CN114525341A - Kit for simultaneously detecting lung cancer and lung infection - Google Patents
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Abstract
Translated fromChinese
Description
Translated fromChinese技术领域technical field
本发明属于生物技术领域，具体涉及一组染色体不稳定区域和病原微生物基因组在制备诊断肺癌和肺部感染的试剂或试剂盒中的应用。The invention belongs to the field of biotechnology, and in particular relates to the application of a set of chromosomal instability regions and pathogenic microorganism genomes in the preparation of reagents or kits for diagnosing lung cancer and pulmonary infection.
背景技术Background technique
原发性肺癌(primary lung cancer，PLC)是世界范围内最常见的恶性肿瘤。从病理和治疗角度，肺癌大致可以分为非小细胞肺癌(non small cell lung cancer，NSCLC)和小细胞肺癌(small cell lung cancer，SCLC)两大类，其中非小细胞肺癌约占80％-85％，其余为小细胞肺癌。Primary lung cancer (PLC) is the most common malignant tumor worldwide. From the perspective of pathology and treatment, lung cancer can be roughly divided into two categories: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), of which non-small cell lung cancer accounts for about 80%- 85% and the rest were small cell lung cancer.
肺部感染是常见的呼吸系统疾病，呼吸道与外界相通，全身血液均流经肺脏，故肺脏易遭受身体内外微生物的侵袭。病毒和细菌引起的呼吸道感染最为常见。既往以肺炎链球菌感染的细菌性肺炎为主，占80-90％，余为链球菌、克莱布斯氏杆菌、葡萄球菌等感染所致。当前细菌感染的情况有所变化，医院外所患肺炎虽仍以肺炎链球菌感染为主，但金黄色葡萄球菌性肺炎、革兰氏阴性杆菌肺炎也明显增多。住院患者所得肺炎(医院内感染所致)中革兰氏阴性杆菌肺炎明显增加，其中以克莱布斯氏杆菌、绿脓杆菌感染最为多见。另真菌感染也增多。由于微生物分离培养技术改进，已能成功地诊断肺炎菌质体、厌氧菌以及新的病原微生物，如军团菌属、细胞巨病毒、肺孢子虫所致的肺部感染。这种病原微生物的变化，可能与患者高龄、病重、免疫疾病增加；肾上腺皮质激素、免疫抑制剂、细胞毒性药物、抗生素的应用；各种插管、气管切开、呼吸机的应用以及器官移植有关。Pulmonary infection is a common respiratory disease. The respiratory tract is connected to the outside world, and the whole body blood flows through the lungs. Therefore, the lungs are vulnerable to the invasion of microorganisms inside and outside the body. Respiratory infections caused by viruses and bacteria are the most common. In the past, bacterial pneumonia was mainly caused by Streptococcus pneumoniae infection, accounting for 80-90%, and the rest were caused by infections such as Streptococcus, Klebsiella, and Staphylococcus. The current situation of bacterial infection has changed. Although Streptococcus pneumoniae is still the main cause of pneumonia outside the hospital, Staphylococcus aureus pneumonia and Gram-negative bacilli pneumonia have also increased significantly. Gram-negative bacilli pneumonia significantly increased in hospitalized patients with pneumonia (due to nosocomial infection), among which Klebsiella and Pseudomonas aeruginosa infections were the most common. Another fungal infection also increased. Due to the improvement of microbial isolation and culture technology, pneumonia mycoplasma, anaerobic bacteria and new pathogenic microorganisms such as Legionella, cytomegalovirus, and Pneumocystis pneumonia have been successfully diagnosed. The changes of this pathogenic microorganism may be related to the patient's advanced age, severe illness, and increased immune diseases; the application of adrenal cortex hormones, immunosuppressants, cytotoxic drugs, and antibiotics; various intubation, tracheotomy, ventilator application, and organ transplant related.
染色体不稳定通常与肿瘤相关，具体包括整个染色体或者染色体片段拷贝的缺失或扩增。含有与肿瘤发生相关的染色体或者染色体片段的扩增和缺失经常是肿瘤发生所特有的，检测肿瘤染色体不稳定区域对于研究肿瘤发生和开发肿瘤诊断技术都至关重要。当前临床上有使用原位荧光杂交的方法对染色体上部分区域的不稳定性进行检测，但缺少对患者整个基因组层面染色体不稳定性的分布特征。Chromosomal instability is often associated with tumors and includes deletion or amplification of copies of entire chromosomes or chromosomal segments. Amplifications and deletions containing chromosomes or chromosomal fragments associated with tumorigenesis are often specific to tumorigenesis, and detection of chromosomally unstable regions in tumors is critical for both tumorigenesis and tumor diagnostic technology. Currently, in situ fluorescence hybridization is used to detect the instability of some regions of chromosomes, but the distribution characteristics of chromosomal instability at the level of the whole genome of patients are lacking.
肺部感染病原微生物确认临床上需要进行培养，对培养条件、操作人员要求高，通量不足，无法满足高通量检测。而且感染病原微生物来源多样，每种微生物培养条件不同，在未知的条件下培养条件无法确定，容易造成部分感染源未被培养得到，造成假阴性。通过测序技术可以对病原进行鉴别，不需要培养，且检测灵敏度高，对浓度低、不易培养的微生物也有很好的检测能力。Pulmonary infection pathogenic microorganisms need to be cultured clinically, and the requirements for culture conditions and operators are high, and the throughput is insufficient to meet high-throughput detection. Moreover, the sources of infectious pathogenic microorganisms are diverse, and the culture conditions of each microorganism are different. Under unknown conditions, the culture conditions cannot be determined, which may easily cause some sources of infection not to be cultured, resulting in false negatives. Pathogens can be identified by sequencing technology without the need for culture, and the detection sensitivity is high, and it also has a good detection ability for microorganisms with low concentration and difficult to cultivate.
现有技术中还未有同时对肺部感染和肺癌进行检测的二代测序试剂盒。There is no next-generation sequencing kit for simultaneous detection of lung infection and lung cancer in the prior art.
发明内容SUMMARY OF THE INVENTION
本发明使用二代测序技术，通过分析肺癌染色体不稳定和病原微生物基因组信息，筛选出适宜表征肺癌的14个区域和微生物基因组，通过该方法可以对肺癌和肺部感染的临床早期诊断和制定个体化治疗方案提供科学依据。通过二代测序可以同时检测人和微生物基因组，通过染色体不稳定区域诊断是否有肿瘤发生，通过检测微生物基因组对感染微生物进行鉴别，使用该方法同时对肺癌和肺部感染进行诊断。The invention uses the next-generation sequencing technology to screen out 14 regions and microbial genomes suitable for characterizing lung cancer by analyzing the chromosomal instability of lung cancer and the genome information of pathogenic microorganisms, and the method can be used for early clinical diagnosis of lung cancer and lung infection and the formulation of individuals. The scientific basis for chemotherapy regimens. Next-generation sequencing can simultaneously detect human and microbial genomes, diagnose whether there is tumorigenesis by chromosomally unstable regions, identify infectious microorganisms by detecting microbial genomes, and use this method to diagnose lung cancer and lung infection at the same time.
本发明中涉及的染色体不稳定区域的编号依据本领域惯用编号规则进行限定，例如不稳定区3q在本领域惯用规则中指第3号染色体的长臂，不稳定区域7p指7号染色体短臂。The numbering of the chromosomal unstable regions involved in the present invention is defined according to the conventional numbering rules in the art. For example, the unstable region 3q refers to the long arm of chromosome 3 in the conventional rules of the art, and the unstable region 7p refers to the short arm of chromosome 7.
本发明中，“基因组”指生物体所有遗传物质的总和。这些遗传物质包括DNA或RNA(病毒RNA)。In the present invention, "genome" refers to the sum total of all genetic material of an organism. This genetic material includes DNA or RNA (viral RNA).
本发明中，“检测”是指对受试者进行是否患病的检测，其检测结果可以是患病或者不患病，可以是一次性检测，也可以是长期连续的检测，可以是局部组织的检测，也可以是系统地检测。In the present invention, "detection" refers to the detection of whether a subject is diseased. The detection result may be diseased or not, it may be a one-time detection, it may be a long-term continuous detection, and it may be a local tissue detection, or systematic detection.
本发明中，“筛查”是指对受试群体进行患病可能进行检测，其并不限于针对有患病症状的群体，同时适用无症状群体，主要目的在于在疾病初期进行检测以及时应对，防止症状的发生，并采取有效的治疗，防止各种后遗症发生。In the present invention, "screening" refers to the detection of the possibility of disease in the subject group, which is not limited to the group with symptoms of the disease, but also applies to the asymptomatic group. The main purpose is to detect at the early stage of the disease and respond in time , to prevent the occurrence of symptoms, and to take effective treatment to prevent the occurrence of various sequelae.
本发明中，“诊断”一般指针对有患病症状的个体或群体进行是否患病确认，但某些情况下也可以是无患病症状的个体或群体。In the present invention, "diagnosing" generally refers to confirming whether an individual or group with symptoms of a disease has the disease, but in some cases, it may also be an individual or group without symptoms of the disease.
本发明中，“预后评估”是指根据经验预测的疾病发展情况，包括对疾病的近期和远期疗效、转归恢复或进展程度的评估。In the present invention, "prognosis assessment" refers to the disease development situation predicted according to experience, including the assessment of the short-term and long-term efficacy, outcome recovery or progression degree of the disease.
本发明中，“病情监测”是指长期地、连续地收集、核对、分析疾病的动态分布和影响因素的资料，并将信息及时上报和反馈，以便及时采取干预措施。In the present invention, "disease monitoring" refers to long-term, continuous collection, verification, and analysis of data on the dynamic distribution of diseases and influencing factors, and reporting and feeding back the information in a timely manner, so that intervention measures can be taken in a timely manner.
一方面，本发明提供了染色体不稳定区域和病原微生物基因组在制备用于肺部疾病的检测、筛查、诊断、预后评估或病情监测的试剂或试剂盒中的用途。In one aspect, the present invention provides the use of chromosomally unstable regions and pathogenic microorganism genomes in the preparation of reagents or kits for detection, screening, diagnosis, prognosis assessment or disease monitoring of pulmonary diseases.
所述的染色体不稳定区域包括：2p、3p、3q、6p、7p、8q、9p、12p、17p、17q、18q、20q、21p、21q；所述的病原微生物基因组包括细菌、真菌、病毒中任意一种或多种的基因组。The chromosomally unstable regions include: 2p, 3p, 3q, 6p, 7p, 8q, 9p, 12p, 17p, 17q, 18q, 20q, 21p, and 21q; the pathogenic microorganism genomes include bacteria, fungi, and viruses. any one or more of the genomes.
具体地，所述的染色体不稳定包括整个染色体或者染色体片段拷贝的缺失或扩增。Specifically, the chromosomal instability includes deletion or amplification of copies of entire chromosomes or chromosome segments.
具体地，所述的7p区域的扩增可导致表皮生长因子EGFR(原癌基因)高表达；所述的9p区域16位点抑癌基因的缺失，导致细胞周期调控的异常；所述的17p缺失导致抑癌基因TP53不能表达，促进肿瘤进展。Specifically, the amplification of the 7p region can lead to high expression of the epidermal growth factor EGFR (proto-oncogene); the deletion of the tumor suppressor gene at site 16 in the 9p region leads to abnormal cell cycle regulation; the 17p Deletion results in the inability to express the tumor suppressor gene TP53, which promotes tumor progression.
具体地，所述的14个不稳定区域在肺癌患者中总的携带率高达89.3％。Specifically, the overall carrier rate of the 14 unstable regions in lung cancer patients was as high as 89.3%.
优选地，所述的细菌包括但不限于：肺炎链球菌、金黄色葡萄球菌、甲型溶血性链球菌、肺炎克雷白杆菌、流感嗜血杆菌、铜绿假单胞菌、埃希大肠杆菌、绿脓杆菌。Preferably, the bacteria include but are not limited to: Streptococcus pneumoniae, Staphylococcus aureus, A-hemolytic Streptococcus, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Escherichia coli, Pseudomonas aeruginosa.
优选地，所述的真菌包括但不限于：白色念珠菌、曲霉、放射菌。Preferably, the fungi include but are not limited to: Candida albicans, Aspergillus, Actinomyces.
优选地，所述的病毒包括但不限于：冠状病毒、腺病毒、流感病毒、巨细胞病毒、单纯疱疹病毒。Preferably, the virus includes but is not limited to: coronavirus, adenovirus, influenza virus, cytomegalovirus, herpes simplex virus.
所述的肺部疾病为肺癌和/或肺部感染。Said lung disease is lung cancer and/or lung infection.
具体地，所述的肺部疾病包括肺癌和肺部感染中的一种情形或两种情形同时发生。Specifically, the pulmonary disease includes one or both of lung cancer and pulmonary infection occurring simultaneously.
具体地，所述的应用为通过二代测序对染色体不稳定区域和微生物基因组进行检测。Specifically, the application described is the detection of chromosomally unstable regions and microbial genomes by next-generation sequencing.
另一方面，本发明提供了染色体不稳定区域和病原微生物基因组在制备用于治疗或辅助治疗肺部疾病的药物中的用途。In another aspect, the present invention provides the use of a chromosomally unstable region and a pathogenic microorganism genome in the preparation of a medicament for the treatment or adjuvant treatment of pulmonary diseases.
所述的染色体区域包括：2p、3p、3q、6p、7p、8q、9p、12p、17p、17q、18q、20q、21p、21q；所述的病原微生物基因组包括细菌、真菌、DNA病毒中任意一种或多种的基因组。The chromosomal regions include: 2p, 3p, 3q, 6p, 7p, 8q, 9p, 12p, 17p, 17q, 18q, 20q, 21p, 21q; the pathogenic microorganism genome includes any of bacteria, fungi, and DNA viruses. one or more genomes.
具体地，所述的染色体不稳定包括整个染色体或者染色体片段拷贝的缺失或扩增。Specifically, the chromosomal instability includes deletion or amplification of copies of entire chromosomes or chromosome segments.
所述的肺部疾病为肺癌和/或肺部感染。Said lung disease is lung cancer and/or lung infection.
具体地，所述的肺部疾病包括肺癌和肺部感染中的一种情形或两种情形同时发生。Specifically, the pulmonary disease includes one or both of lung cancer and pulmonary infection occurring simultaneously.
具体地，所述的应用为通过二代测序对染色体不稳定区域和微生物基因组进行检测。Specifically, the application described is the detection of chromosomally unstable regions and microbial genomes by next-generation sequencing.
又一方面，本发明提供了一种用于肺部疾病的检测、筛查、诊断、预后评估或病情监测的试剂盒。In yet another aspect, the present invention provides a kit for detection, screening, diagnosis, prognosis assessment or condition monitoring of pulmonary diseases.
所述的试剂盒通过二代测序进行检测。The kit is detected by next-generation sequencing.
具体地，所述的试剂盒包括但不限于：诊断肺癌和肺部感染的试剂盒、肺癌和肺部感染早期筛查试剂盒、肺癌和肺部感染病情监测试剂盒、肺癌和肺部感染预后评估试剂盒。Specifically, the kits include but are not limited to: kits for diagnosing lung cancer and lung infections, early screening kits for lung cancer and lung infections, monitoring kits for lung cancer and lung infections, and prognosis for lung cancer and lung infections Evaluation Kit.
所述的试剂盒中包括用于检测染色体不稳定区域和病原微生物基因组的试剂；所述的染色体不稳定区域包括：2p、3p、3q、6p、7p、8q、9p、12p、17p、17q、18q、20q、21p、21q；所述的病原微生物基因组包括细菌、真菌、DNA病毒中任意一种或多种的基因组。The kit includes reagents for detecting chromosomally unstable regions and pathogenic microorganism genomes; the chromosomally unstable regions include: 2p, 3p, 3q, 6p, 7p, 8q, 9p, 12p, 17p, 17q, 18q, 20q, 21p, 21q; the pathogenic microorganism genomes include any one or more genomes of bacteria, fungi, and DNA viruses.
具体地，所述的试剂或试剂盒还包含：阳性参考品、阴性参考品、缓冲液、酶、文库接头、可检测标签、核酸提取试剂、核酸纯化试剂中的一种或多种。Specifically, the reagent or kit further comprises: one or more of positive reference products, negative reference products, buffers, enzymes, library adapters, detectable tags, nucleic acid extraction reagents, and nucleic acid purification reagents.
进一步具体地，所述的酶包含打断酶、DNA聚合酶、DNA连接酶中的一种或多种。More specifically, the enzyme includes one or more of fragmentase, DNA polymerase, and DNA ligase.
进一步具体地，所述的阳性参考品为混合有前述的14个区域变异的细胞系和混合有前述的相关病原的基因组；所述的阴性参考品为无染色体变异、无病原基因组的细胞系。More specifically, the positive reference product is a cell line mixed with the aforementioned 14 regional variations and a genome mixed with the aforementioned related pathogen; the negative reference product is a cell line with no chromosomal mutation and no pathogenic genome.
具体地，所述的试剂盒中还包括测序引物，所述的测序引物包括P7端标签引物和P5端标签引物。Specifically, the kit further includes sequencing primers, and the sequencing primers include a P7 end index primer and a P5 end index primer.
优选地，所述的P7端标签引物选自SEQ ID NO.1-8中的一种或多种，所述的P5端标签引物选自SEQ ID NO.9-16中的一种或多种。Preferably, the P7 end index primer is selected from one or more of SEQ ID NO.1-8, and the P5 end index primer is selected from one or more of SEQ ID NO.9-16 .
可选择地，所述的试剂盒可通过以下方法进行结果检测：数字PCR、原位荧光杂交、核酸探针杂交法等。Optionally, the kit can perform result detection by the following methods: digital PCR, in situ fluorescence hybridization, nucleic acid probe hybridization method, and the like.
可选择地，所述试剂盒还包含临床上用于肺癌的检测、筛查、诊断、预后评估或病情监测和肺部感染的检测、诊断的其它试剂，以辅助或验证通过检测上述14个染色体区域所得到的结果。Optionally, the kit also includes other reagents clinically used for the detection, screening, diagnosis, prognosis assessment or condition monitoring of lung cancer and the detection and diagnosis of pulmonary infection, to assist or verify by detecting the above-mentioned 14 chromosomes. results in the area.
优选地，所述的试剂或试剂盒可检测的临床样品包括但不限于：肺泡灌洗液(冲洗液)、胸腔积液(胸水)等。Preferably, the clinical samples that can be detected by the reagent or kit include, but are not limited to, bronchoalveolar lavage fluid (flush), pleural effusion (pleural effusion), and the like.
又一方面，本发明还提供了前述肺部疾病相关的试剂盒的操作方法。In another aspect, the present invention also provides a method for operating the aforementioned pulmonary disease-related kit.
具体地，所述的操作方法包括以下步骤：Specifically, the operating method includes the following steps:
(1)从检测对象获得待测样品；(1) Obtain the sample to be tested from the test object;
(2)待测样品与检测试剂接触；(2) The sample to be tested is in contact with the detection reagent;
(3)检测前述的14个染色体区域和病原微生物基因组；(3) Detecting the aforementioned 14 chromosomal regions and pathogenic microorganism genomes;
(4)根据检测结果进行肺癌的检测、筛查、诊断、预后评估或病情监测和肺部感染的检测、诊断。(4) Lung cancer detection, screening, diagnosis, prognosis assessment or condition monitoring and detection and diagnosis of lung infection based on the test results.
本发明的有益效果：Beneficial effects of the present invention:
本发明收集临床确诊的肺癌患者、肺部感染患者和健康人的肺泡灌洗液(冲洗液)、胸腔积液(胸水)样本，通过提取人源和微生物的基因组DNA，并进行全基因组测序，进而比较肿瘤患者与对照的染色体异常情况，感染患者和健康人微生物基因组情况。阐述肺癌所特有的染色体不稳定性分布特征，公开了肺癌癌患者14个常见染色体不稳定区域，总的携带率高达83.9％；并同时检测患者感染源，与临床培养结果一致性达到90％，具有非常高的可替代性，这对临床肺癌的检测、筛查、诊断、预后评估或病情监测和肺部感染的检测、诊断的具有很大的意义，为下一步进行早期诊断和制定个体化治疗方案提供科学依据。The present invention collects bronchoalveolar lavage fluid (washing fluid) and pleural effusion (pleural effusion) samples of clinically diagnosed lung cancer patients, pulmonary infection patients and healthy people, extracts human and microbial genomic DNA, and performs whole genome sequencing, The chromosomal abnormalities in tumor patients and controls were then compared, and the microbial genomes of infected patients and healthy people were compared. The chromosomal instability distribution characteristics unique to lung cancer are expounded, and 14 common chromosomal instability regions in patients with lung cancer are disclosed, with a total carrier rate of 83.9%; It has a very high substitutability, which is of great significance for the detection, screening, diagnosis, prognosis evaluation or condition monitoring of clinical lung cancer and the detection and diagnosis of pulmonary infection. Treatment options provide a scientific basis.
附图说明Description of drawings
图1为3q+、7p+、8q+染色体不稳定分析结果图；其中A为3q+，B为7p+，C为8q+。Figure 1 shows the results of chromosomal instability analysis of 3q+, 7p+, and 8q+; A is 3q+, B is 7p+, and C is 8q+.
图2为9p-染色体不稳定分析结果图。Figure 2 is a graph showing the results of 9p-chromosomal instability analysis.
图3为17p-、17q+染色体不稳定分析结果图；其中A为17p-，B为17q+。Figure 3 shows the results of chromosomal instability analysis of 17p- and 17q+; wherein A is 17p- and B is 17q+.
图4为本发明14个染色体区域和病原检测的ROC曲线图，其中AUC为ROC曲线下面积。Figure 4 is a ROC curve diagram of 14 chromosomal regions and pathogen detection in the present invention, wherein AUC is the area under the ROC curve.
需要特别说明的是，图1-3为分析软件直出图，当前条件下可以判定检测结果。It should be noted that, Figure 1-3 is the straight-out diagram of the analysis software, and the detection result can be determined under the current conditions.
具体实施方式Detailed ways
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。The present invention will be described in further detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but are only used to illustrate the present invention. The experimental methods used in the following examples, unless otherwise specified, the experimental methods that do not specify specific conditions in the examples are usually in accordance with conventional conditions, and the materials, reagents, etc. used in the following examples, unless otherwise specified, are all Commercially available.
实施例1肺泡灌洗液人源和微生物基因组DNA提取Example 1 Extraction of human and microbial genomic DNA from bronchoalveolar lavage fluid
本实施例选用的试剂盒购自QIAGEN公司(货号：80204)。The kit selected in this example was purchased from QIAGEN Company (article number: 80204).
a.将肺泡灌洗液转移至50mL离心管中，加入同体积的1×PBS缓冲液，震荡混匀，进行1600g离心10min，小心倒去上清液，再用移液枪小心吸弃剩余的上清液。a. Transfer the bronchoalveolar lavage fluid to a 50mL centrifuge tube, add the same volume of 1×PBS buffer, shake and mix, centrifuge at 1600g for 10min, carefully pour off the supernatant, and then use a pipette to carefully remove the remaining supernatant.
b.加入350μL 1×Buffer RL/DTT至新的1.5mL离心管中，用一次性20号针头的注射器小心抽打裂解液至少5次打散细胞。b. Add 350 μL of 1×Buffer RL/DTT to a new 1.5 mL centrifuge tube, and carefully aspirate the lysate at least 5 times with a disposable 20-gauge needle to disperse the cells.
c.把DNA纯化柱装在2mL收集管中。把细胞裂解液转移至gDNA过滤柱中。14000g离心2min。c. Pack the DNA purification column in a 2mL collection tube. Transfer the cell lysate to a gDNA filter column. Centrifuge at 14000g for 2min.
d.取DNA纯化柱装在2mL收集管中。加入500μL Buffer DW1至柱子中，静置2min。10000g离心30-60s。d. Take the DNA purification column and pack it into a 2mL collection tube. Add 500 μL of Buffer DW1 to the column and let it stand for 2 min. Centrifuge at 10000g for 30-60s.
e.倒弃流出液，把柱子装回收集管中。加入500μL Buffer RW2至柱子中。10000g离心30-60s。e. Discard the effluent and put the column back into the collection tube. Add 500 μL of Buffer RW2 to the column. Centrifuge at 10000g for 30-60s.
f.倒弃流出液，把柱子装回收集管中。加入500μL Buffer RW2至柱子中。10000g离心30-60s。f. Discard the effluent and put the column back into the collection tube. Add 500 μL of Buffer RW2 to the column. Centrifuge at 10000g for 30-60s.
g.倒弃流出液，把柱子套回空的收集管。13000g离心2min。g. Discard the effluent and put the column back into the empty collection tube. Centrifuge at 13000g for 2min.
h.将DNA柱子装在1.5mL离心管中。加入30-50μL预热至65℃无核酸酶水至柱子膜中央。室温静置3min。13000g离心1min。h. Pack the DNA column in a 1.5 mL centrifuge tube. Add 30-50 μL of nuclease-free water preheated to 65°C to the center of the column membrane. Stand at room temperature for 3 min. Centrifuge at 13000g for 1 min.
i.弃去DNA柱，把DNA保存于2-8℃或-20℃。i. Discard the DNA column and store the DNA at 2-8°C or -20°C.
实施例2染色体不稳定区域和病原微生物DNA的检测Example 2 Detection of chromosomally unstable regions and pathogenic microorganism DNA
本实施例中相关建库试剂盒购自NEB公司，货号：E7645S；磁珠购自Beckman公司，货号：A63882。Relevant library building kits in this example were purchased from NEB Company, article number: E7645S; magnetic beads were purchased from Beckman Company, article number: A63882.
包括以下步骤：Include the following steps:
1、基因组片段化：取实施例1中制备的20ng人/微生物基因组DNA，配制如表1所示的酶切反应体系，按照表2程序进行反应。本实施例中人基因组DNA的浓度为2ng/μL，反应体系中需加入10μL。1. Genome fragmentation: Take 20 ng of human/microorganism genomic DNA prepared in Example 1, prepare the enzyme digestion reaction system shown in Table 1, and carry out the reaction according to the procedure in Table 2. In this example, the concentration of human genomic DNA is 2ng/μL, and 10 μL needs to be added to the reaction system.
如果人基因组DNA为无细胞的游离DNA，如血液中的cfDNA，则不需要经过基因组片段化，直接进入下步操作。If the human genomic DNA is cell-free DNA, such as cfDNA in blood, it does not need to undergo genome fragmentation and goes directly to the next step.
表1Table 1
振荡混匀、离心(避免气泡)后在PCR仪上运行以下程序：After vortexing, centrifuging (avoiding air bubbles), run the following program on the PCR machine:
表2Table 2
2、接头连接反应：将接头连接预混液、接头连接增强剂按照所检测的样本数目吸取适当的体积进行混合后，吸取15.5μL混合液加入到上一步的末端处理反应混合液中，振荡混匀、离心后再吸取1.5μL实施例1中制备的混合接头加入到上述反应液中，振荡混匀、离心。最终形成下表3中的反应体系。2. Linker ligation reaction: After the linker ligation premix and the linker ligation enhancer are mixed in an appropriate volume according to the number of samples tested, 15.5 μL of the mixture is added to the end treatment reaction mixture in the previous step, and the mixture is shaken and mixed. After centrifugation, 1.5 μL of the mixed joint prepared in Example 1 was added to the above reaction solution, shaken, mixed and centrifuged. The reaction system in Table 3 below was finally formed.
表3table 3
将上述体系置于PCR仪中，运行程序：20℃，30min，热盖关闭。The above system was placed in a PCR machine, and the running program was: 20° C., 30 min, and the hot lid was closed.
3、连接反应后纯化步骤：3. Purification steps after the ligation reaction:
a、提前从磁珠试剂盒中取出文库纯化磁珠，室温静置至少30min，使用前需混匀。a. Take out the library purification magnetic beads from the magnetic bead kit in advance, let stand at room temperature for at least 30 minutes, and mix well before use.
b、将上述步骤中的接头连接反应液转移到相应编号的1.5mL离心管，加入46μL重悬的文库纯化磁珠，使用适当量程的移液器匀速吸打20次，室温孵育5min。b. Transfer the linker ligation reaction solution in the above step to a 1.5mL centrifuge tube with the corresponding number, add 46μL of resuspended library-purified magnetic beads, use a pipette with an appropriate range to pipette 20 times at a constant speed, and incubate at room temperature for 5min.
c、将离心管置于磁力架上，待溶液澄清后，弃上层清液。c. Place the centrifuge tube on the magnetic stand, and after the solution is clear, discard the supernatant.
d、向其中加入新鲜配置的200μL 80％的乙醇，静置30s后，弃上层清液。d. Add 200 μL of freshly prepared 80% ethanol to it, and after standing for 30s, discard the supernatant.
e、重复步骤d一次。e. Repeat step d once.
f、磁力架上取下离心管，瞬时离心3sec，离心管放回磁力架上，吸弃掉残留的80％乙醇，注意不要吸到磁珠。打开管盖，室温晾干2-10min。f. Remove the centrifuge tube from the magnetic frame, centrifuge briefly for 3 sec, put the centrifuge tube back on the magnetic frame, and discard the residual 80% ethanol. Be careful not to absorb the magnetic beads. Open the cap of the tube and let it dry at room temperature for 2-10min.
g、待磁珠成亚光色，离心管中加入16μL Low TE缓冲液(或者无核酸酶水)，轻微震荡使磁珠重悬，室温孵育5min。g. When the magnetic beads become matt color, add 16 μL of Low TE buffer (or nuclease-free water) to the centrifuge tube, shake slightly to resuspend the magnetic beads, and incubate at room temperature for 5 min.
h、将离心管置于磁力架上，静置2min。待溶液澄清后，取15μL上清留待下一步扩增反应。h. Place the centrifuge tube on a magnetic stand and let it stand for 2 min. After the solution was clear, 15 μL of the supernatant was taken for the next amplification reaction.
4、PCR扩增：依下表4，加入相应试剂至PCR管中：4. PCR amplification: according to Table 4, add the corresponding reagents to the PCR tube:
表4Table 4
其中，P7端标签引物和P5端标签引物经由生工生物(上海)公司合成。具体序列如下：Among them, the P7 end index primer and the P5 end index primer were synthesized by Sangon Bio (Shanghai) Company. The specific sequence is as follows:
表5table 5
P7端标签引物选自SEQ ID NO.1-8，P5端标签引物选自SEQ ID NO.9-16，任意组合均能达到检测效果。本实施例中选用P7-01和P5-01。The P7 end index primers are selected from SEQ ID NO. 1-8, and the P5 end index primers are 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.
将混合好的PCR管放入至PCR仪中，运行以下程序：Put the mixed PCR tube into the PCR machine and run the following program:
表6Table 6
5、PCR产物纯化参考步骤3纯化步骤，其中：5. Refer to step 3 for purification of PCR product, wherein:
步骤b中重悬的文库纯化磁珠的用量为22.5μL；The amount of the resuspended library purification magnetic beads in step b is 22.5 μL;
步骤g中Low TE缓冲液(或者无核酸酶水)加入量为31μL；In step g, the amount of Low TE buffer (or nuclease-free water) added is 31 μL;
步骤h中取30μL上清留待下一步操作。In step h, 30 μL of supernatant was taken and reserved for the next step.
6、文库定量上机6. Library quantification on the machine
上述纯化文库使用Agilent BioAnalyzer生物芯片分析系统对片段大小进行分析，使用
结果分析讨论：Analysis and discussion of the results:
上机测序数据经过本发明所述数据分析方法后，得到染色体测序深度分布图，如图1-3所示。After the on-machine sequencing data is subjected to the data analysis method of the present invention, a chromosome sequencing depth distribution map is obtained, as shown in Figures 1-3.
分析结果由两部分组成：The analysis results consist of two parts:
一部分为染色体变异情况，结果包括染色体编号，染色体分区以及染色体测序深度分布。染色体测序深度分布区域中圆点为染色体小区域拷贝数分布情况，当其在纵轴scores值在-3到3之间时判定无不稳定发生，当其scores值大于3时判定为扩增，当其scores值小于-3时判定为缺失，对于发生扩增或缺失的区域使用灰色背景与正常区域进行区分。通过分析结果查看是否有上述染色体不稳定发生，若发生判定为肿瘤，若未发生判定为非肿瘤。Part of the chromosome variation, the results include chromosome number, chromosome partition and chromosome sequencing depth distribution. The dots in the chromosomal sequencing depth distribution area are the distribution of copy numbers in small chromosomal regions. When the scores on the vertical axis are between -3 and 3, it is judged that no instability occurs. When the scores are greater than 3, it is judged as amplification. When the score value is less than -3, it is judged to be missing, and the region with amplification or deletion is distinguished from the normal region by using a gray background. By analyzing the results, check whether the above chromosomal instability occurs. If it occurs, it is judged as a tumor, and if it does not, it is judged as a non-tumor.
另一部分为微生物检测情况。输出结果包括微生物种属和对应测序支持Reads数，通过检测到微生物致病性和检测Reads数判断感染的是否发生、由哪种微生物感染引起。检测结果见表7。The other part is the microbiological testing situation. The output results include the microbial species and the number of reads supported by the corresponding sequencing. By detecting the pathogenicity of the microorganisms and the number of detected reads, it can be judged whether the infection has occurred and which microorganism is caused by the infection. The test results are shown in Table 7.
表7病原微生物检测结果示例Table 7 Examples of pathogenic microorganism detection results
图4为本发明14个染色体区域和病原检测的ROC曲线图，其中AUC为ROC曲线下面积。Figure 4 is a ROC curve diagram of 14 chromosomal regions and pathogen detection in the present invention, wherein AUC is the area under the ROC curve.
实施例3检测样本验证Embodiment 3 Test sample verification
选取临床样本116例(肺癌患者56例，感染患者60例)和健康样本42例，参考实施例1和实施例2进行检测，结果如下：肿瘤患者检出染色体拷贝数变异者47例，感染患者检出病原微生物与培养结果一致54例，感染患者检出染色体异常5例，健康人检出染色体异常1例、病原微生物感染5例。结果表明：本发明专利对于肺癌检测灵敏度83.9％，特异性94.1％；本试剂盒对于肺部病原微生物感染检测正确检出率90％，特异性88％。综上，本试剂盒可以很好的用于肺癌和肺部病原感染的检测，为临床诊断提供支持。116 clinical samples (56 lung cancer patients, 60 infected patients) and 42 healthy samples were selected and tested with reference to Example 1 and Example 2. The results are as follows: 47 patients with chromosomal copy number variation were detected in tumor patients, and 47 patients with infection 54 cases of pathogenic microorganisms were found to be consistent with the culture results, 5 cases of chromosomal abnormality were detected in infected patients, 1 case of chromosomal abnormality and 5 cases of pathogenic microorganism infection were detected in healthy individuals. The results show that the patent of the present invention has a sensitivity of 83.9% and a specificity of 94.1% for the detection of lung cancer; the correct detection rate of the kit for the detection of pulmonary pathogenic microorganism infection is 90% and the specificity is 88%. In conclusion, this kit can be well used for the detection of lung cancer and pulmonary pathogenic infection, and provides support for clinical diagnosis.
对比例Comparative ratio
参照实施例1和实施例2设置以下对比例：The following comparative examples are set with reference to Example 1 and Example 2:
通过对比例发现，当检测不稳定区域减少(对比例1和2)时，相对本发明检测特异性变化不明显，但灵敏度有显著性下降，临床使用意义显著降低；当检测不稳定区域增加(对比例3)时，灵敏度和特异性变化都不明显，相对本发明并没有显著变化，但检测费用需要增加。因此，本发明染色体异常组合既能保证临床检测性能，同时又不显著增加检测费用，是当前最优的检测组合。Through the comparative example, it is found that when the unstable area of detection is reduced (Comparative Examples 1 and 2), the specificity of detection is not significantly changed compared to the present invention, but the sensitivity is significantly reduced, and the clinical significance is significantly reduced; when the unstable area of detection is increased ( In Comparative Example 3), the sensitivity and specificity did not change significantly, and there was no significant change compared to the present invention, but the detection cost needed to be increased. Therefore, the chromosomal abnormality combination of the present invention can not only ensure the clinical detection performance, but also does not significantly increase the detection cost, and is the current optimal detection combination.
序列表sequence listing
<110> 苏州宏元生物科技有限公司<110> Suzhou Hongyuan Biotechnology Co., Ltd.
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| CN103403182A (en)* | 2010-11-30 | 2013-11-20 | 香港中文大学 | Detection of genetic or molecular aberrations associated with cancer |
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