CN114525341A - Kit for simultaneously detecting lung cancer and lung infection - Google Patents

Kit for simultaneously detecting lung cancer and lung infection Download PDF

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CN114525341A
CN114525341A CN202210173308.0A CN202210173308A CN114525341A CN 114525341 A CN114525341 A CN 114525341A CN 202210173308 A CN202210173308 A CN 202210173308A CN 114525341 A CN114525341 A CN 114525341A
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genome
kit
chromosome
pathogenic microorganism
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钱自亮
徐文胜
王白云
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Suzhou Hongyuan Biological Technology Co ltd
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Abstract

The invention provides a lung disease detection kit. The kit simultaneously detects human genome chromosome instability and pathogenic microorganism genomes by a second-generation sequencing technology, wherein the chromosome instability region comprises the following 14 regions: 2p, 3q, 6p, 7p, 8q, 9p, 12p, 17q, 18q, 20q, 21p, 21q, said pathogenic microorganisms including bacteria, fungi and DNA viruses. The invention has great significance for clinically diagnosing and treating lung cancer and lung infection, and provides scientific basis for further treatment and formulation of individualized treatment schemes for patients.

Description

Kit for simultaneously detecting lung cancer and lung infection
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a group of chromosome unstable regions and pathogenic microorganism genomes in preparation of a reagent or a kit for diagnosing lung cancer and lung infection.
Background
Primary Lung Cancer (PLC) is the most common malignancy worldwide. From the pathological and therapeutic aspects, lung cancer can be roughly classified into non-small cell lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC), wherein the non-small cell lung cancer accounts for about 80% -85%, and the rest are small cell lung cancer.
Pulmonary infection is a common respiratory disease, the respiratory tract is connected with the outside, and the whole blood flows through the lung, so the lung is easily attacked by microorganisms inside and outside the body. Respiratory infections caused by viruses and bacteria are the most common. Bacterial pneumonia caused by streptococcus pneumoniae infection accounts for 80-90% of the past, and the rest is caused by streptococcus, klebsiella, staphylococcus and the like. The current situation of bacterial infection changes, although pneumonia outside hospitals is mainly caused by streptococcus pneumoniae infection, staphylococcus aureus pneumonia and gram-negative bacillary pneumonia are also increased obviously. Gram-negative bacillary pneumonia (caused by nosocomial infection) of hospitalized patients is increased remarkably, and infection of klebsiella and pseudomonas aeruginosa is the most common. Fungal infections also increase. As a result of improvements in microbial isolation and culture techniques, lung infections caused by pneumocystis, anaerobes and new pathogenic microorganisms, such as Legionella, cytomegalovirus and pneumocystis, have been successfully diagnosed. The change of the pathogenic microorganism can be related to the advanced age, serious disease and increased immune diseases of patients; the application of adrenocortical hormone, immunosuppressant, cytotoxic drug and antibiotic; various intubation, tracheotomy, ventilator applications, and organ transplantation.
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. Currently, in-situ fluorescence hybridization is used to detect instability of partial chromosome regions clinically, but the distribution characteristics of chromosome instability on the whole genome level of a patient are lacked.
The lung infection pathogenic microorganisms are confirmed to need to be cultured clinically, the requirements on culture conditions and operators are high, the flux is insufficient, and the high-flux detection cannot be met. Moreover, the sources of the infectious pathogenic microorganisms are various, the culture conditions of each microorganism are different, the culture conditions cannot be determined under unknown conditions, and part of the infectious sources are easily obtained without being cultured, so that false negative is caused. The method can be used for identifying the pathogen by a sequencing technology, does not need to culture, has high detection sensitivity and has good detection capability on microorganisms with low concentration and difficult culture.
No second-generation sequencing kit for simultaneously detecting lung infection and lung cancer exists in the prior art.
Disclosure of Invention
The invention uses the second-generation sequencing technology, screens 14 regions and microbial genomes suitable for representing lung cancer by analyzing the unstable chromosome and pathogenic microbial genome information of the lung cancer, and can provide scientific basis for clinical early diagnosis of lung cancer and lung infection and establishment of an individual treatment scheme by the method. Human and microbial genomes can be detected simultaneously through second-generation sequencing, whether tumorigenesis occurs or not is diagnosed through a chromosome instability region, infected microbes are identified through detecting the microbial genome, and lung cancer and lung infection are diagnosed simultaneously through the method.
The numbering of the chromosomal instability regions involved in the present invention is defined according to the numbering convention customary in the art, for example, the instability region 3q refers to the long arm of chromosome 3 and the instability region 7p refers to the short arm of chromosome 7 in the convention.
In the present invention, "genome" refers to the sum of all genetic material of an organism. These genetic materials include DNA or RNA (viral RNA).
In the present invention, "detection" refers to detection of whether a subject is affected by a disease, and the detection result may be affected or not, and may be a one-time detection, a long-term continuous detection, a local tissue detection, or a systematic detection.
In the present invention, "screening" means to detect the possibility of a disease in a test population, and is not limited to a population with a disease symptom, but is applied to an asymptomatic population, and is mainly aimed at detection and timely treatment at the initial stage of a disease to prevent the occurrence of symptoms, and to prevent the occurrence of various sequelae by taking effective treatment.
In the present invention, "diagnosis" generally refers to confirmation of presence or absence of a disease in an individual or a population having symptoms of a disease, but may be an individual or a population having no symptoms of a disease in some cases.
As used herein, "prognostic assessment" refers to empirically predicted disease progression, including assessment of near and far term efficacy, outcome recovery or degree of progression of the disease.
In the invention, "disease condition monitoring" means that the information of dynamic distribution and influencing factors of diseases is continuously collected, checked and analyzed for a long time, and the information is reported and fed back in time so as to take intervention measures in time.
In one aspect, the invention provides the use of a chromosome instability region and a genome of a pathogenic microorganism in the manufacture of a reagent or kit for the detection, screening, diagnosis, prognostic assessment or monitoring of a pulmonary disease.
The chromosome instability region comprises: 2p, 3q, 6p, 7p, 8q, 9p, 12p, 17q, 18q, 20q, 21p, 21 q; the genome of the pathogenic microorganism comprises the genome of any one or more of bacteria, fungi and viruses.
Specifically, the chromosomal instability includes deletion or amplification of the entire chromosome or a copy of a chromosome fragment.
Specifically, the amplification of the 7p region can lead to high expression of the epidermal growth factor EGFR (proto-oncogene); the deletion of the cancer suppressor gene at the 16 sites of the 9p region causes the abnormal regulation of the cell cycle; the 17p deletion causes that the cancer suppressor gene TP53 can not be expressed, and promotes the tumor progression.
Specifically, the total carrying rate of the 14 unstable regions in lung cancer patients is up to 89.3%.
Preferably, the bacteria include, but are not limited to: streptococcus pneumoniae, staphylococcus aureus, alpha hemolytic streptococcus, klebsiella pneumoniae, haemophilus influenzae, pseudomonas aeruginosa, escherichia coli and pseudomonas aeruginosa.
Preferably, the fungi include, but are not limited to: candida albicans, Aspergillus, and actinomycetes.
Preferably, the viruses include, but are not limited to: coronavirus, adenovirus, influenza virus, cytomegalovirus, herpes simplex virus.
The lung disease is lung cancer and/or lung infection.
Specifically, the lung disease includes one or both of lung cancer and lung infection.
Specifically, the application is to detect a chromosome instability region and a microbial genome through second-generation sequencing.
In another aspect, the invention provides the use of a chromosome instability region and a pathogenic microorganism genome in the manufacture of a medicament for the treatment or co-treatment of a pulmonary disease.
The chromosome region includes: 2p, 3q, 6p, 7p, 8q, 9p, 12p, 17q, 18q, 20q, 21p, 21 q; the genome of the pathogenic microorganism comprises the genome of any one or more of bacteria, fungi and DNA viruses.
Specifically, the chromosomal instability includes a deletion or amplification of a copy of the entire chromosome or a fragment of the chromosome.
The lung disease is lung cancer and/or lung infection.
Specifically, the lung disease includes one or both of lung cancer and lung infection.
Specifically, the application is to detect a chromosome instability region and a microbial genome through second-generation sequencing.
In yet another aspect, the invention provides a kit for the detection, screening, diagnosis, prognostic assessment or monitoring of a pulmonary disease.
The kit is used for detecting through second-generation sequencing.
Specifically, the kit includes but is not limited to: the kit comprises a kit for diagnosing lung cancer and lung infection, a kit for screening early lung cancer and lung infection, a kit for monitoring lung cancer and lung infection conditions, and a kit for evaluating prognosis of lung cancer and lung infection.
The kit comprises reagents for detecting chromosome unstable regions and pathogenic microorganism genomes; the chromosome instability region comprises: 2p, 3q, 6p, 7p, 8q, 9p, 12p, 17q, 18q, 20q, 21p, 21 q; the genome of the pathogenic microorganism comprises the genome of any one or more of bacteria, fungi and DNA viruses.
Specifically, the reagent or the kit further comprises: one or more of a positive reference, a negative reference, a buffer, an enzyme, a library adaptor, a detectable label, a nucleic acid extraction reagent, and a nucleic acid purification reagent.
Further specifically, the enzyme comprises one or more of a disrupting enzyme, a DNA polymerase, a DNA ligase.
More specifically, the positive reference is a cell line mixed with the 14 regional variations and a genome mixed with the related pathogens; the negative reference product is a cell line without chromosome variation and pathogen genome.
Specifically, the kit further comprises a sequencing primer, wherein the sequencing primer comprises a P7 end tag primer and a P5 end tag primer.
Preferably, the P7 terminal tag primer is selected from one or more of SEQ ID NO.1-8, and the P5 terminal tag primer is selected from one or more of SEQ ID NO. 9-16.
Alternatively, the kit can detect the result by the following method: digital PCR, in situ fluorescent hybridization, nucleic acid probe hybridization, and the like.
Optionally, the kit further comprises other reagents clinically used for detection, screening, diagnosis, prognosis evaluation or disease monitoring of lung cancer and detection and diagnosis of lung infection to assist or verify the results obtained by detecting the 14 chromosome regions.
Preferably, the clinical samples detectable by the reagent or kit include, but are not limited to: alveolar lavage (irrigation fluid), pleural effusion (pleural effusion), etc.
In still another aspect, the present invention also provides a method for operating the aforementioned kit related to lung diseases.
Specifically, the operation method comprises the following steps:
(1) obtaining a sample to be detected from a detection object;
(2) contacting a sample to be detected with a detection reagent;
(3) detecting the aforementioned 14 chromosomal regions and the genome of the pathogenic microorganism;
(4) and detecting, screening, diagnosing, prognostically evaluating or monitoring the condition of the lung cancer and detecting and diagnosing the lung infection according to the detection result.
The invention has the beneficial effects that:
the invention collects the alveolar lavage fluid (flushing fluid) and pleural effusion (pleural effusion) samples of clinically confirmed lung cancer patients, lung infection patients and healthy people, extracts the genome DNA of human source and microorganism, and performs whole genome sequencing, thereby comparing the chromosome abnormal conditions of tumor patients and contrast, and the microbial genome conditions of infected patients and healthy people. The specific chromosome instability distribution characteristics of the lung cancer are explained, 14 common chromosome instability regions of the lung cancer patient are disclosed, and the total carrying rate is up to 83.9%; and simultaneously, the infection source of a patient is detected, the consistency with the clinical culture result reaches 90%, and the method has very high substitutability, has great significance for detection, screening, diagnosis, prognosis evaluation or disease condition monitoring of clinical lung cancer and detection and diagnosis of lung infection, and provides scientific basis for the next step of early diagnosis and the establishment of an individualized treatment scheme.
Drawings
FIG. 1 is a graph showing the results of analysis of instability of 3q +, 7p +, and 8q + chromosomes; wherein A is 3q +, B is 7p +, and C is 8q +.
FIG. 2 is a graph showing the results of analysis of instability of 9 p-chromosome.
FIG. 3 is a graph showing the results of analysis of instability of 17p-, 17q + chromosomes; wherein A is 17 p-and B is 17q +.
FIG. 4 is a ROC plot of 14 chromosomal regions and pathogen detection according to the invention, where AUC is the area under the ROC curve.
It should be noted that fig. 1-3 are diagrams illustrating the analysis software, and the detection result can be determined under the current conditions.
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 alveolar lavage fluid extraction of genomic DNA from human and microorganisms
The kit used in this example was purchased from QIAGEN (cat # 80204).
a. The alveolar lavage fluid is transferred to a 50mL centrifuge tube, 1 XPBS buffer solution with the same volume is added, the mixture is shaken and mixed evenly, centrifugation is carried out for 10min at 1600g, the supernatant is carefully poured off, and the rest supernatant is carefully discarded by 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 taken and packed in a 2mL collection tube. Add 500. mu.L Buffer DW1 to the column and let stand for 2 min. 10000g for 30-60 s.
e. The effluent is decanted and the column is returned to the collection tube. Add 500. mu.L of Buffer RW2 to the column. 10000g for 30-60 s.
f. The effluent is decanted and the column is returned to the collection tube. Add 500. mu.L of Buffer RW2 to the column. 10000g for 30-60 s.
g. The effluent is decanted and the column is returned to the empty collection tube. 13000g for 2 min.
h. The DNA column was loaded into 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.
i. The DNA column was discarded and the DNA was stored at 2-8 ℃ or-20 ℃.
Example 2 detection of chromosomal instability regions and pathogenic microorganism DNA
The related library building kit in the embodiment is purchased from NEB company, and has a cargo number: E7645S; magnetic beads were purchased from Beckman corporation, cat # s: A63882.
the method comprises the following steps:
1. genome fragmentation: 20ng of the human/microorganism genomic DNA prepared in example 1 was used to prepare an enzyme digestion reaction system shown in Table 1, and the reaction was carried out according to the procedure shown in Table 2. In this example, the concentration of human genomic DNA was 2 ng/. mu.L, and 10. mu.L was added to the reaction system.
If the human genome DNA is cell-free DNA, such as cfDNA in blood, genome fragmentation is not needed, and the next operation is directly carried out.
TABLE 1
DNA cleavage reaction System of
Genomic DNA 10μL
Breaking enzymes 3μL
Buffer 7μL
Non-nucleic acid water Make up to 35 mu L
After shaking, mixing and centrifugation (avoiding air bubbles), the following procedure was run on the PCR instrument:
TABLE 2
Figure BDA0003519389310000071
2. And (3) joint connection reaction: and sucking a proper volume of the adaptor connection premix and the adaptor connection reinforcing agent according to the number of the detected samples, mixing, sucking 15.5 mu L of the mixed solution, adding the mixed solution into the reaction mixed solution for the terminal treatment in the previous step, sucking 1.5 mu L of the mixed adaptor prepared in the embodiment 1 after oscillation, uniform mixing and centrifugation, adding the mixed adaptor into the reaction solution, oscillation, uniform mixing and centrifugation. The reaction system in table 3 below was finally formed.
TABLE 3
Linker ligation reactions System of
Mixed solution for end repairing reaction (last step) 35μL
Joint connection premix liquid 15μL
Joint connection enhancer 0.5μL
Hybrid joint 1.5μL
General System 52μL
The system is placed in a PCR instrument, and the program is run: at 20 deg.C, 30min, the hot lid was closed.
3. A purification step after the ligation reaction:
a. and taking the library purified magnetic beads out of the magnetic bead kit 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 46 mu L of the resuspended library purification magnetic beads, uniformly sucking and beating for 20 times by using a pipettor with a proper range, and incubating 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, carrying out instantaneous centrifugation for 3sec, putting the centrifugal tube back on the magnetic frame, and removing the residual 80% ethanol by suction, taking care not to absorb the magnetic beads. Opening the tube cover, and air drying at room temperature for 2-10 min.
g. When the beads became sub-bright, 16. mu.L of Low TE buffer (or nuclease-free water) was added to the tube, the beads were resuspended by gentle shaking, and incubated at room temperature for 5 min.
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.
4. And (3) PCR amplification: the corresponding reagents were added to the PCR tubes as in table 4 below:
TABLE 4
PCR reaction System of
Linker connecting purified product 15μL
PCR amplification enzyme premix 25μL
PCR amplification universal primer 5μL
PCR amplification label primer 5μL
General System 50μL
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 5
Figure BDA0003519389310000081
Figure BDA0003519389310000091
The P7 end-tag primer is selected from SEQ ID NO.1-8, the P5 end-tag primer is selected from SEQ ID NO.9-16, and any combination can achieve the detection effect. In this embodiment, P7-01 and P5-01 are selected.
The mixed PCR tube was placed in a PCR instrument and the following procedure was run:
TABLE 6
Figure BDA0003519389310000092
5. PCR product purification reference step 3 purification step, wherein:
the amount of the resuspended library purification magnetic beads in step b was 22.5. 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.
6. Library quantitative processing machine
The purified library was analyzed for fragment size using an Agilent BioAnalyzer biochip analysis System
Figure BDA0003519389310000093
The HS Assay Kit measures the library mass concentration, calculates the library molarity from mass concentration and fragment size, and performs sequencing using Illumina Hiseq X-ten according to the sequencer instructions.
Results analysis discussion:
the on-machine sequencing data is subjected to the data analysis method to obtain a chromosome sequencing depth distribution map, as shown in FIGS. 1-3.
The analysis results consisted of two parts:
some are chromosome variation, and the results include chromosome number, chromosome partition and chromosome sequencing depth distribution. 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 as a tumor if the chromosome instability occurs or judging the chromosome instability as a non-tumor if the chromosome instability does not occur.
The other part is the microorganism detection situation. The output result comprises the species of the microorganism and the corresponding sequencing supported Reads number, and whether the infection occurs or not and which microorganism is caused by the infection are judged by detecting the pathogenicity of the microorganism and detecting the Reads number. The results are shown in Table 7.
TABLE 7 examples of results of detection of pathogenic microorganisms
Figure BDA0003519389310000101
FIG. 4 is a ROC plot of 14 chromosomal regions and pathogen detection according to the invention, where AUC is the area under the ROC curve.
Example 3 assay sample validation
116 clinical samples (56 lung cancer patients and 60 infected patients) and 42 healthy samples were selected and tested according to the following test results in reference example 1 and example 2: 47 cases of patients with tumor with chromosome copy number variation, 54 cases of infected patients with pathogenic microorganisms consistent with the culture results, 5 cases of infected patients with chromosome abnormality, 1 case of healthy people with chromosome abnormality, and 5 cases of infected patients with pathogenic microorganisms. The results show that: the invention has the advantages that the lung cancer detection sensitivity is 83.9 percent, and the specificity is 94.1 percent; the kit has a correct detection rate of 90% and a specificity of 88% for detecting the lung pathogenic microorganism infection. In conclusion, the kit can be well used for detecting lung cancer and lung pathogenic infection, and provides support for clinical diagnosis.
Comparative example
The following comparative examples were set up with reference to example 1 and example 2:
Figure BDA0003519389310000111
the comparative examples show that when the detection unstable region is reduced (comparative examples 1 and 2), the detection specificity is not obviously changed compared with the invention, but the sensitivity is obviously reduced, and the clinical use significance is obviously reduced; when the detection unstable region is increased (comparative example 3), neither the sensitivity nor the specificity is significantly changed, and there is no significant change from the present invention, but the detection cost needs to be increased. Therefore, the chromosome abnormality combination can ensure the clinical detection performance, does not remarkably increase the detection cost, and is the current optimal detection combination.
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Claims (10)

1. Use of a chromosome instability region and a genome of a pathogenic microorganism in the manufacture of a reagent or kit for the detection, screening, diagnosis, prognosis or monitoring of a pulmonary disease, wherein the chromosome instability region comprises: 2p, 3q, 6p, 7p, 8q, 9p, 12p, 17q, 18q, 20q, 21p, 21 q; the genome of the pathogenic microorganism comprises the genome of any one or more of bacteria, fungi and viruses.
2. Use of a chromosomal instability region and a genome of a pathogenic microorganism in the manufacture of a medicament for the treatment or co-treatment of a pulmonary disease, wherein the chromosomal region comprises: 2p, 3q, 6p, 7p, 8q, 9p, 12p, 17q, 18q, 20q, 21p, 21 q; the genome of the pathogenic microorganism comprises the genome of any one or more of bacteria, fungi and viruses.
3. Use according to any one of claims 1 to 2, characterized in that the detection of the chromosomal instability regions and the genome of the microorganism is carried out by means of two-generation sequencing.
4. A kit for detection, screening, diagnosis, prognosis evaluation or disease monitoring of a pulmonary disease, comprising reagents for detecting a chromosomal instability region and a pathogenic microorganism genome; the chromosome instability region comprises: 2p, 3q, 6p, 7p, 8q, 9p, 12p, 17q, 18q, 20q, 21p, 21 q; the genome of the pathogenic microorganism comprises the genome of any one or more of bacteria, fungi and viruses.
5. The kit of claim 4, wherein the kit is detected by secondary sequencing.
6. The kit of claim 4, further comprising one or more of a positive reference, a negative reference, a buffer, an enzyme, a library adaptor, a nucleic acid extraction reagent, a nucleic acid purification reagent.
7. The reagent or kit of claim 6, wherein the enzyme comprises one or more of a disrupting enzyme, a DNA polymerase, and a DNA ligase.
8. The reagent or kit according to claim 6, wherein the positive reference is a cell line mixed with 14 chromosomal instability region variants according to claim 1 and a cell line mixed with the genome of a pathogenic microorganism according to claim 1; the negative reference product is a cell line without chromosome variation and pathogen genome.
9. The kit of claim 6, further comprising a sequencing primer, wherein the sequencing primer comprises a P7 end-tag primer and a P5 end-tag primer.
10. The kit of claim 9, wherein the P7 end-tag primer is selected from one or more of SEQ ID nos. 1-8 and the P5 end-tag primer is selected from one or more of SEQ ID nos. 9-16.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013005107A2 (en) * 2011-07-05 2013-01-10 National Taiwan University Method for predicting response or prognosis of lung adenocarcinoma with egfr-activating mutations
CN103403182A (en) * 2010-11-30 2013-11-20 香港中文大学 Detection of genetic or molecular aberrations associated with cancer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103403182A (en) * 2010-11-30 2013-11-20 香港中文大学 Detection of genetic or molecular aberrations associated with cancer
WO2013005107A2 (en) * 2011-07-05 2013-01-10 National Taiwan University Method for predicting response or prognosis of lung adenocarcinoma with egfr-activating mutations

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KAROLINA H CZARNECKA等: "Allelic imbalance in 1p, 7q, 9p, 11p, 12q and 16q regions in non-small cell lung carcinoma and its clinical association: a pilot study", MOL BIOL REP, vol. 40, no. 12, 31 December 2013 (2013-12-31), pages 6671 - 6684 *
冯倩等: "基因组拷贝数变异与肺癌关系的研究进展", 国际检验医学杂志, vol. 38, no. 12, 30 June 2017 (2017-06-30), pages 1641 - 1644 *
童明庆: "临床检验诊断学", 31 October 2002, 东南大学出版社, pages: 196 - 197 *
蔡东焱等: "应用比较基因组杂交技术分析肺癌染色体异常", 中国癌症杂志, no. 06, 15 June 2008 (2008-06-15), pages 436 - 441 *

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