CN110747193A - Lung cancer polygene joint detection kit quality control product based on second-generation sequencing platform - Google Patents
Lung cancer polygene joint detection kit quality control product based on second-generation sequencing platform Download PDFInfo
- Publication number
- CN110747193A CN110747193A CN201910911924.XA CN201910911924A CN110747193A CN 110747193 A CN110747193 A CN 110747193A CN 201910911924 A CN201910911924 A CN 201910911924A CN 110747193 A CN110747193 A CN 110747193A
- Authority
- CN
- China
- Prior art keywords
- lung cancer
- quality control
- control product
- dna
- rna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a lung cancer polygene joint detection kit quality control product based on a second-generation sequencing platform, which comprises one or more of a lung cancer positive quality control product, a lung cancer negative quality control product, a lung cancer fusion positive quality control product and a lung cancer fusion negative quality control product; the positive quality control product for the lung cancer comprises a plasmid with target gene mutation, wild cell strain DNA and a DNA stabilizer. The DNA/RNA stabilizer contained in the lung cancer quality control product provided by the invention can effectively prevent the degradation of free DNA/RNA, provide a stable environment for the DNA/RNA and prolong the storage time of the lung cancer quality control product; the quality control product provided by the invention has the advantages of low preparation cost, stable performance, easiness in storage and the like, can effectively monitor factors such as personnel operation, instrument state, kit effectiveness and the like, and greatly improves the accuracy and reliability of the detection kit.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a quality control product of a lung cancer polygene joint detection kit based on a second-generation sequencing platform.
Background
Lung Cancer is the Cancer with the highest morbidity and mortality in our country, with Non-Small cell lung Cancer (NSCLC) accounting for approximately 80% of all lung cancers. The main reasons for the poor chemotherapy effect and failure of chemotherapy in some patients in clinic are that tumor cells are not sensitive to anticancer drugs or generate drug resistance. Therefore, it is important to understand the relevant factors of the susceptibility of the patients to the chemotherapeutic drugs and to perform individual treatment on the patients with lung cancer.
The EGFR gene is located at 7p12 and encodes a tyrosine kinase type receptor. The incidence of EGFR gene mutations reaches 50% in asian populations and is higher in non-smokers, women and non-mucinous tumors. EGFR gene mutation discovered so far is mainly located in exons 18-21; the KRAS gene is located at 12p12.1 and is a common oncogene, KRAS mutation is most common in smokers and mucinous lung adenocarcinoma, and KRAS mutation is mainly caused in 30 percent of lung adenocarcinoma and is common in codons of 12, 13 and 61; the BRAF gene is located at 7q34 and is a protooncogene. The BRAF mutation rate in the non-small cell lung cancer is about 3 percent, most of the BRAF mutation rates are lung adenocarcinoma, and 80 to 90 percent of the BRAF mutation rates are V600E mutations; the ERBB2 gene is located in chromosome 17q12, can activate downstream PI3K-AKT and MEK-ERK signaling pathways, and regulates cell proliferation, differentiation and other processes, and the mutation is mainly located on 19 th and 20 th exons of the ERBB2 gene. The ERBB2 mutation rate in NSCLC patients is 2-4%.
The ALK gene is located in the p23 band (2p23) of chromosome 2 and belongs to transmembrane receptor tyrosine kinase. The positive rate of adenocarcinoma ALK of Chinese population is 5.1%. The positive rate of ALK fusion gene in EGFR and KRAS wild adenocarcinoma patients in China is as high as 30-42%; the RET gene is located in the q11.2 region of chromosome 10, is a protooncogene, and when the RET gene is fused with genes such as KIF5B, CCDC6, NCOA4 and the like, the RET tyrosine kinase region and downstream signal pathways such as RAS/ERK, PI3K/AKT, MAPK/JNK and the like are continuously activated, so that the occurrence of tumors is caused. RET gene rearrangement is a newly discovered lung adenocarcinoma driving gene, and the mutation frequency is about 1 percent; the ROS1 gene is located in chromosome 6, region q21, and encodes a receptor tyrosine kinase. When fused with genes such as SLC34A2, CD74 and the like, the fusion protein can continuously activate a ROS1 tyrosine kinase region and signal channels such as downstream JAK/STAT, PI3K/AKT, RAS/MAPK and the like, thereby further causing the occurrence of tumors. The incidence rate of ROS1 gene fusion in the non-small cell lung cancer is about 2-4%;
the update of NCCN clinical guidelines confirms the clinical requirements that NSCLC should detect the three gene states of EGFR, ALK and ROS1 before treatment. Furthermore, the Chinese guidelines for the diagnosis and treatment of progressive lymphoma kinase (ALK) positive non-small cell lung cancer also suggest that ROS1, ALK and EGFR be detected simultaneously, with patient's permission.
The current methods for detecting lung cancer mutant genes mainly comprise 2 types: fluorescent quantitative PCR method, Sanger sequencing method. The fluorescence quantitative PCR method has the advantages that only one mutation can be detected by each pair of primers, the operation for detecting multiple mutation sites is complicated, the sample size is large, and false positive is easy to occur; the Sanger sequencing method can only sequence a certain section of area of one sample at a time, and has the advantages of low detection efficiency, high detection cost and high false negative rate.
At present, no quality control product which is simple to operate and is used for a high-throughput sequencing (NGS) lung cancer polygene combined detection sequencing kit is sold in the market, and the quality control product is particularly easy to degrade if the quality control product is not properly stored; the compact quality control product is an important method and quality index for evaluating and verifying the detection accuracy, stability and other performances of the kit. Therefore, the quality control product of the multi-gene combined detection kit for stably preserving the lung cancer is urgently needed in the field.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a quality control product of a lung cancer polygene combined detection kit based on a second-generation sequencing platform.
The first object of the present invention is to provide a DNA stabilizer.
The second purpose of the invention is to provide the application of any one of the DNA stabilizing agents in DNA preservation.
The third purpose of the invention is to provide an application of any one of the DNA stabilizing agents in preparing a quality control product of a multi-gene combined detection kit for lung cancer.
The fourth purpose of the invention is to provide an RNA stabilizer.
The fifth purpose of the invention is to provide the application of any RNA stabilizer in RNA preservation.
The sixth purpose of the invention is to provide an application of any RNA stabilizer in preparation of a quality control product of a multi-gene combined detection kit for lung cancer.
The seventh purpose of the invention is to provide a quality control product for the combined detection of multiple genes of lung cancer.
The eighth purpose of the invention is to provide a preparation method of the quality control product of the multi-gene combined detection kit for the lung cancer based on the DR-Seq800 gene sequencing platform.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the lung cancer polygene joint detection sequencer kit provided by the invention can be used for simultaneously detecting EGFR, KRAS, BRAF, ERBB2, ALK, ROS1 and RET genes by adopting a high throughput sequencing method (NGS), so that the demand of a sample is greatly reduced and the detection cost is reduced; meanwhile, the sensitivity and the accuracy of detection can be improved.
The invention claims a DNA stabilizer, which contains EDTA-3K, aurin tricarboxylate, glycine and betaine hydrochloride.
Preferably, the DNA stabilizer contains 70-100 g/L of EDTA-3K, 800-1000 g/L of aurin tricarboxylate, 80-100 mg/L of glycine and 8-11 mg/L of betaine hydrochloride.
More preferably, the DNA stabilizer contains 80g/L of EDTA-3K, 900g/L of aurin tricarboxylate, 90mg/L of glycine and 10mg/L of betaine hydrochloride.
The use of any one of the DNA stabilizers in DNA preservation also belongs to the protection scope of the invention.
The application of any DNA stabilizer in the preparation of the quality control product of the multi-gene combined detection kit for lung cancer also belongs to the protection scope of the invention.
An RNA stabilizer comprises EDTA-3K, aurin tricarboxylate, serine inhibitor and betaine hydrochloride.
Preferably, the RNA stabilizer contains 50-80 g/L of EDTA-3K, 600-900 g/L of aurin tricarboxylate, 30-50 mg/L of serine inhibitor and 12-16 mg/L of betaine hydrochloride.
More preferably, the RNA stabilizer contains 60g/L of EDTA-3K, 800g/L of aurin tricarboxylate, 40mg/L of serine inhibitor and 15mg/L of betaine hydrochloride.
The application of any RNA stabilizer in RNA preservation also belongs to the protection scope of the invention.
The application of any RNA stabilizer in the preparation of the quality control product of the multi-gene combined detection kit for lung cancer also belongs to the protection scope of the invention.
A quality control product for multi-gene combined detection of lung cancer comprises one or more of lung cancer positive quality control product, lung cancer negative quality control product, lung cancer fusion positive quality control product, and lung cancer fusion negative quality control product;
the positive quality control material for the lung cancer comprises a plasmid with target gene mutation, wild cell strain DNA and a DNA stabilizer;
the lung cancer negative quality control product comprises DNA without target gene mutation and a DNA stabilizer;
the lung cancer fusion positive quality control product comprises RNA fused with a target gene and an RNA stabilizer;
the lung cancer fusion negative quality control product comprises RNA of a target gene which is not fused and an RNA stabilizer;
the target gene is at least one of EGFR, KRAS, BRAF, ERBB2, ALK, ROS1 and RET gene quality control products.
The invention also claims a preparation method of the multi-gene joint detector box quality control product based on the DR-Seq800 gene sequencing platform for lung cancer, which comprises the steps of preparing a lung cancer positive quality control product, preparing a lung cancer negative quality control product, preparing a lung cancer fusion positive quality control product and/or preparing a lung cancer fusion negative quality control product;
wherein, the preparation of the positive quality control product of the lung cancer comprises the following steps: designing a target gene mutation plasmid; mixing the target gene mutation plasmid and the DNA of the wild cell strain according to a proportion; adding the DNA stabilizer into the mixed DNA solution;
preparing a lung cancer negative quality control product: selecting lung cancer tissue sample DNA which is identified as the target gene and does not generate mutation; adding the DNA stabilizer into a DNA sample;
preparing a lung cancer fusion positive quality control product: selecting the lung cancer tissue sample RNA identified as the target gene with fusion mutation; adding the RNA stabilizer into the RNA sample;
preparing a lung cancer fusion negative quality control product: selecting the lung cancer tissue sample RNA identified as the target gene without fusion mutation; and adding the RNA stabilizing agent into the RNA sample.
Preferably, the volume ratio of the DNA sample to the DNA stabilizer is 25-35: 1.
more preferably, the ratio of the amount of the DNA sample to the amount of the DNA stabilizer is 30: 1.
preferably, the volume ratio of the RNA sample to the RNA stabilizer is 15-25: 1.
more preferably, the ratio of the amount of RNA sample to RNA stabilizer is 20: 1.
compared with the prior art, the invention has the following beneficial effects:
the quality control product of the multi-gene joint detection kit capable of stably storing the lung cancer, which is provided by the invention and is based on the second-generation sequencing platform, particularly the DR-Seq800 gene sequencing platform of Darriy biotechnology GmbH in Guangzhou, aims to ensure the accuracy, stability and reliability of the multi-gene joint detection kit for the lung cancer.
Compared with the prior art, the invention has the advantages that:
(1) the DNA stabilizer contained in the lung cancer quality control product provided by the invention can effectively prevent the degradation of free DNA, provide a stable environment for the DNA and prolong the storage time of the lung cancer quality control product;
(2) the RNA stabilizer contained in the lung cancer fusion quality control product provided by the invention can effectively prevent the degradation of free RNA, provide a stable environment for RNA, and prolong the storage time of the lung cancer fusion quality control product;
(3) the DNA stabilizer provided by the invention has the advantages that the components and the proportion of the used components are obtained by analyzing a large number of experiments and statistical results, and the free DNA is well protected;
(4) the RNA stabilizer provided by the invention has the advantages that the components and the proportion of the used components are obtained by analyzing a large number of experiments and statistical results, and the free RNA can be well protected;
(5) the use proportion of the DNA stabilizer in the lung cancer quality control product is obtained by analyzing a large number of experiments and statistical results of the inventor, so that the lung cancer quality control product has good stability;
(6) the application proportion of the RNA stabilizer in the lung cancer fusion quality control product is obtained by analyzing a large number of experiments and statistical results of the inventor, so that the lung cancer fusion quality control product has good stability;
(7) the DNA quality control product provided by the invention has the advantages of low preparation cost, stable performance, easiness in storage and the like, can effectively monitor factors such as personnel operation, instrument state, kit effectiveness and the like, and greatly improves the accuracy and reliability of the detection kit.
Detailed Description
The present invention is further described in detail in the following description and specific examples, which are provided for the purpose of illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1A DNA stabilizer
Comprises the following components: 80g/L of EDTA-3K, 900g/L of aurin tricarboxylate, 90mg/L of glycine and 10mg/L of betaine hydrochloride.
Example 2A DNA stabilizer
Comprises the following components: 70g/L of EDTA-3K, 800g/L of aurin tricarboxylate, 80mg/L of glycine and 8mg/L of betaine hydrochloride.
Example 3A DNA stabilizer
Comprises the following components: 100g/L of EDTA-3K, 1000g/L of aurin tricarboxylate, 100mg/L of glycine and 11mg/L of betaine hydrochloride.
Example 4 an RNA stabilizer
Contains 60g/L of EDTA-3K, 800g/L of aurin tricarboxylate, 40mg/L of serine inhibitor and 15mg/L of betaine hydrochloride.
Example 5 an RNA stabilizer
Contains 50g/L of EDTA-3K, 600g/L of aurin tricarboxylate, 30mg/L of serine inhibitor and 12mg/L of betaine hydrochloride.
Example 6 an RNA stabilizer
Contains 80g/L of EDTA-3K, 900g/L of aurin tricarboxylate, 50mg/L of serine inhibitor and 16mg/L of betaine hydrochloride.
Example 7 protective Effect of DNA stabilizers on free DNA
First, experiment method
6 DNA samples which are proved to be wild by the lung cancer polygene joint detection kit are extracted and respectively subjected to concentration determination, and the respective concentrations are as follows: sample 1: 23.56 ng/. mu.L, sample 2: 26.78 ng/. mu.L, sample 3: 20.12 ng/. mu.L, sample 4: 18.65 ng/. mu.L, sample 5: 25.72 ng/. mu.L, sample 6: 19.89 ng/. mu.L. Then dividing each sample into 6 equal parts, carrying out experimental design according to the table 1, adding or not adding the DNA stabilizer in the embodiment 1, and preserving for a certain time, wherein freezing and thawing or not are carried out during the period; after 1 month, the DNA sample degradation was known by measuring the DNA concentration of each group of samples with an Agilent 2100 bioanalyzer. Meanwhile, the degradation rate is calculated by the following formula: percent degradation ═ DNA concentration before experiment-DNA concentration after experiment)/DNA concentration before experiment × 100%.
The volume ratio of the sample to the DNA stabilizer is 30: 1.
table 1:
second, experimental results
After 1 month, the concentration of 18 experimental groups and 18 control groups was measured, and the specific results are shown in table 2 below.
Table 2:
according to the experimental results, the DNA degradation rate of the DNA sample added with the DNA stabilizer is obviously lower than that of the DNA sample without the DNA stabilizer under the same conditions; therefore, it can be seen that the DNA stabilizer of the present invention can effectively protect DNA from degradation.
Example 8 protective Effect of RNA stabilizers on free RNA
First, experiment method
6 RNA samples which are proved to be wild type by the lung cancer polygene joint detection kit are extracted and respectively subjected to concentration determination, and the respective concentrations are as follows: sample 1: 24.45 ng/. mu.L, sample 2: 28.34 ng/. mu.L, sample 3: 23.62 ng/. mu.L, sample 4: 25.77 ng/. mu.L, sample 5: 24.21 ng/. mu.L, sample 6: 23.91 ng/. mu.L. Then dividing each sample into 6 equal parts, carrying out experimental design according to the table 3, adding or not adding the RNA stabilizer in the embodiment 4, and preserving for a certain time, wherein freezing and thawing or not freezing and thawing are carried out; after 2 weeks, the RNA concentrations of the samples of each group were measured by an Agilent 2100 bioanalyzer, thereby resolving the RNA sample degradation. Meanwhile, the degradation rate is calculated by the following formula: percent degradation ═ RNA concentration before experiment-RNA concentration after experiment)/RNA concentration before experiment × 100%.
Table 3:
second, experimental results
After 2 weeks, the concentration of 18 experimental groups and 18 control groups was measured, and the results are shown in table 4 below.
Table 4:
according to the experimental results, the RNA degradation rate of the RNA sample added with the RNA stabilizer is obviously lower than that of the RNA sample without the RNA stabilizer under the same conditions; therefore, it can be seen that the RNA stabilizer of the present invention can effectively protect RNA from degradation.
Example 9 DR-Seq800 Gene sequencing-based platform Multi-Gene Joint detection kit quality control product for Lung cancer
A, make up
Contains lung cancer positive quality control material, lung cancer negative quality control material, lung cancer fusion positive quality control material and lung cancer fusion negative quality control material;
the positive quality control material for the lung cancer comprises a plasmid with target gene mutation, wild cell strain DNA and a DNA stabilizer;
the lung cancer negative quality control product comprises DNA without target gene mutation and a DNA stabilizer;
the lung cancer fusion positive quality control product comprises RNA fused with a target gene and an RNA stabilizer;
the lung cancer fusion negative quality control product comprises RNA of a target gene which is not fused and an RNA stabilizer;
the target gene is at least one of EGFR, KRAS, BRAF, ERBB2, ALK, ROS1 and RET gene quality control products.
The DNA stabilizer contains 80g/L of EDTA-3K, 900g/L of aurin tricarboxylate, 90mg/L of glycine and 10mg/L of betaine hydrochloride.
The RNA stabilizer contains 60g/L of EDTA-3K, 800g/L of aurin tricarboxylate, 40mg/L of serine inhibitor and 15mg/L of betaine hydrochloride.
Second, preparation method
Preparing a lung cancer positive quality control product: designing a target gene mutation plasmid; mixing the target gene mutation plasmid and the DNA of the wild cell strain according to a proportion; adding the DNA stabilizer into the mixed DNA solution;
preparing a lung cancer negative quality control product: selecting lung cancer tissue sample DNA which is identified as the target gene and does not generate mutation; adding the DNA stabilizer into a DNA sample;
preparing a lung cancer fusion positive quality control product: selecting the lung cancer tissue sample RNA identified as the target gene with fusion mutation; adding the RNA stabilizer into the RNA sample;
preparing a lung cancer fusion negative quality control product: selecting the lung cancer tissue sample RNA identified as the target gene without fusion mutation; and adding the RNA stabilizing agent into the RNA sample.
Wherein the volume ratio of the DNA sample to the DNA stabilizer is 30: 1; the volume ratio of the RNA sample to the RNA stabilizer is 20: 1.
third, using method
And (3) operating the quality control products according to the specifications of the multi-gene combined detection kit for the lung cancer, and then performing on-machine sequencing by using a DR-Seq800 gene sequencing platform to obtain the result of each quality control product. The detection result of the lung cancer positive quality control product is that mutation occurs, and the lung cancer negative quality control product is wild type; the positive quality control product of lung cancer fusion should be fused, and the negative quality control product of lung cancer fusion should not be fused.
Example 10 quality control stability and accuracy
First, experiment method
Experimental groups: three batches of the quality control product of example 9 were produced, and 1 box was randomly selected from each batch and labeled as test group 1, test group 2, and test group 3.
Control group: three batches of quality control products of the lung cancer polygene combined detection kit without the stabilizer added in the embodiment 1 and the embodiment 2 are produced, 1 box of quality control products is randomly extracted from each batch, and the quality control products are respectively marked as a control group 1, a control group 2 and a control group 3.
Then, according to the operation of the specifications of the multi-gene combined detection kit for lung cancer, sequencing 3 experimental groups and 3 control groups respectively by using a DR-Seq800 gene sequencing platform, and recording the first sequencing result; and finally, sequencing after 3 months, 6 months and 9 months according to the operation, and recording sequencing results. The specific experimental protocol is shown in table 5 below.
Table 5:
| experimental group 1 | 0 month sequencing results | 3 month sequencing results | 6 month sequencing results | 9 month sequencing results |
| Experimental group 2 | 0 month sequencing results | 3 month sequencing results | 6 month sequencing results | 9 month sequencing results |
| Experimental group 3 | 0 month sequencing results | 3 month sequencing results | 6 month sequencing results | 9 month sequencing results |
| Control group 1 | 0 month sequencing results | 3 month sequencing results | 6 month sequencing results | 9 month sequencing results |
| Control group 2 | 0 month sequencing results | 3 month sequencing results | 6 month sequencing results | 9 month sequencing results |
| Control group 3 | 0 month sequencing results | 3 month sequencing results | 6 month sequencing results | 9 month sequencing results |
Second, experimental results
According to the design of the experimental scheme, the experimental results of 0 month, 3 months, 6 months and 9 months are shown in the following table 6.
Table 6:
according to the experimental results, the quality control products of the lung cancer polygene combined detection kit added with the stabilizing agents in the embodiment 1 and the embodiment 2 are consistent in the sequencing results of DR-Seq800 gene sequencing platforms at 0 month, 3 months, 6 months and 9 months, and all meet the specification requirements of the lung cancer polygene combined detection kit; the quality control product of the multi-gene combined detection kit for lung cancer, which is not added with the stabilizing agent described in the embodiment 1 and the embodiment 2, starts to fail in sequencing at 6 months, and fails in 3 batches after 9 months; therefore, the DNA stabilizer and the RNA stabilizer can be applied to the quality control product of the multi-gene combined detection kit for the lung cancer, and simultaneously, the stability and the accuracy of the quality control product of the multi-gene combined detection kit for the lung cancer are improved.
EXAMPLE 11 protective Effect of DNA stabilizer component on free DNA
First, experiment method
The DNA sample which is proved to be wild by the lung cancer polygene joint detection kit is extracted in a large quantity, and then concentration determination is carried out, wherein the concentration is as follows: 25.16 ng/. mu.L. The sample was divided into 18 equal portions, and DNA stabilizers of different compositions were prepared according to table 7, with the sample and each group of DNA stabilizers being respectively used in a dose-to-volume ratio of 30: 1, and then respectively storing at-20 ℃, and after 1 month, respectively measuring the DNA concentration of each group of samples by an Agilent 2100 bioanalyzer so as to know the degradation condition of the DNA samples. Meanwhile, the degradation rate is calculated by the following formula: percent degradation ═ DNA concentration before experiment-DNA concentration after experiment)/DNA concentration before experiment × 100%.
Table 7:
second, experimental results
After 1 month, concentration tests were performed on 1 experimental group and 17 control groups, and the specific results are shown in table 8 below.
Table 8:
according to the experimental results, under the same conditions, the stability of the DNA can be obviously improved only when the components of the DNA stabilizer provided by the invention simultaneously comprise EDTA-3K, aurin tricarboxylate, glycine and betaine hydrochloride (experimental group 1), and the degradation of the components in the storage process is effectively prevented. The components of the DNA stabilizer of the invention are obtained by a great deal of research and statistical analysis of the inventor, and the effect of the invention can not be realized by lacking any one, two or three components (control groups 1-14), or replacing EDTA-3K with other EDTA (control group 15), or replacing glycine with other types of amino acid (control group 16).
EXAMPLE 12 Effect of DNA stabilizer component concentration on protection of free DNA
First, experiment method
The DNA sample which is proved to be wild by the lung cancer polygene joint detection kit is extracted in a large quantity, and then concentration determination is carried out, wherein the concentration is as follows: 24.67 ng/. mu.L. The sample was divided into 20 equal portions, and the experimental design was performed according to table 9, and stored at-20 ℃ for 1 month, and then the DNA concentrations of the samples of each group were measured by an Agilent 2100 bioanalyzer, thereby understanding the degradation of the DNA samples. Meanwhile, the degradation rate is calculated by the following formula: percent degradation ═ DNA concentration before experiment-DNA concentration after experiment)/DNA concentration before experiment × 100%.
Table 9:
second, experimental results
After 1 month, 11 experimental groups and 9 control groups were subjected to concentration measurement, and the specific results are shown in table 10 below.
Table 10:
according to experimental results, under the same conditions, the concentration ratio of EDTA-3K, aurin tricarboxylate, glycine and betaine hydrochloride in the DNA stabilizer provided by the invention is (70-100 g/L): (800-1000 g/L): (80-100 mg/L): (8-11 mg/L) (experimental groups 1-11) can effectively prevent degradation of free DNA in the preservation process, and meanwhile, the concentration ratio of EDTA-3K, aurin tricarboxylate, glycine and betaine hydrochloride is 80 g/L: 900 g/L: 90 mg/L: the effect is optimal when the concentration is 10mg/L (experimental group 1). The proportion of the components of the DNA stabilizer of the invention is obtained by a great deal of research and statistical analysis of the inventor, the preservation effect of the DNA stabilizer on free DNA can be maximized only by the use proportion within the scope of the invention, and the preservation effect of the DNA stabilizer on free DNA can be obviously reduced by increasing or decreasing the use proportion of the components without the scope of the invention (control groups 1-9). The results of experiments on the effect of the amount of other components not within the range of the ratio of the present invention on the preservation effect of DNA stabilizers were similar to those of the present example, and the detailed data were omitted.
EXAMPLE 13 protective Effect of RNA stabilizer component on free RNA
First, experiment method
Performing mass extraction on the RNA sample which is verified to be wild by the lung cancer polygene joint detection kit, and then performing concentration determination, wherein the concentration is as follows: 23.43 ng/. mu.L. The sample was divided into 18 equal portions, RNA stabilizers of different compositions were prepared according to table 11, and the ratio of the sample to each group of RNA stabilizers was 20: 1, respectively storing at-20 ℃, and after 1 month, respectively measuring the RNA concentration of each group of samples by an Agilent 2100 bioanalyzer, thereby solving the degradation condition of the RNA samples. Meanwhile, the degradation rate is calculated by the following formula: percent degradation ═ RNA concentration before experiment-RNA concentration after experiment)/RNA concentration before experiment × 100%.
TABLE 11
Second, experimental results
After 1 month, concentration tests were performed on 1 experimental group and 17 control groups, and the specific results are shown in table 12 below.
TABLE 12
According to the experimental results, under the same conditions, the RNA stabilizer provided by the invention can obviously improve the stability of DNA and effectively prevent the degradation of the DNA in the preservation process when the components simultaneously comprise EDTA-3K, aurin tricarboxylate, glycine and betaine hydrochloride (experimental group 1). The components of the RNA stabilizer of the invention are obtained by a great deal of research and statistical analysis of the inventor, and the effect of the invention can not be realized by lacking any one, two or three components (control groups 1-14), or replacing EDTA-3K with other EDTA (control group 15) and replacing glycine with other types of amino acid (control group 16).
EXAMPLE 14 protective Effect of RNA stabilizer component concentration on free RNA
First, experiment method
Performing mass extraction on the RNA sample which is verified to be wild by the lung cancer polygene joint detection kit, and then performing concentration determination, wherein the concentration is as follows: 23.56 ng/. mu.L. The sample was divided into 22 equal portions, experimental design was performed according to table 13, the samples were stored at-20 ℃ for 1 month, and after that, the RNA concentrations of the samples of each group were measured by an Agilent 2100 bioanalyzer, thereby resolving the degradation of the RNA samples. Meanwhile, the degradation rate is calculated by the following formula: percent degradation ═ RNA concentration before experiment-RNA concentration after experiment)/RNA concentration before experiment × 100%.
Watch 13
Second, experimental results
After 1 month, 13 experimental groups and 9 control groups were subjected to concentration measurement, and the specific results are shown in table 14 below.
TABLE 14
According to experimental results, under the same conditions, the concentration ratio of EDTA-3K, aurin tricarboxylate, glycine and betaine hydrochloride in the RNA stabilizer provided by the invention is (50-80 g/L): (600-900 g/L): (30-50 mg/L): (12-16 mg/L) (experimental group 1-13) can effectively prevent degradation of free RNA in the preservation process, and meanwhile, the concentration ratio of EDTA-3K, aurin tricarboxylate, glycine and betaine hydrochloride is 60 g/L: 800 g/L: 40 mg/L: 15mg/L (Experimental group 1) gave the best results. The proportion of the components of the RNA stabilizer is obtained by a great deal of research and statistical analysis of the inventor, the preservation effect of the RNA stabilizer on free RNA can be maximized only by using the proportion within the range of the invention, and the preservation effect of the RNA stabilizer on the free RNA can be obviously reduced by increasing or decreasing the proportion of the components without the range of the invention (control groups 1-9). The results of experiments on the effect of other components not in the range of the ratio of the present invention on the preservation effect of RNA stabilizers are similar to those of the present example, and the detailed data are omitted.
Claims (10)
1. A DNA stabilizer is characterized by comprising EDTA-3K, aurin tricarboxylate, glycine and betaine hydrochloride.
2. The DNA stabilizer according to claim 1, wherein the DNA stabilizer comprises 70-100 g/L of EDTA-3K, 800-1000 g/L of aurin tricarboxylate, 80-100 mg/L of glycine and 8-11 mg/L of betaine hydrochloride.
3. Use of the DNA stabilizer of any one of claims 1 to 2 for DNA preservation.
4. Use of the DNA stabilizer of any one of claims 1 to 2 in the preparation of a quality control product of a multi-gene combined detection kit for lung cancer.
5. An RNA stabilizer, which is characterized by comprising EDTA-3K, aurin tricarboxylate, a serine inhibitor and betaine hydrochloride.
6. The RNA stabilizer according to claim 5, wherein the RNA stabilizer comprises 50-80 g/L EDTA-3K, 600-900 g/L aurin tricarboxylate, 30-50 mg/L serine inhibitor and 12-16 mg/L betaine hydrochloride.
7. Use of the RNA stabilizer of any one of claims 5 to 6 for RNA preservation.
8. Use of the RNA stabilizer of any one of claims 5 to 6 in the preparation of a quality control product of a multi-gene combined detection kit for lung cancer.
9. A quality control product for multi-gene combined detection of lung cancer is characterized by comprising one or more of a lung cancer positive quality control product, a lung cancer negative quality control product, a lung cancer fusion positive quality control product and a lung cancer fusion negative quality control product;
the positive quality control material for the lung cancer comprises a plasmid with target gene mutation, wild cell strain DNA and a DNA stabilizer;
the lung cancer negative quality control product comprises DNA without target gene mutation and a DNA stabilizer;
the lung cancer fusion positive quality control product comprises RNA fused with a target gene and an RNA stabilizer;
the lung cancer fusion negative quality control product comprises RNA of a target gene which is not fused and an RNA stabilizer;
the target gene is at least one of EGFR, KRAS, BRAF, ERBB2, ALK, ROS1 and RET gene quality control products.
10. A preparation method of a multi-gene combined detection kit quality control product based on a DR-Seq800 gene sequencing platform for lung cancer is characterized by comprising the steps of preparing a lung cancer positive quality control product, preparing a lung cancer negative quality control product, preparing a lung cancer fusion positive quality control product and/or preparing a lung cancer fusion negative quality control product;
wherein, the preparation of the positive quality control product of the lung cancer comprises the following steps: designing a target gene mutation plasmid; mixing the target gene mutation plasmid and the DNA of the wild cell strain according to a proportion; adding the DNA stabilizer of any one of claims 1 to 2 to the mixed DNA solution;
preparing a lung cancer negative quality control product: selecting lung cancer tissue sample DNA which is identified as the target gene and does not generate mutation; adding the DNA stabilizer of any one of claims 1 to 2 to a DNA sample;
preparing a lung cancer fusion positive quality control product: selecting the lung cancer tissue sample RNA identified as the target gene with fusion mutation; adding the RNA stabilizer of any one of claims 5 to 6 to the RNA sample;
preparing a lung cancer fusion negative quality control product: selecting the lung cancer tissue sample RNA identified as the target gene without fusion mutation; adding the RNA stabilizer of any one of claims 5 to 6 to the RNA sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910911924.XA CN110747193B (en) | 2019-09-25 | 2019-09-25 | Lung cancer polygene joint detection kit quality control product based on second-generation sequencing platform |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910911924.XA CN110747193B (en) | 2019-09-25 | 2019-09-25 | Lung cancer polygene joint detection kit quality control product based on second-generation sequencing platform |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110747193A true CN110747193A (en) | 2020-02-04 |
| CN110747193B CN110747193B (en) | 2021-08-31 |
Family
ID=69277066
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910911924.XA Active CN110747193B (en) | 2019-09-25 | 2019-09-25 | Lung cancer polygene joint detection kit quality control product based on second-generation sequencing platform |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110747193B (en) |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005116081A2 (en) * | 2004-05-24 | 2005-12-08 | Genvault Corporation | Stable protein storage and stable nucleic acid storage in recoverable form |
| CN103173433A (en) * | 2011-12-26 | 2013-06-26 | 上海芯超生物科技有限公司 | DNA (Deoxyribonucleic Acid) low-temperature storage solvent and storage method thereof |
| US20140054508A1 (en) * | 2009-02-18 | 2014-02-27 | Streck, Inc. | Preservation of cell-free rna in blood samples |
| CN106065400A (en) * | 2015-04-24 | 2016-11-02 | 上海润腾生物科技有限公司 | A kind of ribonucleic acid protective agent, test kit, application and store method |
| CN106701743A (en) * | 2016-12-26 | 2017-05-24 | 广州和实生物技术有限公司 | Blood collection tube for protecting and stabilizing free DNA |
| CN106701742A (en) * | 2016-12-26 | 2017-05-24 | 广州和实生物技术有限公司 | Blood collection tube for protecting and stabilizing free RNA (Ribonucleic Acid) |
| CN108048529A (en) * | 2017-12-28 | 2018-05-18 | 广州市金圻睿生物科技有限责任公司 | A kind of quality-control product of lung cancer methylated genes detection kit for stablizing preservation and application |
| CN108085391A (en) * | 2017-12-28 | 2018-05-29 | 广州市金圻睿生物科技有限责任公司 | A kind of RNA quality-control products for the lung cancer fusion detection kit for stablizing preservation |
| CN108103054A (en) * | 2017-12-28 | 2018-06-01 | 广州市金圻睿生物科技有限责任公司 | A kind of RNA preserves liquid and application thereof |
| CN108315327A (en) * | 2018-05-15 | 2018-07-24 | 杭州百殷生物科技有限公司 | Nucleic acid preservation liquid and preparation method thereof, the method for preserving nucleic acid using the preservation liquid |
| CN109997843A (en) * | 2019-04-15 | 2019-07-12 | 无锡芯超生物科技有限公司 | A kind of circulation dissociative DNA and haemocyte save medium |
| CN110072999A (en) * | 2017-01-16 | 2019-07-30 | 光谱解决方案有限责任公司 | Nucleic acid preservation solution and production and preparation method thereof |
-
2019
- 2019-09-25 CN CN201910911924.XA patent/CN110747193B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005116081A2 (en) * | 2004-05-24 | 2005-12-08 | Genvault Corporation | Stable protein storage and stable nucleic acid storage in recoverable form |
| US20140054508A1 (en) * | 2009-02-18 | 2014-02-27 | Streck, Inc. | Preservation of cell-free rna in blood samples |
| CN103173433A (en) * | 2011-12-26 | 2013-06-26 | 上海芯超生物科技有限公司 | DNA (Deoxyribonucleic Acid) low-temperature storage solvent and storage method thereof |
| CN106065400A (en) * | 2015-04-24 | 2016-11-02 | 上海润腾生物科技有限公司 | A kind of ribonucleic acid protective agent, test kit, application and store method |
| CN106701743A (en) * | 2016-12-26 | 2017-05-24 | 广州和实生物技术有限公司 | Blood collection tube for protecting and stabilizing free DNA |
| CN106701742A (en) * | 2016-12-26 | 2017-05-24 | 广州和实生物技术有限公司 | Blood collection tube for protecting and stabilizing free RNA (Ribonucleic Acid) |
| CN110072999A (en) * | 2017-01-16 | 2019-07-30 | 光谱解决方案有限责任公司 | Nucleic acid preservation solution and production and preparation method thereof |
| CN108048529A (en) * | 2017-12-28 | 2018-05-18 | 广州市金圻睿生物科技有限责任公司 | A kind of quality-control product of lung cancer methylated genes detection kit for stablizing preservation and application |
| CN108085391A (en) * | 2017-12-28 | 2018-05-29 | 广州市金圻睿生物科技有限责任公司 | A kind of RNA quality-control products for the lung cancer fusion detection kit for stablizing preservation |
| CN108103054A (en) * | 2017-12-28 | 2018-06-01 | 广州市金圻睿生物科技有限责任公司 | A kind of RNA preserves liquid and application thereof |
| CN108315327A (en) * | 2018-05-15 | 2018-07-24 | 杭州百殷生物科技有限公司 | Nucleic acid preservation liquid and preparation method thereof, the method for preserving nucleic acid using the preservation liquid |
| CN109997843A (en) * | 2019-04-15 | 2019-07-12 | 无锡芯超生物科技有限公司 | A kind of circulation dissociative DNA and haemocyte save medium |
Non-Patent Citations (2)
| Title |
|---|
| 曾溢滔: "《遗传病分子基础与基因诊断》", 31 December 2017 * |
| 程亚楠: "靶向高通量测序在非小细胞肺癌中的临床应用", 《中国肿瘤临床》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110747193B (en) | 2021-08-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108374046B (en) | Tumor cell microsatellite unstable state detection system | |
| CN111518800B (en) | Non-quenching oligonucleotide probe for amplifying variant target gene fragment and application thereof | |
| Normanno et al. | Molecular diagnostics and personalized medicine in oncology: challenges and opportunities | |
| Brecqueville et al. | Array comparative genomic hybridization and sequencing of 23 genes in 80 patients with myelofibrosis at chronic or acute phase | |
| CN102912013A (en) | Sequencing primer for qualitative detection of cytochrome oxidase CYP2C19 genetic typing and kit of sequencing primer | |
| Qu et al. | Comparison of four next generation sequencing platforms for fusion detection: Oncomine by ThermoFisher, AmpliSeq by illumina, FusionPlex by ArcherDX, and QIAseq by QIAGEN | |
| Park et al. | Effect of ERCC1 polymorphisms and the modification by smoking on the survival of non-small cell lung cancer patients | |
| Wen et al. | Screening circular RNAs related to acquired gefitinib resistance in non-small cell lung cancer cell lines | |
| CN100374575C (en) | Method of estimating the risk of expression of adverse drug reaction caused by the administration of a compound, which is either metabolized per se by UGT1A1 enzyme or whose metabolic intermediate is | |
| CN106755360B (en) | Nucleic acid, kit and method for detecting human CYP2D6 gene polymorphism | |
| CN104962621A (en) | Primer group used for detecting locus polymorphism of MTHFR genes and MTRR genes, method for detecting same and application of primer group | |
| CN101671740B (en) | Method for detecting gene polymorphism of UGT1A1 and liquid phase chip | |
| CN109234370B (en) | MUT gene mutation detection kit | |
| CN105154568A (en) | Primer pair and kit for detecting CYP3A4 genotyping by pyrosequencing | |
| Mäki‐Nevala et al. | Mutated Ephrin Receptor Genes in Non‐Small Cell Lung Carcinoma and Their Occurrence with Driver Mutations—Targeted Resequencing Study on Formalin‐Fixed, Paraffin‐Embedded Tumor Material of 81 Patients | |
| CN111662986B (en) | Method and probe for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S | |
| CN110747193B (en) | Lung cancer polygene joint detection kit quality control product based on second-generation sequencing platform | |
| Wang et al. | Association between Ataxia Telangiectasia Mutated gene polymorphisms and childhood leukemia in Taiwan | |
| Kimura et al. | No duplicate KRAS mutation is identified on the same allele in gastric or colorectal cancer cells with multiple KRAS mutations | |
| Ge et al. | Detection of IDH1 and TERT promoter mutations with droplet digital PCR in diffuse gliomas | |
| EP2984185B1 (en) | Methods and compositions for treating cancer | |
| CN109306374A (en) | For detecting primer, detection method and the kit of mankind PIK3CA gene E545K mutation | |
| US20190115105A1 (en) | Tandem duplicator phenotype (tdp) as a distinct genomic configuration in cancer and use thereof | |
| CN103667268A (en) | DNA (Deoxyribonucleic Acid) probe library hybridized with BRAF (v-Raf murine sarcoma viral oncogene homolog B1) gene, and method for enriching BRAF gene segments by adopting same | |
| CN114514327A (en) | Assessment of diffuse glioma and responsiveness to treatment using simultaneous marker detection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |