【摘要】： 現(xiàn)代醫(yī)學(xué)的研究表明：許多疾病如癌癥和遺傳病的發(fā)生都與基因的突變有關(guān)，，另外許多傳染性疾病是由環(huán)境中的病毒、病菌或寄生蟲所引起，因此對特定序列DNA的分析以及對DNA鏈中堿基突變的檢測在基因篩選、法醫(yī)學(xué)鑒定、遺傳疾病的早期診斷和治療、病原基因的測定方面具有十分深遠(yuǎn)的意義。近年來，快速可靠的特定序列DNA檢測技術(shù)得以迅速發(fā)展。大量的研究以酶、同位素、熒光、電化學(xué)和化學(xué)發(fā)光等標(biāo)記物標(biāo)記DNA探針，建立高靈敏度、高選擇性的特定序列DNA的檢測技術(shù)。鑒于DNA探針標(biāo)記物的存在，一定程度上復(fù)雜化了傳感器的制備和使用，所以無標(biāo)記型DNA檢測技術(shù)的研究成為病原基因測定和基因疾病診斷領(lǐng)域新的研究熱點之一。 化學(xué)發(fā)光(CL)分析法不需要光源，避免了雜散光的干擾，因此具有極高的靈敏度；且這一方法儀器設(shè)備簡單、操作簡便，因而得到了迅速發(fā)展。目前，CL分析成為一個非�；钴S的研究領(lǐng)域，已成功地用于藥學(xué)、生物學(xué)、分子生物學(xué)、臨床醫(yī)學(xué)和環(huán)境學(xué)中各種物質(zhì)的檢測。2003年，本研究小組首次報道了基于鳥嘌呤特異性化學(xué)發(fā)光的無標(biāo)記(瞬時衍生)DNA檢測新技術(shù)。 本論文旨在發(fā)展了一系列具有創(chuàng)新意義的磁性微球表面瞬時衍生化學(xué)發(fā)光DNA檢測技術(shù)，由以下部分構(gòu)成： 第一章：緒論 第一節(jié)，介紹了特定序列DNA檢測的研究進(jìn)展及其意義，內(nèi)容包括：熒光、電化學(xué)、表面基質(zhì)共振和石英微晶天平法等，并列舉了近年來它們在該分析領(lǐng)域的典型示例；第二節(jié)，介紹了主要的CL體系(如魯米諾體系、過氧化草酸酯體系、吖啶酯體系和堿性磷酸酯酶體系)的原理、特點以及應(yīng)用，尤其是在免疫和DNA分析領(lǐng)域的應(yīng)用。第三節(jié)，闡述了本論文的目的和意義，指出了論文的主要研究內(nèi)容及創(chuàng)新之處。 第二章：基于Poly T修飾的磁性微球表面瞬時衍生特定序列DNA化學(xué)發(fā)光檢測新技術(shù) 1．端粒特定序列檢測新技術(shù) 端粒是染色體3’末端的重復(fù)性序列，由一種核糖核蛋白復(fù)合體——端粒酶在一定條件下合成。端粒和端粒酶具有保護(hù)細(xì)胞不被融合、降解的重要作用，它們量的多少與惡性腫瘤等疾病的發(fā)生密切相關(guān)，因此這一基因片段的測定對重大疾病的早期診斷和防治具有重要意義�；诖判晕⑶虮砻嫠矔r衍生化學(xué)發(fā)光，建立了一種新型的端粒特定序列DNA無標(biāo)記檢測技術(shù)。整個分析過程由以下三個實驗步驟構(gòu)成：(1)dT_(20)修飾的磁性微球，與連接有dA_(20)的捕獲探針雜交；(2)再與端粒特定序列DNA進(jìn)行第二次雜交；(3)磁性分離洗滌，基于3，4，5-三甲氧基苯甲酰甲醛(TMPG)與端粒中富含的鳥嘌呤(G)反應(yīng)產(chǎn)生的特異性CL，進(jìn)行端粒DNA的無標(biāo)記檢測。結(jié)果表明：該法具有操作簡便、分析快速和靈敏度高等特點。目標(biāo)序列濃度在5-100 nM濃度范圍內(nèi)具有良好的線性關(guān)系，相關(guān)系數(shù)為0.9918，最低檢測濃度為0.5 nM。 2．炭疽桿菌特定序列檢測新技術(shù) 作為一種全球性的生化武器，炭疽桿菌引起了公眾以及軍事部門的熱切關(guān)注，建立實時有效的識別、檢測技術(shù)，對控制其傳播具有重要意義。本節(jié)以炭疽相關(guān)的一段含有30個堿基的序列作為研究對象，構(gòu)建了無標(biāo)記CL檢測技術(shù)，并進(jìn)一步發(fā)展了基于G_(30)序列的放大檢測方法。放大檢測技術(shù)可描述如下：首先通過A_(20)-T_(20)雜交反應(yīng)將捕獲探針固定于磁性微球表面；捕獲探針15個核苷酸單位與目標(biāo)序列相應(yīng)序列段雜交結(jié)合；然后加入報告序列與目標(biāo)序列的另外15個核苷酸單位進(jìn)行第三次雜交反應(yīng)；利用TMPG與目標(biāo)序列和報告序列中富含的G堿基反應(yīng)產(chǎn)生的CL來檢測。報告序列含有兩個功能序列段：一是能夠與目標(biāo)序列的15個核苷酸單位雜交的序列段，另一個是富含G的序列段——(T_2G_(15))_2，結(jié)果表明：在6-60 nM的濃度范圍內(nèi)，CL信號呈線性增加(R~2=0.9984)，最低檢測濃度為6 nM；采用G_(30)報告序列放大檢測技術(shù)，目標(biāo)序列在0.45-6 nM濃度范圍內(nèi)，線性良好(R~2=0.9917)，最低檢測濃度為0.45 nM(0.045 pmol)；放大檢測技術(shù)較不放大技術(shù)靈敏度提高了一個數(shù)量級，且對A-A單堿基錯配識別良好。 第三章：基于羧基修飾磁性微球表面瞬時衍生的無標(biāo)記化學(xué)發(fā)光檢測新技術(shù) 本章以炭疽桿菌特定序列DNA為研究對象，采用羧基修飾的磁性微球為分離和預(yù)富集載體，通過羧基——氨基縮合反應(yīng)固定捕獲探針，雜交目標(biāo)序列后利用G堿基的特異性CL反應(yīng)來檢測，并在此基礎(chǔ)上發(fā)展了G_(30)放大檢測技術(shù)。該技術(shù)操作簡單，不放大檢測技術(shù)可表述為：在一定條件下，羧基磁性微球經(jīng)過EDC活化后，結(jié)合炭疽桿菌特定序列的捕獲探針，然后通過雜交反應(yīng)結(jié)合目標(biāo)序列，洗滌轉(zhuǎn)移后利用特異性反應(yīng)試劑TMPG直接測定目標(biāo)序列中的G堿基；G_(30)放大檢測技術(shù)是一種夾心方法，在上述第一步雜交完成后，加入一段富含G的報告序列，與目標(biāo)序列另外15個核苷酸單位進(jìn)行第二次雜交反應(yīng)，洗滌轉(zhuǎn)移后檢測TMPG與目標(biāo)序列和報告序列中富含的G反應(yīng)產(chǎn)生的CL信號。結(jié)果表明：該方法具有準(zhǔn)確可靠、重現(xiàn)性和選擇性好的特點。目標(biāo)序列濃度在2-5 nM范圍內(nèi)，CL信號呈線性增加(R~2=0.9962)，最低可檢測濃度為2 nM；G_(30)放大技術(shù)，目標(biāo)序列濃度在50 pM-4 nM范圍內(nèi)，CL信號線性相關(guān)性良好(R~2=0.9965)，最低檢測濃度為50 pM，較不放大技術(shù)靈敏度提高一個數(shù)量級。此外，該技術(shù)較基于Poly T修飾的磁性微球的不放大及G_(30)放大技術(shù)靈敏度提高2.5和10倍，且A-A單堿基錯配識別良好。 第四章：基于碳納米管放大的特定序列DNA化學(xué)發(fā)光檢測新技術(shù) 本章以羧基修飾的磁性微球作為分離以及預(yù)富集載體，以碳納米管(CNTs)這種新型納米材料作為放大載體，發(fā)展了一種高靈敏度CL新技術(shù)檢測特定序列DNA。該技術(shù)利用夾心雜交檢測法：(1)將氨基修飾的捕獲探針固定于經(jīng)過EDC活化的羧基修飾的磁性微球表面；(2)通過第一步雜交將目標(biāo)序列結(jié)合于磁性微球表面；(3)表面羧基功能化的碳納米管，經(jīng)過EDC活化后結(jié)合大量氨基修飾的報告序列形成放大復(fù)合物，通過第二步雜交反應(yīng)將放大復(fù)合物結(jié)合于磁性微球表面；(4)檢測TMPG與目標(biāo)序列和放大復(fù)合物中的G堿基反應(yīng)產(chǎn)生的CL信號。報告序列由兩個功能序列段組成：一是富含G的序列段，二是和目標(biāo)序列互補的序列段；各類CNTs經(jīng)過濃硫酸-過氧化氫(9：1)的混合溶液氧化處理后形成表面羧基功能化的CNTs-COOH；通過TEM以及粒度／zeta電位測定儀對其性質(zhì)進(jìn)行表征。研究了四種碳納米管的放大效果，并最終選用兩種作為載體進(jìn)行特定序列DNA放大檢測；該方法不需要在測定前將待測組分從分離載體上洗脫下來，也不需要用酶、熒光染料等對核苷酸作進(jìn)一步的標(biāo)記，整個實驗可以在3-4 h內(nèi)完成。實驗結(jié)果表明：該法操作較簡便、結(jié)果準(zhǔn)確、能夠高靈敏度檢測目標(biāo)序列；在20 pM-2 nM(2-200 fmol)濃度范圍內(nèi)線性良好(R~2=0.996)，最低檢測濃度為10 pM(1 fmol)；較不放大的方法靈敏度提高200倍，并且能較好的區(qū)別單堿基錯配，為DNA分析提供了一種有效的途徑。 第五章：基于聚苯乙烯微球作為放大載體的特定序列DNA化學(xué)發(fā)光檢測新技術(shù) 本章以商品化的鏈霉親和素聚苯乙烯微球為放大載體平臺，構(gòu)建了一種省時、省力、高效的放大檢測技術(shù)。與碳納米管作為放大載體的技術(shù)相比，不需對放大載體進(jìn)行表面功能化，節(jié)省分析時間，且利于放大技術(shù)的進(jìn)一步發(fā)展。簡單地來說，該技術(shù)原理是：結(jié)合于磁性微球上的捕獲探針以及通過鏈霉親和素——生物素反應(yīng)自組裝于聚苯乙烯微球上的報告序列，在目標(biāo)序列存在條件下三者夾心雜交結(jié)合，無目標(biāo)序列存在，放大復(fù)合物不能結(jié)合于磁性微球表面。雜交反應(yīng)結(jié)束后，磁性分離去除上清液，利用特異性化學(xué)發(fā)光反應(yīng)試劑TMPG對磁性微球上結(jié)合的組分進(jìn)行檢測。結(jié)果表明：該方法簡單方便、穩(wěn)定可靠、快速靈敏，整個檢測過程在2-3 h內(nèi)完成。在10 pM-1 nM濃度范圍內(nèi)具有良好的線性關(guān)系(R~2=0.995)，最低檢測濃度為5 pM(0.5 fmol)，較不放大方法提高400倍，較CNTs放大技術(shù)提高2倍。錯配識別實驗表明該方法可以較好的區(qū)別目標(biāo)序列和各種錯配序列。綜上所述，該法非常適合于高靈敏度檢測特定序列DNA。 第六章：基于DNAzyme的化學(xué)發(fā)光新技術(shù)檢測特定序列DNA 利用羧基修飾的磁性微球作為高效分離載體，基于DNA催化酶(DNAzyme)催化Luminol和H_2O_2產(chǎn)生化學(xué)發(fā)光的原理，構(gòu)建了一種新型的磁性微球表面瞬時衍生特定序列DNA檢測技術(shù)。包括以下分析步驟：(1)羧基磁性微球由EDC活化后，固定氨基修飾的捕獲探針；(2)捕獲探針與目標(biāo)序列的15個寡聚核苷酸單位(5'-AAT ATT GAT AAGGAT-3’)雜交；(3)目標(biāo)序列的另15個寡聚核苷酸單位(5'-GAG GGA TTA TTG TTA-3’)與報告序列的相應(yīng)序列段進(jìn)行第二次雜交，報告序列含有一段能夠自我折疊形成四聚體的序列——d(TIT GGG TAG GGC GGG TTG GG)，能夠特異性結(jié)合血紅素(Hemin)，形成具有HRP相似催化性能的復(fù)合物(DNAzyme)；(4)用Luminol和H_2O_2在堿性條件與洗滌后的磁性微球反應(yīng)，產(chǎn)生CL進(jìn)行檢測�？疾觳�(yōu)化了各種實驗參數(shù)，包括磁性微球量、雜交時間和CL檢測條件等。在優(yōu)化的實驗條件檢測炭疽桿菌特定序列DNA，結(jié)果表明：該法具有操作簡便和分析快速等特點，目標(biāo)序列濃度在0.2-20 nM濃度范圍內(nèi)具有良好的線性關(guān)系，相關(guān)系數(shù)為0.9987，并在堿基錯配分辨中有一定效果，為DNA檢測提供了一種有價值分析手段。 第七章：基于Lumino-NaIO_4-環(huán)糊精流動注射的表面活性劑分析新技術(shù) 這章內(nèi)容是較為早期的工作，旨在發(fā)展一種通用型的流動注射CL分析技術(shù)，用于檢測各種類型的表面活性劑——陽離子型、陰離子型和非離子型表面活性劑。從工業(yè)制造、科學(xué)研究、家庭用品到環(huán)境污染物，表面活性劑是一類隨處可見的化合物。由于它們涉及到社會生活的方方面面，對于它們的研究顯得尤為重要。其中，發(fā)展相應(yīng)的分析檢測方法是對其研究的一個重要方向。本章發(fā)展的技術(shù)原理是：在堿性溶液中，Luminol-NaIO_4-多羥基化合物(如：環(huán)糊精、葡萄糖和甘油等)體系的CL能夠被各種類型的表面活性劑不同程度的淬滅，在一定濃度范圍內(nèi)，淬滅程度與表面活性劑的濃度線性相關(guān)。我們研究了該現(xiàn)象的反應(yīng)機(jī)制，認(rèn)為淬滅原因在于多羥基化合物改變了CL反應(yīng)的微環(huán)境。據(jù)此發(fā)展了適應(yīng)于三種類型表面活性劑檢測的CL技術(shù)，陽離子型表面活性劑—CTMAB、陰離子型表面活性劑—SDS和非離子型表面活性劑—Triton X-100。結(jié)果發(fā)現(xiàn)：三種表面活性劑在4.0μM-0.4 mM濃度范圍內(nèi)線性相關(guān)性良好，三種物質(zhì)的檢測靈敏度為2μM，重復(fù)測定10μM的表面活性劑所得的相對標(biāo)準(zhǔn)偏差約為3.0％。該技術(shù)為多種表面活性劑經(jīng)過HPLC或CE分離后的柱后衍生分析提供了基礎(chǔ)。
[Abstract]:Modern medical research has shown that many diseases, such as cancer and genetic diseases, are related to gene mutations, and many other infectious diseases are caused by viruses, pathogens or parasites in the environment. Therefore, the analysis of specific DNA sequences and the detection of base mutations in DNA strands are involved in gene screening, forensic identification, genetic diseases. In recent years, rapid and reliable DNA detection techniques for specific sequences have been developed rapidly. A large number of studies have been carried out to label DNA probes with enzymes, isotopes, fluorescence, electrochemistry and chemiluminescence markers to establish highly sensitive and selective detection of specific sequence DNA. In view of the existence of DNA probe markers, to a certain extent, the preparation and use of sensors are complicated. Therefore, the study of unlabeled DNA detection technology has become one of the new research hotspots in the field of pathogenic gene detection and gene disease diagnosis.
Chemiluminescence (CL) analysis method does not need light source and avoids the interference of stray light, so it has very high sensitivity; moreover, this method has been developed rapidly because of its simple equipment and easy operation. At present, CL analysis has become a very active research field and has been successfully used in pharmacy, biology, molecular biology, clinical medicine and so on. Detection of various substances in environmental sciences. In 2003, our team reported for the first time a new technique for the detection of unmarked (transient derivative) DNA based on guanine specific chemiluminescence.
This paper aims to develop a series of innovative techniques for the detection of transient derivatization chemiluminescent DNA on magnetic microspheres.
Chapter 1: Introduction
The first section introduces the research progress and significance of DNA detection for specific sequences, including fluorescence, electrochemistry, surface matrix resonance and quartz microcrystalline balance, and lists their typical examples in this field in recent years; the second section introduces the main CL systems (such as luminol system, oxalate peroxide system, acridine ester system). The principles, characteristics and applications of the system and alkaline phosphatase system, especially in the fields of immunity and DNA analysis. Section 3, the purpose and significance of this paper are described, and the main research contents and innovations are pointed out.
Chapter 2: A New Chemiluminescence Detection Technique for Instantaneous Derivation of Specific DNA Sequences on the Surface of Magnetic Microspheres Modified by Poly T
1. new technology for telomere specific sequence detection
Telomere is a repetitive sequence at the 3'end of chromosome. Telomere is synthesized by a ribonucleoprotein complex, telomerase, under certain conditions. Telomere and telomerase play an important role in protecting cells from fusion and degradation. Their amount is closely related to the occurrence of malignant tumors and other diseases. Therefore, the determination of this gene fragment is of great significance. Based on the surface transient derivatization chemiluminescence of magnetic microspheres, a novel labeless detection technique for telomere-specific DNA sequence was developed. The whole analysis process consists of three experimental steps: (1) dT_ (20) modified magnetic microspheres hybridized with a capture probe connected with dA_ (20); (2) the detection of DNA sequence was carried out using a magnetic microsphere. The results showed that the method was simple, rapid and sensitive. The correlation coefficient was 0.9918 and the minimum detection concentration was 0.5 nM.
2. new technology for detection of anthrax specific sequences
As a global biochemical weapon, Bacillus anthracis has attracted great attention from the public as well as military departments. Establishing real-time and effective identification and detection technology is of great significance to control its spread. The amplification detection method based on G_ (30) sequence is developed. The amplification detection technique can be described as follows: Firstly, the capture probe is fixed on the surface of the magnetic microspheres by A_ (20) - T_ (20) hybridization reaction; 15 nucleotide units of the capture probe are hybridized with the corresponding sequence segments of the target sequence; then the report sequence is added to the other 15 nuclei of the target sequence. The report sequence contains two functional segments: one is a sequence segment capable of crossing 15 nucleotide units of the target sequence, the other is a sequence segment rich in G - (T_2G_ (15)) _2, the result table Ming: In the concentration range of 6-60 nM, the CL signal increases linearly (R~2=0.9984) and the minimum detection concentration is 6 nM; the target sequence is linearly good (R~2=0.9917) and the minimum detection concentration is 0.45 nM (0.045 pmol) in the concentration range of 0.45-6 nM using G_ (30) report sequence amplification detection technology; amplification detection technology is more sensitive than non-amplification technology. It is one order of magnitude higher and has good recognition for A-A single base mismatch.
The third chapter: a new technology of unlabeled chemiluminescence detection based on carboxyl modified magnetic microspheres surface.
In this chapter, the specific sequence DNA of Bacillus anthracis was studied. Carboxyl modified magnetic microspheres were used as separation and preconcentration carriers, fixed capture probes were used by carboxyl-amino condensation reaction, hybridized target sequences were detected by G-base specific CL reaction. On this basis, G_ (30) amplification detection technology was developed. Simple, non-amplified detection technique can be described as follows: under certain conditions, carboxyl magnetic microspheres activated by EDC, combined with the capture probe of specific sequence of Bacillus anthracis, then combined with the target sequence by hybridization reaction, washed and transferred using specific reaction reagent TMPG to directly determine the G base in the target sequence; G_ (30) amplified detection technique It is a sandwich method. After the first step of hybridization, a G-rich report sequence is added to the target sequence for the second hybridization. After washing and transferring, CL signals generated by the G-rich reaction between TMPG and target sequence and report sequence are detected. In the range of 2-5 nM, the CL signal increases linearly (R~2=0.9962) and the lowest detectable concentration is 2 nM. In G_ (30) amplification technique, the CL signal has good linear correlation (R~2=0.9965) and the lowest detectable concentration is 50 pM, which improves the sensitivity of non-amplification technique. In addition, the sensitivity of this technique is 2.5 and 10 times higher than that of non-amplification and G_ (30) amplification based on poly T modified magnetic microspheres, and the A-A single base mismatch recognition is good.
The fourth chapter: a new DNA chemiluminescence detection technology based on the specific amplification of carbon nanotubes.
In this chapter, carboxyl-modified magnetic microspheres were used as separation and preconcentration carriers, and carbon nanotubes (CNTs) were used as amplification carriers to develop a novel CL technique with high sensitivity for the detection of specific DNA sequences. (3) Carboxylated carbon nanotubes (CNTs) were activated by EDC and combined with a large number of amino-modified report sequences to form amplified complexes. The amplified complexes were bonded to the surface of magnetic microspheres by the second-step hybridization reaction; (4) Check the surface of the magnetic microspheres. CL signals produced by the reaction of TMPG with the target sequence and the G base in the amplified complex were measured. The report sequence consists of two functional segments: one is a G-rich sequence segment, the other is a complementary sequence segment with the target sequence, and all kinds of CNTs were oxidized by concentrated sulfuric acid-hydrogen peroxide (9:1) mixed solution to form surface carboxyl functionalized CNTs. - COOH; characterized by TEM and particle size/zeta potentiometer. The amplification effect of four kinds of carbon nanotubes was studied. Finally, two kinds of carriers were selected as carriers for amplification detection of specific DNA sequences. This method does not need to elute the components from the separation carrier before determination, nor does it need enzymes, fluorescent dyes and so on. The experimental results show that the method is simple, accurate and can detect target sequences with high sensitivity; the linearity is good in the concentration range of 20 pM-2 nM (2-200 fmol) (R~2=0.996), and the minimum detection concentration is 10 pM (1 fmol); the sensitivity is improved compared with the method without amplification. It is 200 times higher and can better distinguish single base mismatch, thus providing an effective way for DNA analysis.
The fifth chapter: a new DNA chemiluminescence detection technology based on polystyrene microspheres as amplification vectors.
In this chapter, a time-saving, labor-saving and efficient amplification detection technique based on commercial streptavidin polystyrene microspheres is proposed. Compared with carbon nanotubes as amplification carriers, there is no need for surface functionalization of amplification carriers, which saves analysis time and is conducive to further development of amplification technology. The principle of this technique is that the capture probe on magnetic microspheres and the report sequence self-assembled on polystyrene microspheres by streptavidin-biotin reaction are combined by sandwich hybridization in the presence of the target sequence. No target sequence exists and the amplified complex can not be combined on the surface of magnetic microspheres. The results showed that the method was simple, reliable, rapid and sensitive. The whole detection process was completed within 2-3 hours. There was a good linear relationship between the concentration of 10 pM-1 nM (R~2=0.995) and the minimum detection. The detection concentration is 5 pM (0.5 fmol), 400 times higher than that of the non-amplification method and 2 times higher than that of the CNTs amplification technique. Mismatch recognition experiments show that the method can distinguish the target sequence from the mismatch sequence.
The sixth chapter: a new chemiluminescence technology based on DNAzyme to detect specific sequence DNA.
Based on the principle of chemiluminescence of Luminol and H_2O_2 catalyzed by DNA zyme, a novel technique for the detection of specific sequence DNA on the surface of magnetic microspheres was developed by using carboxyl-modified magnetic microspheres as efficient separation carriers. (3) Another 15 oligonucleotide units of the target sequence (5'-GAG GGA TTA TTA-3') were hybridized with the corresponding sequence segments of the report sequence for the second time, and the report sequence contained a segment that could self-fold to form tetramers. The sequence D (TIT GGG TAG GGC GGG TTG GG) specifically binds hemin to form a complex (DNA zyme) with HRP-like catalytic properties; (4) CL is produced by the reaction of Luminol and H_2O_2 with washed magnetic microspheres in alkaline conditions. Various experimental parameters, including the amount of magnetic microspheres and impurity, are investigated and optimized. The results showed that the method was simple and rapid. The linear relationship between the concentration of target sequence and the concentration of target sequence was good in the range of 0.2-20 nM. The correlation coefficient was 0.9987. The method was effective in the discrimination of base mismatch and was suitable for DNA detection. A kind of measurement is provided.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R346

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