CN113025721A - Prostate cancer diagnosis and prognosis evaluation kit - Google Patents
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Abstract
The invention provides a combination of a set of chromosome instability regions and gene variation regions, wherein the chromosome instability regions and the gene variation regions comprise: 2q, 3q, 5q, 6q, 7p, 7q, 8p, 8q, 9p, 12q, 13q, 14q, 16q, 18q, PCA3, ERG. The 16 chromosome unstable regions and the gene variation regions reach 91.5 percent of prostate cancer patients, and have high sensitivity and specificity when being applied to a reagent or a kit for screening and/or diagnosing and/or prognostically evaluating the prostate cancer, thereby having good clinical application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit for screening, diagnosing and prognostically evaluating prostate cancer.
Background
Prostate Cancer (PC) refers to an epithelial malignancy that occurs in the prostate gland and is the most common malignancy of the male genitourinary system. The pathological types of the prostate cancer comprise adenocarcinoma (acinar adenocarcinoma), ductal adenocarcinoma, urothelial carcinoma, squamous cell carcinoma and adenosquamous carcinoma, wherein the adenocarcinoma accounts for more than 95 percent. Prostate cancer is commonly referred to as prostate adenocarcinoma.
Current screening and diagnosis of prostate cancer relies primarily on PSA (prostate specific antigen), digital rectal examination, and needle biopsy. Among them, PSA screening is most widely used, but benign prostatic hyperplasia also has signs of increased PSA, so specificity is high, resulting in high PSA misdiagnosis rate. The rectal digital diagnosis is simple and easy to implement, but the diagnosis sensitivity is low, and the rectal digital diagnosis is easily influenced by factors such as doctor level and experience. Rectal ultrasound-guided downlink needle biopsy is the standard for clinical diagnosis of prostate cancer, but the needle operation is invasive and has the possibility of infection. Therefore, a prostate cancer screening and diagnosing method with high sensitivity and specificity, simple and convenient detection and no wound is urgently needed in clinic at present, so that the early diagnosis rate of the prostate cancer is improved, and unnecessary biopsy is reduced.
Chromosomal instability is often associated with tumors, and specifically includes deletion or amplification of entire chromosomal or chromosomal segment copies. The amplification and deletion of chromosomes or chromosome fragments containing a gene associated with tumorigenesis is often unique to tumorigenesis, and detection of regions of chromosomal instability in tumors is critical for both the study of tumorigenesis and the development of diagnostic techniques for tumors.
Chinese patent 201910712588.6 discloses the use of a set of chromosome instability regions in the preparation of a reagent or kit for diagnosing multiple myeloma, wherein the chromosome instability regions comprise the following 7 regions: 1p, 1q, 6q, 11q13.3, 13q, 14q, 17 p. The total carrying rate of 7 common chromosome unstable regions in the multiple myeloma reaches 89.3%, so that the method has great significance for clinical diagnosis, treatment and prognosis evaluation of a monitor, and provides scientific basis for the next step of early diagnosis and the establishment of an individual treatment scheme.
Currently, in clinical practice, the instability of partial chromosome regions is detected by using an in situ fluorescence hybridization method, but the distribution characteristics of the instability of the chromosome at the whole genome level of a prostate cancer patient are lacked, so that the method is not beneficial to comprehensively guiding the diagnosis and prognosis of the prostate cancer.
The PCA3 gene is a biomarker of prostate cancer, which is not expressed in tissues other than prostate, and is expressed at a low level in normal prostate tissue, Benign Prostatic Hyperplasia (BPH) cells, and is expressed at an elevated level in PCA cells and metastatic necrotic lesions. Most prostate cancer patients have overexpression of the PCA3 gene. In most prostate cancer tissues, ERG genes belonging to the ETS transcription factor family can be fused with other genes, and nonrandom gene rearrangements are the most common forms of genetic variation in human malignancies. Therefore, PCA3 gene overexpression and ERG gene fusion have important significance for prostate cancer screening, early diagnosis and treatment.
Multiple regions and gene variation regions on a genome can be simultaneously detected through second-generation sequencing, and the method can be well applied to the simultaneous detection and analysis of the whole genome or a large number of regions on the genome.
Disclosure of Invention
In order to solve the problems, the invention discloses a specific process of an optimized genome-wide second-generation sequencing method, a chromosome instability region and a gene variation region on the basis of researching a specific chromosome instability region and a related molecular marker of the prostate cancer, finds 16 chromosome instability regions and gene variation regions which are common to the occurrence of the prostate cancer, and provides scientific basis for clinical screening, early diagnosis, personalized treatment scheme formulation and prognosis evaluation of the prostate cancer by applying a second-generation sequencing technology.
The numbering of the chromosomal instability region and the genetic variation region referred to in the present invention is defined according to the numbering convention in the art, for example, the instability region 2q refers to the long arm of chromosome 2 in the convention in the art, and the instability region 7p refers to the short arm of chromosome 7.
In one aspect, the invention provides a set of combinations of chromosomal instability regions and genetic variation regions.
The chromosome instability region and the gene variation region comprise: 2q, 3q, 5q, 6q, 7p, 7q, 8p, 8q, 9p, 12q, 13q, 14q, 16q, 18q, PCA3, ERG.
The chromosomal instability comprises a deletion or amplification of the entire chromosome or a copy of a chromosome fragment; the gene variation comprises PCA3 gene overexpression and ERG gene fusion.
The 7p amplification leads to high expression of epidermal growth factor EGFR (proto-oncogene), and promotes tumor progression.
The 8q amplification leads to high expression of a proto-oncogene MYC, and promotes tumor progression.
The loss or amplification loss of the 8p11.21, 13q24, 16q and 17q12 regions also leads to low expression of related cancer suppressor genes and promotes tumor progression.
The PCA3 is prostate cancer gene 3, is located in autosome 9q21-22, and is expressed in prostate epithelial cell non-coding mRNA.
The PCA3 can regulate the survival and apoptosis of prostate cancer cells and is closely related to the invasiveness of tumors.
The ERG gene is located on chromosome 21q22, belongs to ETS transcription factor family, is a protooncogene frequently overexpressed in prostate cancer cells, and is related to the occurrence and development of prostate cancer.
The total carrying rate of the 16 chromosome unstable regions and the gene variation regions in the prostate cancer patients is 91.5%.
In another aspect, the invention provides a library adaptor.
The sequence of the library joint is selected from SEQ ID NO.1 and/or SEQ ID NO. 2.
The library adaptors are used to detect combinations of the aforementioned chromosomal instability regions and genetic variation regions.
In a further aspect, the present invention provides the use of the aforementioned combination of chromosome instability regions and gene variation regions and/or library adaptors in the preparation of a reagent or kit for screening and/or diagnosing and/or prognostically assessing prostate cancer.
In yet another aspect, the present invention provides a reagent or kit for screening and/or diagnosing and/or prognostically assessing prostate cancer.
The reagent or kit includes but is not limited to: a reagent or a kit for screening the prostatic cancer, a reagent or a kit for early diagnosing the prostatic cancer, and a reagent or a kit for monitoring the prostatic cancer.
The reagent is used for detecting the combination of the chromosome unstable region and the gene variation region in the second-generation sequencing.
The kit comprises a reagent for detecting the combination of the chromosome instability region and the gene variation region in the second-generation sequencing.
The kit also comprises the library adaptor.
The kit also comprises one or more of a positive reference substance, a negative reference substance, a buffer solution, an enzyme, a detectable label, a nucleic acid extraction reagent and a nucleic acid purification reagent.
Further, the enzyme comprises one or more of a breaking enzyme, a DNA polymerase, a DNA ligase, an RNA digesting enzyme, a DNA digesting enzyme and a reverse transcriptase.
Further, the positive reference is a cell line mixed with the 16 regional variations; the negative reference substance is a cell line without chromosome variation.
Optionally, the kit further comprises other reagents clinically used for screening, diagnosis, treatment selection, monitoring and prognosis evaluation of prostate cancer to assist or verify the results obtained by detecting the 16 chromosome instability and gene variation regions.
In yet another aspect, the invention provides methods of using the aforementioned reagents or kits.
The method of use includes but is not limited to digital PCR, in situ fluorescence hybridization, nucleic acid probe hybridization, and the like.
The clinical samples detectable by the reagent or the kit include but are not limited to: body fluids, needle flushes, tissues, cells, and the like.
The operation method of the kit comprises the following steps:
(1) obtaining a sample to be detected from a detection object;
(2) contacting a sample to be detected with the detection reagent of the invention;
(3) detecting the 16 chromosome unstable and gene variation areas of the sample to be detected;
(4) and carrying out screening, diagnosis, treatment scheme selection, monitoring and prognosis evaluation on the prostate cancer according to the detection result.
In yet another aspect, the present invention also provides a method of constructing a gene library using the aforementioned library adaptors.
The method for constructing the gene library comprises the step of constructing the library by using the library adaptor.
In still another aspect, the invention also provides the application of the library adaptor and the method for constructing the gene library in the preparation of a reagent or a kit for screening, diagnosing, prognostically evaluating or monitoring the disease condition of the prostate.
Specifically, the kit includes but is not limited to: a reagent or a kit for screening the prostatic cancer, a reagent or a kit for early diagnosing the prostatic cancer, and a reagent or a kit for monitoring the prostatic cancer.
The invention has the beneficial effects that:
1. 16 chromosome instability regions and gene variation regions up to 91.5% in prostate cancer patients;
2. the 16 chromosome unstable regions and the gene variation regions are used as analysis marks, and the diagnosis of the prostate cancer by the second-generation sequencing has high sensitivity and specificity.
Drawings
FIG. 1 is a diagram showing the distribution of 16 chromosome instability regions and genetic variations on chromosome 2 and chromosome 3. The figure is a direct graph of the analysis software, and if the shadow part is artificially modified, the result is not easy to interpret, so that the gray picture is reserved.
FIG. 2 is a distribution diagram of 16 chromosome instability regions and genetic variations on chromosome 7. The figure is a direct graph of the analysis software, and if the shadow part is artificially modified, the result is not easy to interpret, so that the gray picture is reserved.
FIG. 3 is a diagram showing the distribution of 16 chromosome instability regions and genetic variations on chromosome 8 and chromosome 9. The figure is a direct graph of the analysis software, and if the shadow part is artificially modified, the result is not easy to interpret, so that the gray picture is reserved.
FIG. 4 is a diagram showing the distribution of 16 chromosome instability regions and genetic variations on chromosome 12, chromosome 14 and chromosome 18. The figure is a direct graph of the analysis software, and if the shadow part is artificially modified, the result is not easy to interpret, so that the gray picture is reserved.
FIG. 5 is a schematic representation of ERG gene fusion.
FIG. 6 is a ROC curve obtained by calculating the sensitivity and specificity of the present analytical method.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
Example 1 screening of chromosomal instability region and Gene variation region
The nucleic acids of 106 clinically and pathologically diagnosed prostate patients and 113 healthy people are subjected to library construction, the whole genome and transcriptome high-throughput sequencing or target region targeted capture sequencing is carried out on the nucleic acids, the chromosome copy number and the genetic variation are analyzed, and the regions with chromosome instability and genetic variation are identified. Analysis and statistics of differences in chromosome instability and gene variation between tumor patients and control groups.
Through the above operations, 16 common chromosomal instability regions and gene variation regions with high prostate association were found, including 2q, 3q, 5q, 6q, 7p, 7q, 8p, 8q, 9p, 12q, 13q, 14q, 16q, 18q, PCA3, ERG.
From the frequency of the gene transfer, 16 chromosomal instability regions and gene mutation regions were found to be 91.5% in prostate cancer patients, and 97 patients in total carried the chromosomal instability regions and gene mutation regions. The gene variation PCA3 gene is over-expressed, the occurrence frequency is 48.11%, and the occurrence frequency of ERG gene fusion is 25.47%. Chromosomal instability was most common at 18q, occurring at a frequency of 49.06%; followed by 8q, 7q, 9p, which occurred at 35.85%, 23.58% and 14.15%, respectively.
Example 2 an exosome obtaining method
The kit selected in this example was purchased from SBI corporation, cat #: SBI.EXTOCC 10A-1.
a. Transferring 5mL of urine into a 15mL centrifuge tube, centrifuging 3000g for 15min, and removing cells or cell debris;
b. transferring the supernatant to a sterilizing tube, and adding 1mL of ExoQuick reagent;
c. turning the centrifuge tube upside down, and mixing uniformly; standing overnight at 4 deg.C or room temperature;
d.1500g, centrifuging for 30min, and absorbing supernatant when light yellow to white precipitate is visible at the bottom of the tube;
e.1500g, centrifuging for 5min again, and removing residual supernatant by suction;
f. sterile distilled water or PBS was added and the exosome pellet was resuspended.
Example 3 Co-extraction of genomic DNA from urine exfoliated cells with Total RNA
The kit used in this example was purchased from QIAGEN (cat. No. 80204).
a. The urine sample obtained was transferred to a 15mL centrifuge tube, centrifuged at 1600g for 10min, the supernatant was carefully decanted, and the remaining supernatant was carefully discarded with a pipette.
b. Add 350. mu.L of 1 XBuffer RL/DTT to a new 1.5mL centrifuge tube and carefully aspirate the lysate with a disposable 20 gauge needle syringe at least 5 times to break up the cells.
c. The DNA purification column was packed in a 2mL collection tube. The cell lysate was transferred to a gDNA filter column. Centrifuge at 14000g for 2 min.
d. The DNA purification column was retained and the extraction was performed as n-s.
e. Adding 70% ethanol with equal volume to the filtrate, and sucking with pipette 3-5 times.
f. The RNA purification column was packed in a 2mL collection tube. Transferring less than or equal to 750 mu L of mixed solution to a column, and centrifuging 12000g for 30-60 s.
(optionally: when the mixture exceeds 750. mu.L) the filtrate is discarded and the column is returned to the collection tube. The remaining mixture was transferred to the column and centrifuged at 12000g for 30-60 s.
h. The filtrate was decanted and the column was returned to the collection tube. Add 500. mu.L Buffer RW1 to the column and centrifuge at 12000g for 30-60 s.
i. The filtrate was decanted and the column was returned to the collection tube. Add 500. mu.L Buffer RW2 to the column and centrifuge at 12000g for 30-60 s.
j. The filtrate was decanted and the column was returned to the collection tube. Add 500. mu.L Buffer RW2 to the column and centrifuge at 12000g for 30-60 s.
k. The effluent is decanted and the column is returned to the collection tube. Centrifuge at 12000g for 2 min.
L. transfer the column to a 1.5mL centrifuge tube and add 20-100. mu.L of RNase free water to the center of the column membrane. Standing at room temperature for 2 min. Centrifuge at 12000g for 1 min.
m. discard the binding column and store RNA at-20 ℃.
n. taking DNA purification column (step d) and packing in 2mL collection tube. Add 500. mu.L Buffer DW1 to the column and let stand for 2 min. 10000g for 30-60 s.
And o, pouring out the effluent, and putting the column back into the collecting pipe. Add 500. mu.L of Buffer RW2 to the column. 10000g for 30-60 s.
p, pouring out the effluent, and putting the column back into the collecting pipe. Add 500. mu.L of Buffer RW2 to the column. 10000g for 30-60 s.
And q, pouring out the effluent, and sleeving the column back to the empty collecting pipe. 13000g for 2 min.
r. the DNA column was packed in a 1.5mL centrifuge tube. Add 30-50. mu.L of nuclease-free water pre-heated to 65 ℃ to the center of the column membrane. Standing at room temperature for 3 min. 13000g for 1 min.
s. discard the DNA column, store the DNA at 2-8 ℃ or-20 ℃.
The urine-shed cells in this example can be replaced with a puncture needle rinse or exosomes prepared in example 2.
Example 4 urine supernatant free DNA and free RNA extraction
The kit used in this example was purchased from QIAGEN (cat # 55184).
a. The urine sample obtained was transferred to a 15mL centrifuge tube and centrifuged at 1600g for 10 min.
b. Carefully aspirate 4mL of the supernatant from the previous step into two 2mL centrifuge tubes and centrifuge at 16000g for 10 min.
c. 600. mu.L of buffer RPL was added to a new 15mL centrifuge tube.
d. 4mL of the supernatant was added to the reaction solution in the previous step, and vortexed for 5 seconds to mix. Standing at room temperature for 3 min.
e. Add 400. mu.L of buffer RPP, vortex for 20s and mix well, incubate for 3min on ice.
f.3000g, and centrifugation for 10 min.
g. Transfer 4mL of supernatant to a new 15mL centrifuge tube (on ice).
h. Add 4mL of glacial isopropanol and vortex and mix well.
i. Putting the RNA purification column into a 15mL collecting tube, transferring 4mL of the mixed solution into the RNA purification column, centrifuging for 1min at 5000g, and pouring off the waste liquid.
j. The remaining mixture was repeated once per step i.
k. Add 4mL buffer RWT to the column, 5000g, centrifuge for 1min, and discard the waste.
l. add 2.5mL buffer RPE to the column, 5000g, centrifuge for 5min, and discard the waste.
m. place the purification column into a new 15mL collection tube. Carefully add 200. mu.L of RNase-free water to the center of the purification cartridge membrane.
n. centrifuging at 20000g for 1min, and eluting nucleic acid.
The urine supernatant in this example can be replaced with the resuspended exosomes prepared in example 2.
Example 5 preparation of library adaptors
The linker sequence of this example was synthesized via bio (Shanghai) corporation.
The sequence of the linker-1 is SEQ ID NO.1, and the sequence of the linker-2 is SEQ ID NO. 2.
The 5 'end of the linker-1 is modified by phosphorylation, and the 3' end of the linker-2 is modified by phosphorothioate between G and T.
The dry powders of linker-1 and linker-2 were diluted to 10 μ M with Annealing buffer (Annealing buffer), respectively, and the dilutions were mixed in equal proportion, and the resulting mixture was placed in a PCR instrument, and the procedure was as shown in table 1:
TABLE 1
Example 616 chromosome instability regions and gene variation regions were constructed as genomic DNA libraries for screening and diagnosing prostate cancer as analytical markers.
The relevant kit in this example was purchased from NEB corporation, cat no: E7645S.
1. Genome fragmentation: 20ng of human genomic DNA (obtained from a clinically confirmed sample) was used to prepare an enzyme digestion reaction system shown in Table 2, and the reaction was carried out according to the procedure shown in Table 3. In this example, the concentration of human genomic DNA was 2 ng/. mu.L, and 10. mu.L was added to the reaction system.
TABLE 2
| Components | System/. mu.L |
| Genomic DNA | 10 |
| Fragmentation mix enzyme | 10 |
| Fragmentation buffer | 5 |
| Non-nucleic acid water | Supplement to 50 |
| General System | 50 |
After shaking, mixing and centrifugation (avoiding air bubbles), the following procedure was run on the PCR instrument:
TABLE 3
2. Terminal addition of A
If the human genome DNA is cell-free DNA, such as cfDNA in blood, the non-modified A is directly carried out without genome fragmentation, 20ng of cfDNA is taken to prepare an enzyme digestion reaction system shown in Table 4, and the reaction is carried out according to the procedure in Table 6. In this example, the concentration of cfDNA was 2 ng/. mu.L, and 10. mu.L was added to the reaction system. The reaction product of the previous step was prepared as shown in Table 5.
TABLE 4
| cfDNA end-modification A reaction | System/. mu.L |
| cfDNA | 10 |
| Non-modified enzyme A | 3 |
| Buffer solution A was not added | 7 |
| Nuclease-free water | Supplement to 60 |
| |
60 |
TABLE 5
After shaking, mixing and centrifugation (avoiding air bubbles), the following procedure was run on the PCR instrument:
TABLE 6
3. And (3) joint connection reaction: after adaptor ligase (5. mu.L/sample) and adaptor ligation enhancer (30. mu.L/sample) were mixed by pipetting an appropriate volume based on the number of samples to be tested, 35. mu.L of the mixed solution was pipetted and added to the reaction product in the previous step, 5. mu.L of the mixed adaptor prepared in example 5 was pipetted and added to the reaction solution, and then the mixture was shaken, homogenized and centrifuged. The reaction system in table 7 below was finally formed.
TABLE 7
| Components | System/. mu.L |
| |
60 |
| Joint connection premix liquid | 30 |
| |
1 |
| Hybrid joint | 2.5 |
| General System | 93.5 |
The system is placed in a PCR instrument, and the program is run: at 20 deg.C, 30min, the hot lid was closed.
4. A purification step after the ligation reaction:
a. taking out the library purified magnetic beads in advance, standing at room temperature for at least 30min, and mixing uniformly before use.
b. Transferring the joint connection reaction liquid in the steps to a 1.5mL centrifuge tube with the corresponding number, adding 84 mu L of the resuspended library purified magnetic beads, sucking and beating the library purified magnetic beads for 20 times at a constant speed by using a pipettor with a proper range, and incubating the library for 5min at room temperature.
c. Placing the centrifuge tube on a magnetic frame, and discarding the supernatant after the solution is clarified.
d. To this, 200. mu.L of 80% ethanol was added in a fresh state, and after standing for 30 seconds, the supernatant was discarded.
e. Repeating the step d once.
f. And taking off the centrifugal tube from the magnetic frame, performing instantaneous centrifugation for 3s, putting the centrifugal tube back on the magnetic frame, and removing the residual 80% ethanol by suction, taking care not to suck the magnetic beads. Opening the tube cover, and air drying at room temperature for 2-10 min.
g. When the magnetic beads become sub-bright, 17. mu.L of nuclease-free water is added into the centrifuge tube, the magnetic beads are resuspended by slight shaking, and the incubation is carried out for 5min at room temperature.
h. Placing the centrifuge tube on a magnetic frame, and standing for 2 min. After the solution was clarified, 15. mu.L of the supernatant was collected for the next amplification reaction.
5. And (3) PCR amplification: the corresponding reagents were added to the PCR tubes as in table 8 below:
TABLE 8
| Components | System/. mu.L |
| Purification of ligation product in the previous step | 15 |
| PCR amplification premix solution | 25 |
| P7 end-tag primer | 5 |
| P5 end-tag primer | 5 |
| General System | 50 |
Among them, the P7 end-tag primer and the P5 end-tag primer were synthesized by bio (shanghai) corporation. The specific sequence is as follows:
TABLE 9
| Numbering | Tag primer sequence 5 '-3' |
| P7-01 | SEQ ID NO.3 |
| P7-02 | SEQ ID NO.4 |
| P7-03 | SEQ ID NO.5 |
| P7-04 | SEQ ID NO.6 |
| P7-05 | SEQ ID NO.7 |
| P7-06 | SEQ ID NO.8 |
| P7-07 | SEQ ID NO.9 |
| P7-08 | SEQ ID NO.10 |
| P5-01 | SEQ ID NO.11 |
| P5-02 | SEQ ID NO.12 |
| P5-03 | SEQ ID NO.13 |
| P5-04 | SEQ ID NO.14 |
| P5-05 | SEQ ID NO.15 |
| P5-06 | SEQ ID NO.16 |
| P5-07 | SEQ ID NO.17 |
| P5-08 | SEQ ID NO.18 |
The mixed PCR tube was placed in a PCR instrument and the following procedure was run:
watch 10
6. PCR product purification reference step 3 purification step, wherein:
the amount of the resuspended library purification magnetic beads in step b was 45. mu.L;
adding 31 mu L of Low TE buffer solution (or nuclease-free water) in the step g;
in step h, 30. mu.L of the supernatant was collected for further processing.
7. Library quantitative processing machine
The purified library was analyzed for fragment size using the Qseq Biofragment analysis System
The mass concentration of the library was measured by the dsDNA HS Assay Kit (purchased from Saimer Feishi technologies), and the molar concentration of the library was calculated from the mass concentration and the fragment size, according to the aboveSequencer instructions sequencing was performed using Illumina Hiseq X-ten.
Example 716 chromosome instability regions and Gene variation regions as markers for analysis and screening, diagnosis of prostate cancer
The kit selected in this example was purchased from NEB corporation under the following cargo number: e6310, E7770.
1. Hybridization of the RNA sample to the probe: taking 10-50ng of RNA, taking out probe hybridization buffer solution, melting on an ice box, preparing probe hybridization premix solution according to the probe hybridization buffer solution (3 mu L/sample) and rRNA probe mixed solution (H/M/R) (2 mu L/sample), preparing a reaction system (operating on the ice box) in the table 11, gently blowing and uniformly mixing by using a pipette, and carrying out instantaneous centrifugation.
TABLE 11
X represents the volume of 10-50ng RNA sample (for example: RNA concentration 25 ng/. mu.L, 50ng RNA is added, X-50/25-2. mu.L).
Placed in a PCR instrument and run the following program:
TABLE 12
After hybridization, the sample was immediately removed from the PCR machine and placed on an ice box for further processing.
2. RNase H digestion: 10 XRNase H Buffer was removed from the ice and thawed before preparing RNase H reaction premix according to the system shown in Table 13 below.
| Components | System/. mu.L |
| 10×RNase H Buffer | 2 |
| RNase H | 2 |
| Nuclease- |
1 |
| General System | 5 |
And (3) adding 5 mu L of RNase H reaction premixed solution into the reaction solution in the step 1 to ensure that the RNase H reaction system reaches 20 mu L, gently blowing and uniformly mixing by a pipette, and carrying out instantaneous centrifugation.
The reaction system is placed in a PCR instrument (the hot cover is more than or equal to 45 ℃) for 30min at 37 ℃ to carry out RNase H reaction. After the digestion of RNase H was completed, the sample was immediately taken out of the PCR machine and placed on an ice box for the next operation.
3. DNase I digestion: the 10 XDNase I Buffer was removed from the ice and thawed, and the DNase I reaction premix was prepared according to the system in Table 14 below.
TABLE 14
| Components | System/. mu.L |
| 10×DNase I Buffer | 5 |
| DNase I | 2.5 |
| Nuclease-free water | 22.5 |
| General System | 30 |
Adding 30 mu L of DNase I reaction premixed solution into the reaction solution in the step 2 to ensure that the DNase I reaction system reaches 50 mu L, gently blowing and beating the mixed solution by a pipette, and performing instantaneous centrifugation.
The reaction system is placed in a PCR instrument (the hot cover is more than or equal to 45 ℃) for 30min at 37 ℃ for DNase I reaction. After the digestion of DNase I, the samples were immediately removed from the PCR machine and placed on an ice box for immediate purification.
4. RNA purification to remove rRNA:
a. taking out the RNA purified magnetic beads from 2-8 ℃ in advance, standing and balancing for 30min to room temperature, and uniformly mixing by vortex or oscillation before use;
after the DNase I reaction is finished, adding 110 mu L of RNA purified magnetic beads (2.2 x) into each reaction tube, and blowing, beating and uniformly mixing;
c. standing at room temperature for 5min, transferring to magnetic frame for 5min until the solution becomes clear, and carefully removing supernatant;
d. keeping the centrifugal tube on a magnetic frame, adding 200 microliter of 80% ethanol, standing for 30s, and discarding all supernatants;
e. repeating the step d, and washing the magnetic beads with 80% ethanol for 1 time. Thoroughly sucking the residual liquid by using a 10-mu-L gun head;
f. drying the magnetic beads for 2-3min, adding 7 μ L of nuclease-free water after the alcohol is completely volatilized, and blowing, beating and mixing uniformly;
g. standing at room temperature for 2min, mounting on a magnetic frame for 1min, carefully sucking 5 μ L of supernatant into another new centrifuge tube when the solution becomes clear;
h. add 5. mu.L fragmentation buffer to the supernatant tube, blow and mix well, perform RNA disruption according to the following procedure (hot lid 105 ℃ C.) in Table 15 (free RNA does not need this fragmentation, add water directly to 10. mu.L for the next step):
watch 15
When the temperature was lowered to 4 ℃, it was removed, subjected to instantaneous centrifugation and then placed on ice, and immediately subjected to the next first strand cDNA synthesis.
5. First strand cDNA synthesis: taking out the RT chain specific reagent to melt at room temperature, preparing the system shown in the table 16 on ice, lightly blowing and uniformly mixing by a pipette, and performing instantaneous centrifugation.
TABLE 16
| Components | System/. mu.L |
| Post-disrupted mRNA | 10 |
| RT chain |
8 |
| First Strand synthetase mixture | 2 |
| |
20 |
Placed in a PCR instrument and run the following program:
TABLE 17
6. Second strand cDNA synthesis: the reaction system was prepared on the ice box according to the following table, gently blown and beaten by a pipette, mixed well, and centrifuged instantaneously.
Watch 18
| Components | System of |
| |
20 |
| Second Strand |
8 |
| Second Strand synthetase mixture | 4 |
| Enzyme-free water | 48 |
| General System | 80 |
The PCR was run in a PCR machine at 16 ℃ for 60min with the hot lid closed.
Immediately after the reaction, purification was carried out by the following steps:
a. taking out the RNA purified magnetic beads from 2-8 ℃ in advance, standing and balancing for 30min to room temperature, and uniformly mixing by vortex or oscillation before use;
b. 144 μ L of RNA purified magnetic beads (2.2X) were added to each reaction tube, and the mixture was pipetted and mixed;
c. standing at room temperature for 5min, transferring to magnetic frame for 5min until the solution becomes clear, and carefully removing supernatant;
d. keeping the centrifugal tube on a magnetic frame, adding 200 microliter of 80% ethanol, standing for 30s, and discarding all supernatants;
e. repeating the step d, and washing the magnetic beads with 80% ethanol for 1 time. Thoroughly sucking the residual liquid by using a 10-mu-L gun head;
f. drying the magnetic beads for 2-3min, adding 39 μ L of nuclease-free water after the alcohol is completely volatilized, and blowing, beating and uniformly mixing;
g. after standing at room temperature for 2min and on a magnetic stand for 1min, after the solution became clear, 37. mu.L of the supernatant was carefully pipetted into another new centrifuge tube.
7. End repair plus a: taking out the unmodified buffer solution in advance, melting on an ice box, preparing a premixed solution according to the unmodified buffer solution (10 mu L/sample) and the unmodified enzyme (3 mu L/sample), adding 13 mu L of the premixed solution into the purified product in the previous step, blowing and mixing by a pipette, and performing instantaneous centrifugation (operation on the ice box). The concrete system is as follows:
watch 19
| Components | System/. mu.L |
| Double-stranded cDNA purification product | 37 |
| Buffer for final repair | 10 |
| Un-modified enzyme | 3 |
| General System | 50 |
Placed in a PCR instrument and run the following program:
watch 20
8. Connecting a joint: after the ligation buffer (16.5. mu.L/sample) and ligase (3. mu.L/sample) were mixed by pipetting an appropriate volume in accordance with the number of samples to be detected, 19.5. mu.L of the mixed solution was pipetted and added to the reaction product of the previous step, 2.5. mu.L of the prepared mixed adaptor was pipetted and added to the above reaction solution, and the mixture was shaken, mixed and centrifuged instantaneously. The reaction system of Table 21 below was finally formed.
TABLE 21
| Components | System/. mu.L |
| Reaction product of the last step | 50 |
| Ligation buffer | 16.5 |
| Ligase | 3 |
| Joint | 2.5 |
| General System | 72 |
Place in PCR, run at 22 deg.C for 15min, and close the hot lid.
Purifying a joint connection product:
a. taking out the RNA purification magnetic beads from 2-8 ℃ in advance, standing at room temperature for at least 30min, and mixing uniformly before use.
b. Transferring the joint connection reaction liquid in the steps to a 1.5mL centrifuge tube with the corresponding number, adding 56 mu L of resuspended magnetic beads, uniformly sucking and stirring the mixture by using a pipettor with a proper range at a constant speed, and incubating the mixture for 5min at room temperature.
c. Placing the centrifuge tube on a magnetic frame, and discarding the supernatant after the solution is clarified.
d. To this, 200. mu.L of 80% ethanol was added in a fresh state, and after standing for 30 seconds, the supernatant was discarded.
e. Repeating the step d once.
f. And taking off the centrifugal tube from the magnetic frame, performing instantaneous centrifugation for 3s, putting the centrifugal tube back on the magnetic frame, and sucking away the residual 80% ethanol without sucking the magnetic beads. Opening the tube cover, and air drying at room temperature for 2-10 min.
g. When the magnetic beads become sub-bright, 21. mu.L of nuclease-free water is added into the centrifuge tube, the magnetic beads are resuspended by slight shaking, and the incubation is carried out for 5min at room temperature.
h. Placing the centrifuge tube on a magnetic frame, and standing for 2 min. After the solution was clarified, 20. mu.L of the supernatant was collected for the next amplification reaction.
9. And (3) PCR amplification: the reaction system was prepared according to the following table 22, mixed well and centrifuged instantaneously.
TABLE 22
| Components | System/. mu.L |
| Purification of ligation product in the |
20 |
| PCR amplification premix solution | 25 |
| P7 end-tag primer | 2.5 |
| P5 end-tag primer | 2.5 |
| General System | 50 |
Put into a PCR instrument and run the following program:
TABLE 23
And (3) PCR product purification:
a. taking out RNA purified magnetic beads from 2-8 ℃ in advance, standing at room temperature for at least 30min, and mixing uniformly before use.
b. Transferring the joint connection reaction liquid in the steps to a 1.5mL centrifuge tube with a corresponding number, adding 40 mu L of resuspended magnetic beads, uniformly sucking and stirring the mixture by using a pipettor with a proper range at a constant speed, and incubating the mixture for 5min at room temperature.
c. Placing the centrifuge tube on a magnetic frame, and discarding the supernatant after the solution is clarified.
d. To this, 200. mu.L of 80% ethanol was added in a fresh state, and after standing for 30 seconds, the supernatant was discarded.
e. Repeating the step d once.
f. And taking off the centrifugal tube from the magnetic frame, performing instantaneous centrifugation for 3s, putting the centrifugal tube back on the magnetic frame, and sucking away the residual 80% ethanol without sucking the magnetic beads. Opening the tube cover, and air drying at room temperature for 2-10 min.
g. When the magnetic beads become sub-bright, 42. mu.L of nuclease-free water is added into the centrifuge tube, the magnetic beads are resuspended by slight shaking, and the incubation is carried out for 5min at room temperature.
h. Placing the centrifuge tube on a magnetic frame, and standing for 2 min. After the solution was clarified, 40. mu.L of the supernatant was collected for the next amplification reaction.
10. Performing quality inspection on the library: use of the purified library
The dsDNA HS Assay Kit measures the library mass concentration, the Qseq biological fragment analysis system analyzes the fragment size, the library molar concentration is calculated according to the mass concentration and the fragment size, and the Illumina Hiseq X-ten is used for sequencing according to the instruction of the sequencer.
Results analysis discussion:
the sequencing data from the machine was analyzed to obtain chromosome instability and genetic variation profiles, as shown in FIGS. 1-4.
The chromosome instability analysis result consists of three parts: chromosome number, chromosome partitioning, and chromosome sequencing depth profile. The dots in the chromosome sequencing depth distribution region are the distribution of the copy number of the chromosome small region, and are judged to have no instability when the score value of the vertical axis is between-3 and 3, are judged to be amplified when the score value of the vertical axis is larger than 3, are judged to be deleted when the score value of the vertical axis is smaller than-3, and are distinguished from the normal region by using a gray background for the region in which amplification or deletion occurs. And checking whether the chromosome instability occurs or not according to the analysis result, and judging the chromosome instability to be positive if the chromosome instability occurs or judging the chromosome instability to be negative if the chromosome instability does not occur.
Analysis of gene variation: the PCA3 gene is overexpressed, the relative reads number of PCA3 and PSA is firstly calculated, then the PCA3 score is calculated to be (PCA3 relative reads number/PSA relative reads number) multiplied by 1000, and when the PCA3 score is higher than 25, the PCA3 gene is overexpressed; and (3) ERG gene fusion, wherein the screened sequencing data is compared with the whole genome sequence, the ERG gene is positioned at 21q22, and if the comparison result is at other positions, the ERG gene fusion is judged. A schematic representation of ERG gene fusion is shown in FIG. 5.
The second generation analysis results were compared with the pathological results, and the sensitivity and specificity of the analysis method were calculated to obtain the ROC curve, as shown in fig. 6. The AUC area of the ROC curve of the method reaches 0.918, which shows that the 16 chromosome instability regions and the gene variation regions in the method are used as analysis marks, and the diagnosis of the prostate cancer by the second-generation sequencing has high sensitivity and specificity.
Sequence listing
<110> Suzhou Macro Biotechnology Ltd
<120> prostate cancer diagnosis and prognosis evaluation kit
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
caagcagaag acggcatacg agatcaatag ccgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 6
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
caagcagaag acggcatacg agatgaatcc gtgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 7
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
caagcagaag acggcatacg agatagctac cagtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 8
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
caagcagaag acggcatacg agatggttga acgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 9
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
caagcagaag acggcatacg agatgcgtta gagtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 10
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
caagcagaag acggcatacg agataaccag aggtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 11
<211> 70
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
aatgatacgg cgaccaccga gatctacact tgcttgcaca ctctttccct acacgacgct 60
cttccgatct 70
<210> 12
<211> 70
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
aatgatacgg cgaccaccga gatctacacg agaggttaca ctctttccct acacgacgct 60
cttccgatct 70
<210> 13
<211> 70
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
aatgatacgg cgaccaccga gatctacaca cctggttaca ctctttccct acacgacgct 60
cttccgatct 70
<210> 14
<211> 70
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
aatgatacgg cgaccaccga gatctacaca agcggaaaca ctctttccct acacgacgct 60
cttccgatct 70
<210> 15
<211> 70
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
aatgatacgg cgaccaccga gatctacacc ggaacaaaca ctctttccct acacgacgct 60
cttccgatct 70
<210> 16
<211> 70
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
aatgatacgg cgaccaccga gatctacacg gtaagctaca ctctttccct acacgacgct 60
cttccgatct 70
<210> 17
<211> 70
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
aatgatacgg cgaccaccga gatctacact gtggcataca ctctttccct acacgacgct 60
cttccgatct 70
<210> 18
<211> 70
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
aatgatacgg cgaccaccga gatctacaca ctacggaaca ctctttccct acacgacgct 60
cttccgatct 70
Claims (10)
1. A set of combinations of chromosomal instability regions and genetic variation regions, wherein the chromosomal instability regions and genetic variation regions comprise: 2q, 3q, 5q, 6q, 7p, 7q, 8p, 8q, 9p, 12q, 13q, 14q, 16q, 18q, PCA3, ERG.
2. The combination of a chromosomal instability region and a genetic variation region according to claim 1, wherein the chromosomal instability comprises deletion or amplification of the entire chromosome or a copy of a chromosomal segment; the gene variation comprises PCA3 gene overexpression and ERG gene fusion.
3. A library adaptor having a sequence selected from SEQ ID No.1 and/or SEQ ID No.2 for use in detecting a combination of a chromosomal instability region and a genetic variation region according to claim 1.
4. Use of the library adaptor of claim 3 in the construction of a gene library.
5. A method for constructing a gene library, which comprises constructing the library adaptor according to claim 3.
6. Use of a combination of a chromosomal instability region and a genetic variation region according to any of claims 1-2 and/or a library adaptor according to claim 3 and/or a method of constructing a genetic library according to claim 5 for the preparation of a reagent or kit for screening and/or diagnosing and/or prognostically assessing prostate cancer.
7. A reagent or kit for screening and/or diagnosis and/or prognostic evaluation of prostate cancer, wherein the reagent is used for detecting the chromosome instability region and the gene variation region according to any one of claims 1 to 2; the kit comprises a reagent for detecting the combination of the chromosome instability region and the gene variation region according to any one of claims 1 to 2.
8. The kit of claim 7, further comprising the library adaptor of claim 3.
9. The kit of claim 7, further comprising one or more of a positive reference, a negative reference, a buffer, an enzyme, a detectable label, a nucleic acid extraction reagent, and a nucleic acid purification reagent; the enzyme comprises one or more of a breaking enzyme, a DNA polymerase, a DNA ligase, an RNA digestive enzyme, a DNA digestive enzyme and a reverse transcriptase.
10. The kit of claim 7, wherein the positive reference is a cell line mixed with 16 region variants of claim 1; the negative reference substance is a cell line without chromosome variation.
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| CN113621705A (en) * | 2021-07-28 | 2021-11-09 | 苏州宏元生物科技有限公司 | Female reproductive system tumor screening, diagnosis and prognosis evaluation kit |
| CN117106911A (en) * | 2023-08-14 | 2023-11-24 | 首都医科大学附属北京天坛医院 | Gene set for detecting schwannoma and multiplex PCR-high flux sequencing detection kit thereof |
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| CN117106911A (en) * | 2023-08-14 | 2023-11-24 | 首都医科大学附属北京天坛医院 | Gene set for detecting schwannoma and multiplex PCR-high flux sequencing detection kit thereof |
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