Probe composition and kit for detecting lung cancer mutant gene based on NGS method
阅读说明：本技术 一种基于ngs方法检测肺癌突变基因的探针组合物及试剂盒 (Probe composition and kit for detecting lung cancer mutant gene based on NGS method ) 是由 丁岩 于 2019-11-25 设计创作，主要内容包括：本发明公开了一种基于NGS方法检测肺癌基因突变的探针组合物及试剂盒。所述探针组合物选自核酸序列如SEQ ID NO 1～75所示的探针中的至少一种,其试剂盒适用于FFPE、组织及外周血ctDNA的基于NGS方法的肺癌基因突变检测,进而实现肺癌突变基因早筛和实时监测复发等目的。本发明独特设计的UMI双分子标签可以有效降低背景噪音,杜绝痕迹污染,去除假阳性,保证结果的准确性,使ctDNA检测中灵敏度达到0.1％。在组织检测中使用通用Short-Y接头,其检测灵敏度可达到2％。给患者精准靶向治疗提供了更多的可能性。组织样本与血浆样本建库工作流程相似,保证流程的简便性,省时高效,易操作。(The invention discloses a probe composition and a kit for detecting lung cancer gene mutation based on an NGS method. The probe composition is at least one selected from probes with nucleic acid sequences shown as SEQ ID NO 1-75, and the kit is suitable for lung cancer gene mutation detection of FFPE, tissues and peripheral blood ctDNA based on an NGS method, so that the purposes of early screening of lung cancer mutation genes, real-time monitoring of recurrence and the like are achieved. The uniquely designed UMI bimolecular label can effectively reduce background noise, stop trace pollution, remove false positive, ensure the accuracy of results and enable the sensitivity in ctDNA detection to reach 0.1%. The universal Short-Y joint is used in tissue detection, and the detection sensitivity can reach 2%. Providing more possibilities for precise targeted treatment of patients. The working process of constructing the database of the tissue sample and the plasma sample is similar, the simplicity and the convenience of the process are ensured, the time is saved, the efficiency is high, and the operation is easy.)
1. A probe composition characterized by: at least one probe selected from the probes with nucleic acid sequences shown as SEQ ID NO. 1-75.
2. A kit for detecting lung cancer genes is characterized in that: comprising the probe composition of claim 1.
3. The kit of claim 2, wherein: the linker is a linker with a UMI bimolecular label or a Short-Y linker; wherein the linker with the UMI bimolecular label is formed by adding 6-8 base sequences randomly on the basis of an Illumina universal linker.
4. The kit of claim 2 or 3, characterized in that: the kit is suitable for molecular detection of FFPE, tissues and ctDNA of peripheral blood.
5. The kit of claim 2 or 3, characterized in that: the kit is used for lung cancer gene detection based on an NGS method.
Technical Field
The invention relates to a lung cancer mutant gene detection composition, in particular to a group of probe compositions and a kit for detecting lung cancer genes based on an NGS method.
Background
NGS has significant advantages in clinical tumor gene detection. The second-generation sequencing can simultaneously detect common and rare mutations, gene fusion, gene amplification and other different mutations of genes, guide targeted medication and reveal a drug resistance mechanism. Meanwhile, the liquid biopsy technology based on the NGS can realize dynamic monitoring and prompt the recurrence and metastasis of the tumor earlier.
Free DNA in blood is cfdna (cell free DNA), and DNA of apoptotic cells or detached cancer cell body cells is called ctDNA. ctDNA has the same genetic mutations or characteristics as primary cancer cell DNA. This makes ctDNA have extremely high specificity, and can be used as a highly sensitive biomarker. There are many limiting factors due to the tissue biopsy technique. ctDNA is therefore in more urgent need in the tumor field, such as for aiding diagnosis, assessing therapeutic efficacy and revealing resistance mechanisms to monitoring relapse into tumors and early screening. Plays an important role in the diagnosis and treatment of tumor patients.
At present, all domestic CFDA approved kits are kits aiming at FFPE samples and used for detecting small Panle gene mutation in tissues, and simultaneously, kits aiming at liquid biopsy, fresh combination and FFPE tissues and kits meeting the requirement of early screening of lung cancer are in a vacant state in the market at present. Therefore, how to simultaneously satisfy the detection of multiple types of samples, namely, one kit for multiple detections is provided, and the early detection of lung cancer is realized, is a challenge faced by the current lung cancer gene detection kit.
Disclosure of Invention
The invention aims to provide a probe composition, which is used for a molecular diagnosis technology for accurate treatment of lung cancer patients, supports tissue detection and ctDNA liquid biopsy, and can ensure the detection sensitivity and result accuracy by combining ultrahigh sequencing depth. The UMI bimolecular label in the kit can effectively remove background noise and stop trace pollution, so that the sensitivity in ctDNA detection reaches 0.1 percent, and the blank of liquid biopsy in domestic markets is really filled.
The probe composition is at least one selected from probes with nucleic acid sequences shown as SEQ ID NO 1-75, and more preferably n selected from probes with nucleic acid sequences shown as SEQ ID NO 1-75, wherein n is an integer of 2, 3, 4, 5 … … -75. The lung cancer gene targeted by the probe composition covers a plurality of possible mutation types, including point mutations (SNPs), insertions and deletions (Indels), gene amplification (CNV) and gene Fusion (Fusion).
It is another object of the present invention to provide a kit comprising the probe composition described above.
Preferably, the kit comprises the probe composition with the probe sequences shown in SEQ ID NO. 1-NO 75, and a linker; the joint is a UMI bimolecular label joint or a short-Y joint; wherein the UMI bimolecular label joint is designed by adding 6-8 base sequences on the basis of an Illumina universal joint. A total of 4^ (6-8) base species with UMI bimolecular tag adaptors used in the adaptor ligation step in the detection of plasma free ctDNA; the UMI sequence has uniqueness, each DNA molecule corresponds to a unique UMI label, and after PCR amplification or sequencing, a messenger analyst can trace back to a DNA original template, so that background noise is reduced, and the possibility of false positive mutation is eliminated. The short-Y joint is a commercial product Cat: 201664199, a linker step for FFPE and tissue sample detection.
The kit provided by the invention can be suitable for FFPE (fringe field labeling), molecular detection of tissue samples and ultralow frequency molecular detection of plasma ctDNA (deoxyribonucleic acid) samples, and can be used for lung cancer gene detection.
The kit can be used for lung cancer gene detection based on an NGS method, and the detection process comprises the following steps: collecting samples, extracting gDNA or ctDNA, building a UMI molecular label library, performing Panel hybridization, performing NGS sequencing, analyzing the sequenced data, and judging the genotype of the samples. Wherein the Panel hybridization step comprises a probe composition with the probe sequence shown as SEQ ID NO. 1-75 and a linker.
The invention has the beneficial effects that:
1. the kit aims at the detection of lung cancer patients, and simultaneously meets the requirements of FFPE tissues, fresh tissues and whole blood samples.
2. The probe is designed aiming at the highly related region of each lung cancer gene mutation, the coverage is high and uniform, and the high specificity of the probe is ensured. Meanwhile, 35 lung cancer mutant genes can be detected at one time, targeted drug genes are covered, and the detection cost is reduced.
3. The sensitivity in ctDNA detection can reach 0.1% by adopting the UMI bimolecular label, and the sensitivity in tissue detection can reach 2% by adopting the Short-Y joint. Effectively removing background noise and avoiding trace pollution, and combining ultrahigh deep sequencing to ensure result accuracy and realize early screening of lung cancer mutant genes and real-time monitoring of the problem of recurrence after prognosis.
4. The design of the probe composition is that according to NCCN guidelines and FDA/CFDA guidelines, a plurality of authoritative databases are combined, 35 lung cancer drug-taking related genes are selected, and the lung cancer drug-taking related genes comprise all related genes of approved target drugs of lung cancer. The probe capture is highly uniform while detecting the full exons and intronic regions involved in gene Fusion, including point mutations (SNPs), insertions and deletions (Indels), gene amplification (CNV), gene Fusion (Fusion) and copy number variation. The method is prospective, prepares for product upgrading in the future, and improves more possibilities for accurate targeting and scientific research of the existing patients.
5. The probe composition shown by SEQ ID NO 1-75 covers various possible mutation types, and can be used for simultaneously detecting known or unknown mutation of a sample. For enterprises, one Panel can be used for solving the problems of different organization types provided by different patients, thereby saving both cost and time.
6. The method is simple, is beneficial to realizing the conversion of the product from a laboratory to industrial application, and has good application prospect.
Detailed Description
The following detailed description is to be read in connection with specific embodiments of the invention, but it should be understood that the scope of the invention is not limited to the specific embodiments.
The Allprep DNA/RNA FFPE kit (Cat:80324) was used for the extraction of genomic gDNA from FFPE samples in the examples described below; apostle miniMax was used for extracting plasma blood free ctDNATMThe Minima high-efficiency free DNA separation and enrichment kit (Cat: A17622).
Qubit quantification uses the Qubit dsDNA HS Assay Kit (Cat: Q32854).
The name of the kit is: NuoCancerTMLung Cancer 35 Gene For Tissue/NuoCancerTMLung Cancer 35 Gene For cfDNA (Lung Cancer 35 Gene detection kit, commercially available from Jien Biotechnology Ltd.), the following experiments were performed according to the kit instructions.