying a detection result: the abnormality of CNV was detected in only 3 samples of 5 patients with bladder cancer who had been exfoliated from urine, and further data comparison revealed that 3 samples of CNV abnormality in exfoliated urine (S1, S3, S4) were consistent with the abnormal sites of urinary exosomes CNV (T1, T3, T4). The urinary exosomes CNV of 5 patients of bladder cancer tested in example 3 were all abnormal; CNV detection of urine exfoliated cells was abnormal in only 3 of the cases, and was not detected in the other two cases. The data illustrates that: compared with the detection of urine exfoliative Cells (CNV), the detection method of urine exosomes (CNV) has higher accuracy for the diagnosis of urinary system tumors.

The above description is only a part of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made within the spirit of the invention, and any changes and modifications made are within the scope of the invention.