TWI745939B - A sensitive detection method of egfr mutations on circulating tumor cells in cancer patients - Google Patents
A sensitive detection method of egfr mutations on circulating tumor cells in cancer patients Download PDFInfo
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Abstract
一種偵測周邊血液中EGFR基因突變腫瘤細胞之方法,利用血球分離技術及使用螢光接合抗體純化出標的細胞,接著再採用即時定量PCR(real-time PCR)結合溫控模組對標的細胞進行檢測,即可偵測血液中循環腫瘤細胞的EGFR基因突變。 A method for detecting EGFR gene mutation tumor cells in the surrounding blood, using blood cell separation technology and the use of fluorescent binding antibodies to purify the target cells, and then using real-time PCR (real-time PCR) combined with a temperature control module to perform the target cells Detection can detect EGFR gene mutations in circulating tumor cells in the blood.
Description
本發明係關於一種偵測血液中微量腫瘤細胞之方法,特別是針對周邊血液中帶有EGFR基因突變的腫瘤細胞之偵測方法。 The present invention relates to a method for detecting trace tumor cells in blood, especially for detecting tumor cells with EGFR gene mutations in peripheral blood.
已知血液中不到百分之一是廣義白血球,如單核球,淋巴球,嗜中性球等等,最終可能不到萬分之一的比例是標的腫瘤細胞;因此,想要獲得標的腫瘤細胞就需要經過層層篩選。循環腫瘤細胞(circulating tumor cell,CTC)在癌症病患血液中非常稀少,循環腫瘤細胞在癌症病患血液中的數量多寡與特性被認為與癌症轉移及癌症復發息息相關。習知係利用免疫化學方式辨識及定量全血中之循環腫瘤細胞,以評估癌症程度與治療反應,其中,辨識包括正選擇(positive selection)及負選擇(negative selection)。 It is known that less than 1% of the blood is generalized white blood cells, such as monocytes, lymphocytes, neutrophils, etc. In the end, less than one in 10,000 may be target tumor cells; therefore, you want to obtain the target. Tumor cells need to go through layers of screening. Circulating tumor cells (CTC) are very rare in the blood of cancer patients. The number and characteristics of circulating tumor cells in the blood of cancer patients are thought to be closely related to cancer metastasis and cancer recurrence. The conventional knowledge system uses immunochemical methods to identify and quantify circulating tumor cells in whole blood to assess the degree of cancer and treatment response. Among them, the identification includes positive selection and negative selection.
實務上,正選擇經常搭配負選擇合併使用。使用的免疫辨識套組為上皮細胞黏合蛋白(EpCAM)、細胞角質蛋白(cytokeratin)8(CK8)、細胞角質蛋白18(CK18)、細胞角質蛋白19(CK19)及CD45。從全血中計算EpCAM+、CK8+、CK18+、CK19+和CD45-表現的循環腫瘤細胞數目,但此方法因有大量血球細胞干擾,且並非所有腫瘤細胞皆會表現EpCAM+、CK8+、CK18+及CK19+,所以腫瘤細胞的捕捉效率較低、專一性亦不佳,且耗費大量抗體導致實驗費用高昂。另一相關技術係將血液中 血球細胞去除,再利用免疫化學方式辨識循環腫瘤細胞,再經由磁性流體捕捉具免疫辨識之腫瘤細胞,其使用之免疫抗體套組為EpCAM、表皮生長因素受體(EGFR)、人類表皮生長因素受體(HER2)及鈣粘蛋白(N-cadherin)辨識捕捉腫瘤細胞。由磁性流體方式僅能捕捉腫瘤細胞,但無法直接提供科學化的定量方式。 In practice, positive selection is often combined with negative selection. The immune identification kit used is Epithelial Cell Adhesion Protein (EpCAM), Cytokeratin 8 (CK8), Cytokeratin 18 (CK18), Cytokeratin 19 (CK19) and CD45. Calculate the number of circulating tumor cells expressed by EpCAM+, CK8+, CK18+, CK19+ and CD45- from whole blood, but this method has a large number of blood cell interference, and not all tumor cells will express EpCAM+, CK8+, CK18+ and CK19+, so the tumor The cell capture efficiency is low, the specificity is not good, and the large amount of antibodies is consumed, which leads to high experiment costs. Another related technology department will be in the blood The blood cells are removed, and the circulating tumor cells are identified by immunochemical methods, and then the tumor cells with immunological identification are captured by magnetic fluid. The immune antibody kits used are EpCAM, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor Body (HER2) and cadherin (N-cadherin) identify and capture tumor cells. The magnetic fluid method can only capture tumor cells, but it cannot directly provide a scientific quantitative method.
上述偵測技術皆以抗原抗體直接辨識腫瘤細胞,但偵測到的循環腫瘤細胞數目並不多,在數量上若有些微誤差,將容易導致偽陽性或偽陰性的結果,造成錯誤判斷等問題。由於周邊血液中腫瘤細胞稀少,故開發有效偵測周邊血液中循環腫瘤細胞的技術,使檢驗結果的靈敏度(sensitivity)及特異性(specificity)大幅度提升有其必要性。 The above detection techniques all use antigen and antibody to directly identify tumor cells, but the number of circulating tumor cells detected is not large. If there is a slight error in the number, it will easily lead to false positive or false negative results and cause misjudgment and other problems. . Due to the scarcity of tumor cells in the peripheral blood, it is necessary to develop a technology to effectively detect circulating tumor cells in the peripheral blood to greatly improve the sensitivity and specificity of the test results.
為改善先前技術之缺失,本發明提供一種偵測周邊血液中EGFR基因突變腫瘤細胞之方法,該方法係利用去除血球細胞技術以及使用螢光接合專一抗體來純化周邊血液中循環腫瘤細胞,再採用即時定量PCR(real-time PCR)搭配溫控模組進行檢測。經由本發明偵測周邊血液循環腫瘤細胞之表皮生長因子受體(EGFR)突變分析,有助於提供臨床癌症病患用藥參考與治療追踨。 In order to improve the lack of the prior art, the present invention provides a method for detecting EGFR gene mutation tumor cells in the peripheral blood. Real-time PCR (real-time PCR) with temperature control module for detection. The detection of epidermal growth factor receptor (EGFR) mutation analysis of peripheral blood circulating tumor cells through the present invention is helpful to provide clinical reference for medication and treatment of cancer patients.
本發明為一種偵測周邊血液中EGFR基因突變腫瘤細胞之方法,包括: The present invention is a method for detecting EGFR gene mutation tumor cells in peripheral blood, including:
步驟a. 取得周邊血液檢體; Step a. Obtain peripheral blood samples;
步驟b. 去除血液中之紅血球; Step b. Remove red blood cells from the blood;
步驟c. 使用CD45抗體及CD235a抗體排除檢體中非標的細 胞之白血球; Step c. Use CD45 antibody and CD235a antibody to exclude non-standard details from the sample Cell white blood cells;
步驟d. 添加至少一種辨識腫瘤細胞的螢光接合專一抗體至血液中,將上述抗體與步驟c收集得到表現微弱或不表現CD45及CD235a之白血球細胞結合,並純化出表現螢光的標的細胞; Step d. Add at least one fluorescent binding specific antibody that recognizes tumor cells to the blood, combine the above antibody with step c to collect the white blood cells that show weak or no CD45 and CD235a, and purify the target cells that show fluorescence;
步驟e. 將上述表現螢光的標的細胞進行去氧核醣核酸的萃取,並在即時定量PCR(real-time PCR)中結合溫控模組,對上述細胞使用至少三組由高溫以預定時間下降預定溫度至低溫,接著再從低溫以預定時間上升預定溫度回到高溫,而形成至少三段式的溫差區間變化; Step e. Extract the deoxyribonucleic acid from the above-mentioned fluorescent target cells, and combine the temperature control module in real-time PCR, and use at least three sets of the above-mentioned cells to decrease from high temperature in a predetermined time A predetermined temperature to a low temperature, and then a predetermined temperature rise from a low temperature to a high temperature within a predetermined time, forming at least a three-stage temperature difference interval change;
步驟f. 偵測到EGFR基因突變腫瘤細胞。 Step f. Detect EGFR gene mutation tumor cells.
藉由上述方法,本發明之效果能提供準確偵測循環腫瘤細胞,且有效排除非目標細胞之干擾;此外,若純化出的循環腫瘤細胞含有微量突變DNA,可被本發明所設計的EGFR基因突變DNA增幅反應偵測到;若純化出的循環腫瘤細胞不含有微量突變DNA,則不會有非專一性的訊號產生。 Through the above method, the effect of the present invention can provide accurate detection of circulating tumor cells, and effectively eliminate the interference of non-target cells; in addition, if the purified circulating tumor cells contain a small amount of mutant DNA, the EGFR gene designed by the present invention The mutant DNA amplification reaction is detected; if the purified circulating tumor cells do not contain traces of mutant DNA, there will be no non-specific signals.
S1:步驟a S1: Step a
S2:步驟b S2: Step b
S3:步驟c S3: Step c
S4:步驟d S4: Step d
S5:步驟e S5: Step e
S6:步驟f S6: Step f
S11:第一段溫差區間步驟 S11: Steps for the first section of temperature difference interval
S12:第二段溫差區間步驟 S12: Steps for the second temperature difference interval
S13:第三段溫差區間步驟 S13: Steps for the third temperature difference interval
〔圖1〕係本發明偵測周邊血液中EGFR基因突變腫瘤細胞之方法流程圖 [Figure 1] is the flow chart of the method for detecting EGFR gene mutation tumor cells in peripheral blood of the present invention
〔圖2〕係本發明揭露的三段式溫差區間變化之方法流程圖 [Figure 2] is a flow chart of the three-stage temperature difference interval change method disclosed in the present invention
〔圖3〕係本發明使用區間溫度以SEQ ID NO:1及SEQ ID NO:2針對EGFR exon 18基因突變去氧核醣核酸增幅反應之結果 [Figure 3] is the result of the amplified reaction of the EGFR exon 18 gene mutation deoxyribonucleic acid with SEQ ID NO:1 and SEQ ID NO:2 using the interval temperature of the present invention
〔圖4〕係本發明使用單一溫度以SEQ ID NO:1及SEQ ID NO:2針對EGFR exon 18基因突變去氧核醣核酸增幅反應之結果 [Figure 4] is the result of the amplified reaction of SEQ ID NO: 1 and SEQ ID NO: 2 against the EGFR exon 18 gene mutation DNA amplification reaction using a single temperature in the present invention
〔圖5〕係本發明使用單一溫度以SEQ ID NO:1及SEQ ID NO:2針對EGFR exon 18基因突變去氧核醣核酸增幅反應之結果 [Figure 5] is the result of the amplified reaction of SEQ ID NO: 1 and SEQ ID NO: 2 against the EGFR exon 18 gene mutation DNA amplification reaction in the present invention using a single temperature
〔圖6〕係本發明使用單一溫度以SEQ ID NO:3及SEQ ID NO:4針對EGFR exon 19基因突變去氧核醣核酸增幅反應之結果 [Figure 6] is the result of the present invention using a single temperature with SEQ ID NO: 3 and SEQ ID NO: 4 to target the EGFR exon 19 gene mutation DNA amplification reaction result
〔圖7〕係本發明使用區間溫度以SEQ ID NO:5及SEQ ID NO:6針對EGFR exon 20基因突變去氧核醣核酸增幅反應之結果
[Figure 7] is the result of the amplified reaction of the
〔圖8〕係本發明使用單一溫度以SEQ ID NO:5及SEQ ID NO:6針對EGFR exon 20基因突變去氧核醣核酸增幅反應之結果
[Figure 8] is the result of the amplification reaction of SEQ ID NO: 5 and SEQ ID NO: 6 against the
〔圖9〕係本發明使用單一溫度以SEQ ID NO:5及SEQ ID NO:6針對EGFR exon 20基因突變去氧核醣核酸增幅反應之結果
[Figure 9] is the result of the amplified reaction of SEQ ID NO: 5 and SEQ ID NO: 6 against the
〔圖10〕係本發明使用區間溫度以SEQ ID NO:7及SEQ ID NO:8針對EGFR exon 21基因突變去氧核醣核酸增幅反應之結果 [Figure 10] is the result of the amplification reaction of the EGFR exon 21 gene mutation deoxyribonucleic acid with SEQ ID NO: 7 and SEQ ID NO: 8 using the interval temperature of the present invention
〔圖11〕係本發明使用單一溫度以SEQ ID NO:7及SEQ ID NO:8針對EGFR exon 21基因突變去氧核醣核酸增幅反應之結果 [Figure 11] is the result of the amplification reaction of SEQ ID NO: 7 and SEQ ID NO: 8 against the EGFR exon 21 gene mutation DNA amplification reaction in the present invention using a single temperature
〔圖12〕係本發明使用單一溫度以SEQ ID NO:7及SEQ ID NO:8針對EGFR exon 21基因突變去氧核醣核酸增幅反應之結果 [Figure 12] is the result of the amplification reaction of SEQ ID NO: 7 and SEQ ID NO: 8 against the EGFR exon 21 gene mutation DNA amplification reaction in the present invention using a single temperature
充分瞭解本發明之目的、特徵及功效,茲藉由下述具體之實施例,並配合所附之圖式,對本發明做一詳細說明,說明如後: With a full understanding of the purpose, features, and effects of the present invention, the following specific embodiments are used in conjunction with the accompanying drawings to give a detailed description of the present invention. The description is as follows:
參閱圖1,揭露一種偵測周邊血液中EGFR基因突變腫瘤細胞之方法,包括: Referring to Figure 1, a method for detecting EGFR gene mutation tumor cells in peripheral blood is disclosed, including:
步驟a S1. 取得周邊血液檢體。可利用EDTA採取周邊血液 檢體約10ml左右。 Step a S1. Obtain peripheral blood samples. EDTA can be used to collect peripheral blood The sample is about 10ml.
步驟b S2. 去除血液中之紅血球。先添加固定密度之液體(如ficoll-paque)再離心上述周邊血液檢體,離心可將紅血球及血漿分開形成分層,其中間會分離出一層膚色血球層(buffy coat),取出膚色血球層(buffy coat),其成分為周邊血液單核球細胞(peripheral blood monocytic cell,PBMC),可利用物理分離(physical separation)或化學去除的方式裂解紅血球,在裂解紅血球的部分,可利用滲透壓原理,紅血球屬於無核脆弱之細胞,配置適當濃度的反應試劑可裂解紅血球,僅保留PBMC。物理分離的步驟包括:1.取一可造成細胞密度差異分離之物質,如蔗糖或其他多醣類,將含有PBMC的血液緩慢加入此物質,使其交界液面不受到擾動。這一混合液中,此物質與血液的體積比為1:1;2.離心(3000g)30分鐘,使該混合液分層,最上層為血清層,中層為透明溶液層,底層為紅血球層;3.取血清層與透明溶液層之間乳白色交界的PBMC;4.以細胞培養液沖數次後,獲得不含紅血球之血液。而化學去除係利用滲透壓(osmotic pressure)原理,使血液中無細胞核的紅血球脹破,有細胞核的白血球保留,步驟包括:1.配製紅血球裂解緩衝液1000g(8.30g的氯化銨(NHCl4)、1.18g的碳酸氫鈉(NaHCO3)、1.00g 0.1重量莫爾濃度的乙二胺四乙酸(EDTA)、加入無菌去離子水(ddH2O)至1000g),調整pH值至7.3,並以無菌過濾膜過濾;2.混合紅血球裂解緩衝液與含有PBMC的血液,其體積比為10:1,靜置5分鐘;3.離心(400g)10分鐘;4.去除上清液後加入10毫升之細胞培養液沖洗一次;5.再次離心(400g)10分鐘;6.去除上清液後,剩餘的即為白血球細胞。 Step b S2. Remove red blood cells from the blood. First add a fixed density liquid (such as ficoll-paque) and then centrifuge the above peripheral blood sample. Centrifugation can separate the red blood cells and plasma to form layers, in which a buffy coat will be separated, and the skin blood cell layer will be taken out ( buffy coat), which is composed of peripheral blood monocytic cells (PBMC). The red blood cells can be lysed by physical separation or chemical removal. In the part where red blood cells are lysed, the principle of osmotic pressure can be used. Red blood cells are non-nuclear and fragile cells. The appropriate concentration of reagents can lyse the red blood cells, leaving only PBMC. The physical separation steps include: 1. Take a substance that can cause cell density differential separation, such as sucrose or other polysaccharides, and slowly add blood containing PBMC to this substance so that the junction level is not disturbed. In this mixed solution, the volume ratio of this substance to blood is 1:1; 2. Centrifuge (3000g) for 30 minutes to separate the mixed solution. The uppermost layer is the serum layer, the middle layer is the transparent solution layer, and the bottom layer is the red blood cell layer. 3. Take PBMC at the milky white junction between the serum layer and the clear solution layer; 4. After washing several times with cell culture solution, obtain blood without red blood cells. The chemical removal system uses the principle of osmotic pressure to rupture the red blood cells without nuclei in the blood and retain the white blood cells with nuclei. The steps include: 1. Preparation of red blood cell lysis buffer 1000g (8.30g ammonium chloride (NHCl4)) , 1.18g of sodium bicarbonate (NaHCO3), 1.00g of ethylenediaminetetraacetic acid (EDTA) with a molar concentration of 0.1 weight, add sterile deionized water (ddH2O) to 1000g), adjust the pH to 7.3, and filter aseptically Membrane filtration; 2. Mix the red blood cell lysis buffer and the blood containing PBMC at a volume ratio of 10:1 and let stand for 5 minutes; 3. Centrifuge (400g) for 10 minutes; 4. Remove the supernatant and add 10 ml of cells Rinse the culture solution once; 5. Centrifuge again (400g) for 10 minutes; 6. After removing the supernatant, the remaining is white blood cells.
步驟c S3. 使用CD45抗體及CD235a抗體排除檢體中非標的細胞之白血球。由於PBMC仍有多種細胞,且大部分的細胞屬於非標的細胞;因此,需再加入5-100μl「奈米磁珠結合CD(cluster of differentiation)45抗體」及5-100μl「奈米磁珠結合CD235a抗體」排除非標的細胞。CD45抗體係用以辨識非標的細胞(表現CD45之白血球),換言之,CD45之四尾雙聚體辨識白血球,用以去除血液檢體中所有表現CD45的白血球細胞,經過磁鐵或磁場處理,所有被「奈米磁珠-CD45抗體」吸引的細胞將會被其抓住,未被抓住的細胞可通過磁場,通過磁場的細胞即包括標的細胞。同時使用CD235a抗體作為輔助,能針對紅血球進行與CD45抗體相通原理之處置,即使用CD235a之四尾雙聚體辨識紅血球,以排除紅血球干擾。此外,PBMC常表現上皮細胞黏合蛋白,而使用細胞角質蛋白是因為此蛋白常表現於表皮細胞上,通常腫瘤細胞起源於表皮細胞。上述中,可再加入細胞核染劑更進一步地排除紅血球,如DAPI、Hoechst、Drag 5或Syto 62,細胞核染劑可確認有核細胞,因此可排除紅血球,但不會排除其他細胞。
Step c S3. Use CD45 antibody and CD235a antibody to eliminate white blood cells of non-target cells in the specimen. Because PBMC still has many kinds of cells, and most of the cells are non-standard cells; therefore, it is necessary to add 5-100μl "Nanomagnetic Bead Binding CD (cluster of differentiation)45 antibody" and 5-100μl "Nanomagnetic Bead Binding" "CD235a antibody" excludes non-targeted cells. The CD45 anti-system is used to identify non-target cells (white blood cells expressing CD45). In other words, the four-tailed dimer of CD45 identifies white blood cells and removes all white blood cells expressing CD45 in the blood sample. After magnet or magnetic field treatment, all the white blood cells are The cells attracted by the "Nanomagnetic Beads-CD45 Antibody" will be caught by it. Uncaught cells can pass through the magnetic field, and the cells that pass through the magnetic field include the target cells. At the same time, using CD235a antibody as an aid, it can treat red blood cells in the same principle as CD45 antibody, that is, use the four-tailed dimer of CD235a to identify red blood cells to eliminate red blood cell interference. In addition, PBMC often express epithelial cell adhesion protein, and cytokeratin is used because this protein is often expressed on epidermal cells, and tumor cells usually originate from epidermal cells. In the above, nuclear stains can be added to further exclude red blood cells, such as DAPI, Hoechst,
步驟d S4. 添加至少一種辨識腫瘤細胞的螢光接合專一抗體至血液中,將上述抗體與步驟c S3收集得到表現微弱或不表現CD45及CD235a之白血球細胞結合,並利用微流體多參數細胞分選儀(On-Chip Sort)定量、純化與分離表現螢光接合抗體之標的細胞。利用螢光接合專一抗體加強辨識標的細胞及挑選標的細胞,亦即將螢光接合專一抗體與不表現CD45和CD235a之標的細胞結合,其中該螢光接合專一抗體包括波型蛋白(Vimentin)、鈣粘蛋白(Cadherin)、CD133、CD44、纖連蛋白(Fibronectin)、p16(INK4A)、Snail1、Snail2(Slug)、上皮細胞黏合蛋白(EpCAM)或細胞角 質蛋白(cytokeratin,CK)。上述所提到的微流體多參數細胞分選儀係用於分選細胞的儀器,可對細胞表面或內部表現抗原蛋白質用特殊螢光標誌染色,細胞通過流道時可被特定波長的雷射激發螢光,再由接收器接收螢光。本發明所使用的微流體多參數細胞分選儀之特點在於採用拋棄式微流道晶片,樣本會接觸到的範圍就只有在此晶片內,因此可形成完全封閉的空間,適合分析、分選少量細胞。分選係以空氣壓控制液體流動(flow shift)方式,僅在晶片內進行細胞分選:以鞘液挾帶樣本流動,在雷射檢出目標螢光訊號時,橫向的空氣壓會將液體推入收集槽的系統。此系統可將細胞回收損失最小化,特別是對於目標細胞在極少數的情況。由步驟d S4所得之螢光接合專一抗體結合不表現CD45和CD235a之標的細胞,利用微流體多參數細胞分選儀將發出螢光的細胞以及沒有發出螢光的細胞分流,專一純化出具螢光表現之標的細胞集中回收至微量分裝管中,接著在微量分裝管中加入核酸萃取試劑,進行標的細胞去氧核醣核酸之萃取。 Step d S4. Add at least one fluorescent binding specific antibody that recognizes tumor cells to the blood, collect the above antibody with step c S3 to obtain the white blood cell binding with weak or no CD45 and CD235a, and use microfluidic multi-parameter cell analysis On-Chip Sort quantifies, purifies and separates target cells that express fluorescently conjugating antibodies. The fluorescent binding specific antibody is used to enhance the identification and selection of target cells, that is, the fluorescent binding specific antibody binds to cells that do not express CD45 and CD235a targets. The fluorescent binding specific antibody includes Vimentin and cadherin. Cadherin, CD133, CD44, Fibronectin, p16 (INK4A), Snail1, Snail2 (Slug), Epithelial Cell Adhesion Protein (EpCAM) or Cytokeratin Quality protein (cytokeratin, CK). The above-mentioned microfluidic multi-parameter cell sorter is an instrument for sorting cells. It can stain the surface or inside of cells with special fluorescent markers. The cells can be lasered by a specific wavelength when they pass through the flow channel. The fluorescent light is excited, and then the fluorescent light is received by the receiver. The microfluidic multi-parameter cell sorting instrument used in the present invention is characterized in that it adopts a disposable micro-channel chip, and the range that the sample will touch is only within the chip, so it can form a completely enclosed space, which is suitable for analysis and sorting a small amount cell. The sorting system uses air pressure to control the liquid flow (flow shift) method, and only performs cell sorting in the chip: the sheath liquid is used to entrain the sample to flow. When the laser detects the target fluorescent signal, the lateral air pressure will cause the liquid Push into the collection tank system. This system can minimize the loss of cell recovery, especially for the rare cases of target cells. The fluorescent binding specific antibody obtained in step d S4 binds to cells that do not express CD45 and CD235a targets, and uses a microfluidic multi-parameter cell sorter to separate the fluorescent cells and the non-fluorescent cells to specifically purify and produce fluorescence The target cells of the performance are collected to the micro-packaging tube, and then the nucleic acid extraction reagent is added to the micro-packing tube to extract the target cell DNA.
步驟e S5. 將上述表現螢光的標的細胞進行去氧核醣核酸的萃取,並在即時定量PCR(real-time PCR)檢測突變基因分型。生長因素受體(epidermal growth factor receptor,EGFR)的基本型態受體若接受一分子的細胞成長激素刺激,可啟動一分子的成長效果,而突變型態的EGFR可能接受一分子的細胞成長激素刺激,卻啟動一百個分子的成長效果,亦即下游產生過度的訊息傳遞,造成過度活化。EGFR的突變情況有多種,本發明係針對DNA表現序列(exon)18、19、20及21的突變進行測定,使用以EVA Green螢光染劑為基礎的即時定量PCR可檢測到微量的細胞DNA存在,其原理是使用非專一性螢光物質接合在DNA片段上,借由機器的激發發出螢光,而 未接合於DNA片段上的螢光物質則不會發出螢光。利用即時定量PCR的方式,搭配聚合酶連鎖反應器及本發明所設計之核酸引子進行DNA增幅反應,並結合溫控模組,對上述細胞使用至少三組由高溫以預定時間下降預定溫度至低溫,接著再從低溫以預定時間上升預定溫度回到高溫,而形成至少三段式的溫差區間變化。上述中,即時定量PCR係利用核酸引子(primer)的專一性,辨識核酸序列(DNA sequence)的特定位置,藉由PCR的核酸放大技術,放大特定片段,而同時利用即時定量PCR的螢光呈現強度反映放大出來的核酸片段數量。一組核酸引子包含一前置引子(forward primer)及一反置引子(reverse primer),認定為一片段的頭尾,頭尾所包圍的一段核酸即放大的目標特定片段。 Step e S5. Extract the deoxyribonucleic acid from the above-mentioned fluorescent target cells, and detect the mutation genotyping by real-time PCR. If the basic type of epidermal growth factor receptor (EGFR) receptor is stimulated by a molecule of cell growth hormone, it can initiate a molecule of growth effect, while the mutant form of EGFR may receive a molecule of cell growth hormone. The stimulus activates the growth effect of a hundred molecules, that is, excessive message transmission is generated downstream, resulting in excessive activation. There are many mutations of EGFR. The present invention is based on the detection of mutations in DNA expression sequences (exon) 18, 19, 20 and 21. Real-time quantitative PCR based on EVA Green fluorescent dye can detect a small amount of cellular DNA. Existence, its principle is to use non-specific fluorescent substances to bind to DNA fragments, which are excited by the machine to emit fluorescence, and The fluorescent substance that is not attached to the DNA fragment will not fluoresce. Using real-time quantitative PCR, with the polymerase chain reactor and the nucleic acid primer designed in the present invention to perform DNA amplification reaction, and combined with the temperature control module, use at least three sets of the above-mentioned cells from high temperature to low temperature in a predetermined time , And then rise from the low temperature to the high temperature within a predetermined time, and form at least a three-stage temperature difference interval change. Among the above, real-time quantitative PCR utilizes the specificity of nucleic acid primers to identify specific positions of nucleic acid sequences (DNA sequences), and amplifies specific fragments by the nucleic acid amplification technology of PCR, and at the same time utilizes fluorescent display of real-time quantitative PCR The intensity reflects the number of amplified nucleic acid fragments. A set of nucleic acid primers includes a forward primer and a reverse primer, which are identified as the head and tail of a fragment. The nucleic acid surrounded by the head and tail is the amplified target specific fragment.
步驟f S6. 偵測到EGFR基因突變腫瘤細胞。 Step f S6. Detected EGFR gene mutation tumor cells.
參閱圖2,揭露所述至少三段式的溫差區間之步驟包括: Referring to FIG. 2, the step of exposing the at least three-stage temperature difference interval includes:
第一段溫差區間步驟S11:先升溫至第一溫度定值並控溫維持第一預定時間後,溫度下降至第二溫度定值作為起點,並以每個循環下降第一預定溫度定值至第三溫度定值,維持在所述第三溫度定值進行至少30次循環,所述第一溫度定值高於所述第二溫度定值,所述第二溫度定值高於所述第三溫度定值。 Step S11 of the first temperature difference interval: After the temperature is raised to the first fixed value and the temperature is maintained for the first predetermined time, the temperature drops to the second fixed value as the starting point, and the first predetermined temperature is lowered to the fixed value with each cycle The third temperature setting value is maintained at the third temperature setting value for at least 30 cycles, the first temperature setting value is higher than the second temperature setting value, and the second temperature setting value is higher than the first temperature setting value. Three temperature settings.
第二段溫差區間步驟S12:所述第三溫度定值上升至第四溫度定值並維持第二預定時間,所述第四溫度定值的溫度小於所述第一溫度定值,且所述第四溫度定值大於所述第三溫度定值。 Step S12 of the second temperature difference interval: the third temperature setting value rises to a fourth temperature setting value and maintained for a second predetermined time, the temperature of the fourth temperature setting value is less than the first temperature setting value, and the The fourth temperature setting value is greater than the third temperature setting value.
第三段溫差區間步驟S13:所述第四溫度定值升溫至所述第一溫度定值並持續第三預定時間,接著再以每秒下降第二預定溫度定值至 65℃並維持第四預定時間,接著再以每秒上升第三預定溫度定值至所述第一溫度定值,最後再以每秒下降第四預定溫度定值至40℃並維持第五預定時間。 Step S13 of the third temperature difference interval: the fourth temperature setting value is increased to the first temperature setting value for a third predetermined time, and then the second predetermined temperature setting value is lowered to the second predetermined temperature per second 65℃ and maintain the fourth predetermined time, then increase the third predetermined temperature value to the first temperature value per second, and finally decrease the fourth predetermined temperature value to 40℃ and maintain the fifth predetermined value per second time.
其中,所述第一溫度定值為92~98℃範圍內的其中一溫度,所述第二溫度定值為56~72℃範圍內的其中一溫度,所述第三溫度定值為56~72℃範圍內的其中一溫度,所述第四溫度定值為60~78℃範圍內的其中一溫度;所述第一預定時間為2~15分鐘,所述第二預定時間為低於5分鐘內的任一時間,所述第三預定時間為10~60秒範圍內的其中一時間,所述第四預定時間為10~60秒範圍內的其中一時間,所述第五預定時間為低於5分鐘內的任一時間;所述第一預定溫度定值為0.5~2℃範圍內的其中一溫度,所述第二預訂溫度定值為1~5℃範圍內的其中一溫度,所述第三預訂溫度定值0.1~4℃範圍內的其中一溫度,所述第四預定溫度定值0.1~4℃範圍內的其中一溫度。 Wherein, the fixed value of the first temperature is one temperature in the range of 92~98℃, the fixed value of the second temperature is one temperature in the range of 56~72℃, and the fixed value of the third temperature is 56~ One of the temperatures within the range of 72°C, the fourth temperature is set to one of the temperatures within the range of 60 to 78°C; the first predetermined time is 2 to 15 minutes, and the second predetermined time is less than 5 Any time within minutes, the third predetermined time is a time in the range of 10 to 60 seconds, the fourth predetermined time is a time in the range of 10 to 60 seconds, and the fifth predetermined time is Less than any time within 5 minutes; the first predetermined temperature is set to a temperature in the range of 0.5 to 2°C, the second predetermined temperature is set to be a temperature in the range of 1 to 5°C, The third predetermined temperature is set at one temperature within the range of 0.1-4°C, and the fourth predetermined temperature is set at one temperature within the range of 0.1-4°C.
偵測EGFR突變基因: Detection of EGFR mutation genes:
EGFR的突變情況有四種,分別為EGFR exon18、19、20及21的基因突變,因此本發明係針對DNA表現序列(exon)18、19、20及21的突變進行測定,使用以EVA Green螢光染劑為基礎的即時定量PCR可檢測到微量的細胞DNA存在。 There are four mutations of EGFR, namely EGFR exon18, 19, 20, and 21 gene mutations. Therefore, the present invention is based on the DNA expression sequence (exon) 18, 19, 20, and 21 mutations. Light dye-based real-time quantitative PCR can detect the presence of trace amounts of cellular DNA.
針對EGFR exon 18、19、20及21基因突變之偵測,本發明設計以下檢驗EGFR基因之核酸引子,如核苷酸序列表所記載。序列表中,SEQ ID NO:1係檢驗EGFR exon 18之前置引子;SEQ ID NO:2係檢驗EGFR exon 18之反置引子;SEQ ID NO:3係檢驗EGFR exon 19之前置引子;SEQ ID NO:4
係檢驗EGFR exon 19之反置引子;SEQ ID NO:5係檢驗EGFR exon 20之前置引子;SEQ ID NO:6係檢驗EGFR exon 20之反置引子;SEQ ID NO:7係檢驗EGFR exon 21之前置引子;SEQ ID NO:8係檢驗EGFR exon 21之反置引子。
For the detection of
參閱圖3及以下表1所示,針對EGFR exon 18的部分,本發明以健康人之白血球為wild type DNA來源,以含有G719C突變之質體為突變DNA來源。在兩者混和後相加的總量約為100ng時,混和不同比例之突變DNA(0.01-100%)。以SEQ ID NO:1及SEQ ID NO:2針對EGFR exon 18基因突變去氧核醣核酸增幅反應之結果。當標的細胞含有0.01%以上之突變DNA時,可被本發明所設計的EGFR基因突變去氧核醣核酸增幅反應偵測到。當標的細胞不含突變DNA時,不會有非專一性的訊號產生。exon 18使用59-66℃的區間溫度反應,已經沒有突變的組別F未跑出Ct值(陰性),而A~E組的Ct值皆有<30(陽性),因此均可以鑑別出突變訊號。 Referring to Figure 3 and Table 1 below, for the part of EGFR exon 18, the present invention uses white blood cells of healthy people as the source of wild type DNA, and plastids containing the G719C mutation as the source of mutant DNA. When the total amount added after mixing the two is about 100ng, mix different proportions of mutant DNA (0.01-100%). Take SEQ ID NO: 1 and SEQ ID NO: 2 against the EGFR exon 18 gene mutation deoxyribonucleic acid amplification reaction results. When the target cell contains more than 0.01% of mutant DNA, it can be detected by the EGFR gene mutant deoxyribonucleic acid amplification reaction designed in the present invention. When the target cell does not contain mutated DNA, no non-specific signal will be generated. exon 18 uses an interval temperature response of 59-66℃. Group F, which has no mutations, did not achieve a Ct value (negative), while the Ct values of groups A to E are all <30 (positive), so mutations can be identified. Signal.
在此實施例中: In this example:
第一段溫差區間步驟S11:先將表現螢光的標的細胞升溫至至95℃維持5分鐘;完成上述升溫步驟後進行控溫至95℃持續15秒,接著進行64~58℃反應。起始於64℃持續30秒,隨後每個循環降低0.5℃,直至58℃持續30秒。接著以58℃重複此溫控模組共計35次循環。 Step S11 of the first temperature difference interval: first raise the temperature of the target cell exhibiting fluorescence to 95°C for 5 minutes; after completing the above heating step, control the temperature to 95°C for 15 seconds, and then perform a 64-58°C reaction. Start at 64°C for 30 seconds, then decrease by 0.5°C per cycle until 58°C for 30 seconds. Then repeat this temperature control module at 58°C for a total of 35 cycles.
第二段溫差區間步驟S12:完成前述溫控模組後,在72℃環境中維持30秒。 Step S12 of the second temperature difference interval: After completing the aforementioned temperature control module, maintain it in an environment of 72° C. for 30 seconds.
第三段溫差區間步驟S13:最後進行升溫後迅速降溫,將完成上述步驟之樣品升溫至95℃持續30秒,再以4.4℃/秒的速率降溫度至65℃持續30秒,再以2.2℃/秒的速率升溫度至95℃,最後以0.14℃/秒的速率降 溫度至40℃,持續10秒。 Step S13 of the third temperature difference interval: After the temperature is raised, the temperature is quickly lowered. The sample after the above steps is raised to 95°C for 30 seconds, and then the temperature is lowered at a rate of 4.4°C/s to 65°C for 30 seconds, and then 2.2°C The temperature rises to 95°C at a rate of 0.14°C/sec, and finally decreases at a rate of 0.14°C/sec. The temperature reaches 40°C for 10 seconds.
表1:
參閱圖4及圖5及以下表1-1、表1-2所示,在此實施例中係採用單一溫度將表現螢光的標的細胞進行PCR反應,先將表現螢光的標的細胞升溫至95℃維持10分鐘;完成上述升溫步驟後進行控溫至95℃持續15秒,接著進行59℃或63℃持續25秒。接著以59℃或63℃重複此溫控模組共計50次循環。完成前述溫控模組後,在72℃環境中維持28秒;最後進行升溫後迅速降溫,將完成上述步驟之樣品升溫至95℃持續30秒,再以4.4℃/秒的速率降溫度至65℃持續30秒,再以2.2℃/秒的速率升溫度至95℃,最後以0.14℃/秒的速率降溫度至40℃,持續10秒。從圖4及表1-1中可看出,使用63℃單一溫度進行測試,整體的螢光強度非常低,換言之,整體放大效率不佳,Ct數值也>30(偽陰性)。而在圖5及表1-2中,使用59℃單一溫度進行測試,已沒有突變DNA的組別F來看Ct值落在30(偽陰性),僅有A與B組的Ct值有<30(陽性),可以鑑別出突變訊號。 Refer to Figure 4 and Figure 5 and the following Table 1-1 and Table 1-2. In this embodiment, a single temperature is used to perform a PCR reaction on target cells that exhibit fluorescence, and the target cells that exhibit fluorescence are first heated to Maintain the temperature at 95°C for 10 minutes; after completing the above heating step, control the temperature to 95°C for 15 seconds, and then proceed to 59°C or 63°C for 25 seconds. Then repeat this temperature control module at 59°C or 63°C for a total of 50 cycles. After completing the aforementioned temperature control module, maintain it in an environment of 72°C for 28 seconds; at the end, increase the temperature and quickly cool down. The sample after completing the above steps is heated to 95°C for 30 seconds, and then the temperature is reduced to 65 at a rate of 4.4°C/sec. The temperature lasts for 30 seconds, then the temperature is increased to 95°C at a rate of 2.2°C/sec, and finally the temperature is decreased to 40°C at a rate of 0.14°C/sec for 10 seconds. It can be seen from Figure 4 and Table 1-1 that when a single temperature of 63°C is used for testing, the overall fluorescence intensity is very low. In other words, the overall amplification efficiency is not good, and the Ct value is also >30 (false negative). In Figure 5 and Table 1-2, a single temperature of 59°C was used for testing. Group F, where there is no mutant DNA, shows that the Ct value falls at 30 (false negative). Only the Ct values of groups A and B have < 30 (positive), the mutation signal can be identified.
表1-1:
表1-2:
從表1-3可看出,在exon18使用59-66℃溫度區間進行PCR,相較於以59℃或63℃的固定溫度測試,約可以提升10~1000倍的靈敏度。 It can be seen from Table 1-3 that using exon18 to perform PCR in the temperature range of 59-66°C, compared to testing at a fixed temperature of 59°C or 63°C, the sensitivity can be increased by approximately 10 to 1000 times.
表1-3:
參閱圖6及以下表2所示,針對EGFR exon 19的部分,本發明以健康人之白血球為wild type DNA來源,以含有EGFR exon 19 deletion之細胞株H1650為突變DNA來源。在兩者混和後相加的總量約為100ng時,混和不同比例之突變DNA(0.01-100%)。以SEQ ID NO:3及SEQ ID NO:4針對EGFR exon 19基因突變去氧核醣核酸增幅反應之結果。當標的細胞含有0.01%以上之突變DNA時,可被本發明所設計的EGFR基因突變去氧核醣核酸增幅反應偵測到。當標的細胞不含突變DNA時,不會有非專一性的訊號產生。exon 19使用58℃單一固定溫度反應。而在圖6及表2中,使用58℃單一溫度進行測試,已沒有突變DNA的組別F未跑出Ct值(陰性),A~E組的Ct值皆<40(陽性),因此均可以鑑別出突變訊號。 Referring to Figure 6 and Table 2 below, for the part of EGFR exon 19, the present invention uses white blood cells of healthy people as the source of wild type DNA, and the cell line H1650 containing EGFR exon 19 deletion as the source of mutant DNA. When the total amount added after mixing the two is about 100ng, mix different proportions of mutant DNA (0.01-100%). Take SEQ ID NO: 3 and SEQ ID NO: 4 against the EGFR exon 19 gene mutation DNA amplification reaction results. When the target cell contains more than 0.01% of mutant DNA, it can be detected by the EGFR gene mutant deoxyribonucleic acid amplification reaction designed in the present invention. When the target cell does not contain mutated DNA, no non-specific signal will be generated. exon 19 uses a single fixed temperature reaction of 58°C. In Figure 6 and Table 2, a single temperature of 58°C was used for testing. Group F, which has no mutant DNA, did not achieve a Ct value (negative), and the Ct values of groups A to E were all <40 (positive), so both The mutation signal can be identified.
在此實施例中: In this example:
第一段溫差區間步驟S11:將待測樣品升溫至95℃維持10分鐘;完成上述升溫步驟後進行控溫至95℃持續15秒,接著進行58℃持續60秒。接著以58℃重複此溫控模組共計35次循環。 Step S11 of the first temperature difference interval: increase the temperature of the sample to be tested to 95°C for 10 minutes; after completing the above heating step, control the temperature to 95°C for 15 seconds, and then proceed to 58°C for 60 seconds. Then repeat this temperature control module at 58°C for a total of 35 cycles.
第二段溫差區間步驟S12:完成前述溫控模組後,在60℃環境中維持1分鐘。 Step S12 of the second temperature difference interval: After completing the aforementioned temperature control module, maintain it in an environment of 60° C. for 1 minute.
第三段溫差區間步驟S13:最後進行升溫後迅速降溫,將完 成上述步驟之樣品升溫至95℃持續30秒,再以4.4℃/秒的速率降溫度至65℃持續30秒,再以2.2℃/秒的速率升溫度至95℃,最後以0.14℃/秒的速率降溫度至40℃,持續10秒。 Step S13 of the third section of temperature difference zone: the temperature is finally raised and then the temperature is quickly lowered. After completing the above steps, the sample is heated to 95°C for 30 seconds, then lowered to 65°C at a rate of 4.4°C/sec for 30 seconds, then raised to 95°C at a rate of 2.2°C/sec, and finally at a rate of 0.14°C/sec Decrease the temperature to 40°C at a rate of 10 seconds.
表2:
參閱圖7及以下表3,針對EGFR exon 20的部分,本發明以健康人之白血球為wild type DNA來源,以含有EGFR exon 20 T790M突變之細胞株H1975為突變DNA來源。在兩者混和後相加的總量約為100ng時,混和不同比例之突變DNA(0.01-100%)。以SEQ ID NO:5及SEQ ID NO:6針對EGFR exon 20基因突變去氧核醣核酸增幅反應之結果示。當標的細胞含有0.01%以上之突變DNA時,可被本發明所設計的EGFR基因突變去氧核醣核酸增幅反應偵測到。當標的細胞不含突變DNA時,不會有非專一性的訊號產生。exon 20使用67-72℃溫度區間反應,已沒有突變DNA的組別F未未跑出Ct值(陰性),而A~E組的Ct值皆有<40(陽性),因此均可以鑑別出突變訊號。
Referring to Figure 7 and Table 3 below, for the part of
在此實施例中: In this example:
第一段溫差區間步驟S11:將待測樣品升溫至95℃維持5分 鐘;完成上述升溫步驟後進行控溫至95℃持續15秒,接著進行66~59℃反應。起始於66℃持續30秒,隨後每個循環降低0.5℃,直至59℃持續30秒。接著以59℃重複此溫控模組共計36次循環。 Step S11 of the first temperature difference interval: increase the temperature of the sample to be tested to 95°C for 5 minutes 钟; After completing the above-mentioned heating step, the temperature is controlled to 95°C for 15 seconds, and then the reaction is carried out at 66~59°C. Start at 66°C for 30 seconds, then decrease by 0.5°C per cycle until 59°C for 30 seconds. Then repeat the temperature control module at 59°C for a total of 36 cycles.
第二段溫差區間步驟S12:完成前述溫控模組後,在72℃環境中維持30秒。 Step S12 of the second temperature difference interval: After completing the aforementioned temperature control module, maintain it in an environment of 72° C. for 30 seconds.
第三段溫差區間步驟S13:最後進行升溫後迅速降溫,將完成上述步驟之樣品升溫至95℃持續30秒,再以4.4℃/秒的速率降溫度至65℃持續30秒,再以2.2℃/秒的速率升溫度至95℃,最後以0.14℃/秒的速率降溫度至40℃,持續10秒。 Step S13 of the third temperature difference interval: After the temperature is raised, the temperature is quickly lowered. The sample after the above steps is raised to 95°C for 30 seconds, and then the temperature is lowered at a rate of 4.4°C/s to 65°C for 30 seconds, and then 2.2°C The temperature was increased to 95°C at a rate of 0.14°C/sec, and finally decreased to 40°C at a rate of 0.14°C/sec for 10 seconds.
表3:
參閱圖8及圖9及以下表3-1、表3-2所示,在此實施例中係採用單一溫度將表現螢光的標的細胞進行PCR反應,將表現螢光的標的細胞升溫至95℃維持10分鐘;完成上述升溫步驟後進行控溫模組:控溫至95℃持續15秒,接著進行67℃或69℃持續25秒。接著以67℃或69℃重複此溫控模 組共計35次循環。完成前述溫控模組後,在72℃環境中維持28秒;最後進行升溫後迅速降溫,將完成上述步驟之樣品升溫至95℃持續30秒,再以4.4℃/秒的速率降溫度至65℃持續30秒,再以2.2℃/秒的速率升溫度至95℃,最後以0.14℃/秒的速率降溫度至40℃,持續10秒。從圖8及表3-1中可看出,使用69℃單一溫度進行測試,已沒有突變DNA的組別F來看Ct值落在34(偽陽性),僅有A與B組的Ct值有<30(陽性),可以鑑別出突變訊號。而在圖9及表3-2中,使用67℃單一溫度進行測試,已沒有突變DNA的組別F來看Ct值落在30(偽陽性),僅有A、B、C組的Ct值有<30(陽性),可以鑑別出突變訊號。 Refer to Figure 8 and Figure 9 and the following Table 3-1 and Table 3-2. In this embodiment, a single temperature is used to perform a PCR reaction on the target cells that exhibit fluorescence, and the temperature of the target cells that exhibit fluorescence is raised to 95 The temperature is maintained for 10 minutes; the temperature control module is performed after the above heating step is completed: the temperature is controlled to 95°C for 15 seconds, and then 67°C or 69°C for 25 seconds. Then repeat this temperature control mode at 67℃ or 69℃ The group has a total of 35 cycles. After completing the aforementioned temperature control module, maintain it in an environment of 72°C for 28 seconds; at the end, increase the temperature and quickly cool down. The sample after completing the above steps is heated to 95°C for 30 seconds, and then the temperature is reduced to 65 at a rate of 4.4°C/sec. The temperature lasts for 30 seconds, then the temperature is increased to 95°C at a rate of 2.2°C/sec, and finally the temperature is decreased to 40°C at a rate of 0.14°C/sec for 10 seconds. From Figure 8 and Table 3-1, it can be seen that using a single temperature of 69℃ for testing, the Ct value of group F, which has no mutated DNA, falls at 34 (false positive), and only the Ct values of groups A and B If there is <30 (positive), the mutation signal can be identified. In Figure 9 and Table 3-2, using a single temperature of 67°C for testing, the Ct value of group F that has no mutant DNA is 30 (false positive), and only the Ct values of groups A, B, and C If there is <30 (positive), the mutation signal can be identified.
表3-1:
表3-2:
從表3-3可看出,在exon 20使用67-72℃溫度區間進行PCR,相較於以67℃或69℃的固定溫度測試,約可以提升10~1000倍的靈敏度。
It can be seen from Table 3-3 that using the 67-72°C temperature range for PCR on
表3-3:
參閱圖10及以下表4所示,針對EGFR exon 21的部分,本發明以健康人之白血球為wild type DNA來源,以含有EGFR exon 21 L858R突變之細胞株H1975為突變DNA來源。在兩者混和後相加的總量約為100ng時,混和不同比例之突變DNA(0.01-100%)。以SEQ ID NO:7及SEQ ID NO:8針對EGFR exon 21基因突變去氧核醣核酸增幅反應之結果。當標的細胞含有0.01%以上之突變DNA時,可被本發明所設計的EGFR基因突變去氧核醣核酸增幅反應所偵測到。當標的細胞不含突變DNA時,不會有非專一性的 訊號產生。Exon 21使用63-70℃溫度區間反應,已沒有突變DNA的組別F未未跑出Ct值(陰性),而A~E組的Ct值皆有<40(陽性),因此均可以鑑別出突變訊號。 Referring to Figure 10 and Table 4 below, for the part of EGFR exon 21, the present invention uses the white blood cells of healthy people as the source of wild type DNA, and the cell line H1975 containing the EGFR exon 21 L858R mutation as the source of mutant DNA. When the total amount added after mixing the two is about 100ng, mix different proportions of mutant DNA (0.01-100%). Take SEQ ID NO: 7 and SEQ ID NO: 8 against the EGFR exon 21 gene mutation DNA amplification reaction results. When the target cell contains more than 0.01% mutant DNA, it can be detected by the EGFR gene mutant DNA amplification reaction designed by the present invention. When the target cell does not contain mutant DNA, there will be no non-specific The signal is generated. Exon 21 uses a temperature range of 63-70°C. Group F, which has no mutated DNA, did not show a Ct value (negative), while the Ct values of groups A to E are all <40 (positive), so they can all be identified Mutation signal.
在此實施例中: In this example:
第一段溫差區間步驟S11:將待測樣品升溫至95℃維持5分鐘;完成上述升溫步驟後進行控溫至95℃持續15秒,接著進行70~63℃反應。起始於70℃持續30秒,隨後每個循環降低0.5℃,直至63℃持續30秒。接著以63℃重複此溫控模組共計41次循環。 Step S11 of the first temperature difference interval: increase the temperature of the sample to be tested to 95°C for 5 minutes; after completing the above-mentioned heating step, control the temperature to 95°C for 15 seconds, and then perform a 70-63°C reaction. Start at 70°C for 30 seconds, then decrease by 0.5°C per cycle until 63°C for 30 seconds. Then repeat the temperature control module at 63°C for a total of 41 cycles.
第二段溫差區間步驟S12:完成前述溫控模組後,在72℃環境中維持30秒。 Step S12 of the second temperature difference interval: After completing the aforementioned temperature control module, maintain it in an environment of 72° C. for 30 seconds.
第三段溫差區間步驟S13:最後進行升溫後迅速降溫,將完成上述步驟之樣品升溫至95℃持續30秒,再以4.4℃/秒的速率降溫度至65℃持續30秒,再以2.2℃/秒的速率升溫度至95℃,最後以0.14℃/秒的速率降溫度至40℃,持續10秒。 Step S13 of the third temperature difference interval: After the temperature is raised, the temperature is quickly lowered. The sample after the above steps is raised to 95°C for 30 seconds, and then the temperature is lowered at a rate of 4.4°C/s to 65°C for 30 seconds, and then 2.2°C The temperature was increased to 95°C at a rate of 0.14°C/sec, and finally decreased to 40°C at a rate of 0.14°C/sec for 10 seconds.
表4:
參閱圖11及圖12及以下表4-1、表4-2所示,在此實施例中係採用單一溫度將表現螢光的標的細胞進行PCR反應,將表現螢光的標的細胞升溫至95℃維持10分鐘;完成上述升溫步驟後進行控溫至95℃持續15秒,接著進行63℃或67℃持續25秒。接著以63℃或67℃重複此溫控模組共計50次循環。完成前述溫控模組後,在72℃環境中維持28秒;最後進行升溫後迅速降溫,將完成上述步驟之樣品升溫至95℃持續30秒,再以4.4℃/秒的速率降溫度至65℃持續30秒,再以2.2℃/秒的速率升溫度至95℃,最後以0.14℃/秒的速率降溫度至40℃,持續10秒。從圖11及表4-1中可看出,使用67℃單一溫度進行測試,整體的螢光強度非常低,整體放大效率不佳,Ct數值也都>30(偽陰性)。而在圖12及表4-2中,使用63℃單一溫度進行測試,已沒有突變DNA的組別F來看Ct值落在32(偽陽性),僅有A、B、C組的Ct值有<32(陽性),可以鑑別出突變訊號。 Refer to Figure 11 and Figure 12 and the following Table 4-1 and Table 4-2. In this embodiment, a single temperature is used to perform a PCR reaction on the target cells that exhibit fluorescence, and the temperature of the target cells that exhibit fluorescence is raised to 95 The temperature is maintained for 10 minutes; the temperature is controlled to 95°C for 15 seconds after the above-mentioned heating step is completed, and then 63°C or 67°C for 25 seconds. Then repeat the temperature control module at 63°C or 67°C for a total of 50 cycles. After completing the aforementioned temperature control module, maintain it in an environment of 72°C for 28 seconds; at the end, increase the temperature and quickly cool down. The sample after completing the above steps is heated to 95°C for 30 seconds, and then the temperature is reduced to 65 at a rate of 4.4°C/sec. The temperature lasts for 30 seconds, then the temperature is increased to 95°C at a rate of 2.2°C/sec, and finally the temperature is decreased to 40°C at a rate of 0.14°C/sec for 10 seconds. It can be seen from Figure 11 and Table 4-1 that using a single temperature of 67°C for the test, the overall fluorescence intensity is very low, the overall amplification efficiency is not good, and the Ct value is also >30 (false negative). In Figure 12 and Table 4-2, using a single temperature of 63°C for testing, the Ct value of group F, which has no mutated DNA, fell to 32 (false positive), and only the Ct values of groups A, B, and C If there is <32 (positive), the mutation signal can be identified.
表4-1:
表4-2:
從表4-3可看出,在exon 20使用63-70℃溫度區間進行PCR,相較於以63℃或67℃的固定溫度測試,約可以提升10~1000倍的靈敏度
It can be seen from Table 4-3 that using
表4-3:
本發明之方法提高了周邊血液循環腫瘤細胞分離後的純度,利於後續的基因突變分析。在微流體多參數細胞分選儀純化出周邊血液循環腫瘤細胞後,再以即時定量PCR進行去氧核醣核酸增幅反應,專一偵測EGFR基因突變,將其運用在癌症診斷與預後治療評估,有利於癌症病人預後治療。 The method of the present invention improves the purity of tumor cells in peripheral blood circulation after separation, which is beneficial to subsequent gene mutation analysis. After the peripheral blood circulating tumor cells are purified by the microfluidic multi-parameter cell sorter, real-time quantitative PCR is used to perform the DNA amplification reaction to specifically detect EGFR gene mutations and use it in cancer diagnosis and prognostic treatment evaluation. For the prognostic treatment of cancer patients.
本發明在上文中以較佳實施例揭露,然所屬技術領域中具有通常知識者應理解的是,該實施例僅用於描述本發明,而不應解讀為限制本發明之範圍。舉凡與該實施例等效之變化與置換,均應設為涵蓋於本發明之範疇內。因此,本發明之保護範圍應當以申請專利範圍所界定者為準。 The present invention is disclosed in a preferred embodiment above. However, those skilled in the art should understand that the embodiment is only used to describe the present invention and should not be construed as limiting the scope of the present invention. All changes and substitutions equivalent to this embodiment should be included in the scope of the present invention. Therefore, the protection scope of the present invention should be defined by the scope of the patent application.
<110> 長庚醫療財團法人林口長庚紀念醫院 <110> Linkou Chang Gung Memorial Hospital
<120> 周邊血液中EGFR基因突變腫瘤細胞偵測之方法 <120> Methods of detecting EGFR gene mutation tumor cells in peripheral blood
<160> 8 <160> 8
<210> 1 <210> 1
<211> 21 <211> 21
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 檢驗EGFR exon 18之前置引子 <223> Test the primer before EGFR exon 18
<400> 1 <400> 1
<210> 2 <210> 2
<211> 22 <211> 22
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 檢驗EGFR exon 18之反置引子 <223> Test the reverse primer of EGFR exon 18
<400> 2 <400> 2
<210> 3 <210> 3
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 檢驗EGFR exon 19之前置引子 <223> Test the primer before EGFR exon 19
<400> 3 <400> 3
<210> 4 <210> 4
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 檢驗EGFR exon 19之反置引子 <223> Test the reverse primer of EGFR exon 19
<400> 4 <400> 4
<210> 5 <210> 5
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 檢驗EGFR exon 20之前置引子
<223> Test the primer before
<400> 5 <400> 5
<210> 6 <210> 6
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 檢驗EGFR exon 20之反置引子
<223> Test the reverse primer of
<400> 6 <400> 6
<210> 7 <210> 7
<211> 19 <211> 19
<212> DNA <212> DNA
<213> 檢驗EGFR exon 21之前置引子 <213> Test the primer before EGFR exon 21
<400> 7 <400> 7
<210> 8 <210> 8
<211> 18 <211> 18
<212> DNA <212> DNA
<213> 檢驗EGFR exon 21之反置引子 <213> Test the reverse primer of EGFR exon 21
<400> 8 <400> 8
S1:步驟a S1: Step a
S2:步驟b S2: Step b
S3:步驟c S3: Step c
S4:步驟d S4: Step d
S5:步驟e S5: Step e
S6:步驟f S6: Step f
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