阅读说明：本技术 一种基于二代测序平台的肺癌多基因联合检测试剂盒质控品 (Lung cancer polygene joint detection kit quality control product based on second-generation sequencing platform ) 是由 吴英松 周其伟 刘志鹏 杨学习 李明 于 2019-09-25 设计创作，主要内容包括：本发明公开了一种基于二代测序平台的肺癌多基因联合检测试剂盒质控品,本质控品含有肺癌阳性质控品、肺癌阴性质控品、肺癌融合阳性质控品、肺癌融合阴性质控品中的一种或几种；其中,所述肺癌阳性质控品包括已发生目标基因突变的质粒、野生型细胞株DNA以及DNA稳定剂。本发明提供的肺癌质控品中所含DNA/RNA稳定剂可以有效防止游离DNA/RNA的降解,为DNA/RNA提供稳定的环境,延长肺癌质控品的保存时间；本发明提供质控品具有制备成本低、性能稳定和易保存等优点,可有效地监控人员操作、仪器状态以及试剂盒有效性等因素,极大地提高了检测试剂盒的准确性和可靠性。(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.)

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.

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.