本文關(guān)鍵詞：基于封閉式卡盒的現(xiàn)場傳染病病原體檢測系統(tǒng)的研制與應(yīng)用　出處：《東南大學》2016年博士論文　論文類型：學位論文

  更多相關(guān)文章： 封閉式卡盒 現(xiàn)場檢測 3D打印技術(shù) 多重PCR 核酸檢測系統(tǒng)

【摘要】：近年來,由于環(huán)境變化、自然災(zāi)害、物種多樣性變化等因素,導致各種病毒引起的傳染性疾病不斷爆發(fā)并重復(fù)性出現(xiàn),如SARS病毒、H1N1、H7N9禽流感病毒、中東呼吸綜合征冠狀病毒以及這兩年來談之色變的埃博拉病毒,寨卡病毒等。而且隨著全球化進程的加快以及傳染病病原體傳播途徑的多變性,使得這些傳染病的爆發(fā)不再是局部地域出現(xiàn),更是在全球范圍內(nèi)迅速流行,給人類健康和社會帶來極大的危害。因此,對傳染性疾病的防控、診斷和治療已成為全球公共衛(wèi)生領(lǐng)域的熱點和重點。然而傳統(tǒng)的傳染性病原體檢測方法大都只適用于實驗室部署環(huán)境,具有檢測過程分散、繁瑣、實驗操作復(fù)雜、對環(huán)境敏感等缺點,并且對實驗員的實驗技能要求較高。這些弊端都大大限制了傳統(tǒng)檢測技術(shù)在現(xiàn)場傳染病檢測中的應(yīng)用。傳染病病原體現(xiàn)場快速檢測要求具有操作簡單、成本低、便攜和易于實現(xiàn)自動化等特點,因此,急需尋找一種新的技術(shù)平臺以滿足傳染病的現(xiàn)場檢測需求。本論文首先利用3D打印技術(shù),設(shè)計并制作了封閉式的檢測卡盒,在此基礎(chǔ)上研發(fā)了全自動的便攜式核酸檢測系統(tǒng),并對儀器進行了各項性能測試及實際現(xiàn)場傳染病的檢測,成功地完成了現(xiàn)場傳染病從樣本輸入到結(jié)果輸出的自動化檢測過程。具體內(nèi)容包括:1.基于3D打印技術(shù)的封閉式檢測卡盒的設(shè)計與制造首先根據(jù)病原體核酸檢測的實驗流程明確了封閉式卡盒的設(shè)計需求,提出了基于柔性滑片的封閉式卡盒結(jié)構(gòu)及其內(nèi)部各功能模塊。整個設(shè)計與制造過程采用了高精度的3D打印技術(shù)及醫(yī)用級PC-ABS材料,共經(jīng)歷5個批次的快速迭代試制過程,設(shè)計和優(yōu)化了卡盒7個特殊零件的結(jié)構(gòu),組裝完成的卡盒尺寸為140mm× 90 mm × 30 mm。其內(nèi)部集成移液模塊、樣本處理及檢測區(qū)域,樣本處理范圍為20～1000μL,可同時進行1～6項實時熒光PCR檢測。液體操作范圍為2～200μL,實測在10 μL和100μL處的移液相對誤差分別為1.71%和0.76%,重復(fù)性CV值分別為2.3%和0.77%,均優(yōu)于國標規(guī)定。同時,對卡盒內(nèi)的磁珠法核酸提取及PCR配制過程進行了優(yōu)化。最后,實驗證明了上游核酸提取試劑的揮發(fā)對于下游PCR檢測并未造成抑制影響,說明了在封閉式卡盒內(nèi)進行一體化核酸檢測是可行的。2.基于封閉式卡盒的現(xiàn)場病原體檢測系統(tǒng)的研發(fā)以封閉式卡盒為基礎(chǔ),研發(fā)了與之配套的自動化驅(qū)動系統(tǒng),其內(nèi)部由移液控制、磁分離加熱、熱循環(huán)、熒光采集及輔助電路等功能模塊組成。其中,移液控制模塊的最大操作量程為260 μL,精度為0.118 μL,內(nèi)含濾芯以防止交叉污染;磁分離加熱模塊采用傾斜式滑動結(jié)構(gòu),可程序化控制磁富集的位置,且溫控精度為±0.5℃,可在2min內(nèi)升至目標溫度;熱循環(huán)模塊可提供3通道的PCR反應(yīng)條件,其升降溫速度分別為4.6℃/s及4.0℃/s,控制精度為±0.3℃;熒光采集模塊提供雙色熒光通道(FAM/SYBR Green和Texas Red波長的范圍的),采用單色LED作為激發(fā)光源,波動性小于1μW,單次熒光信號的采集在2.5 s內(nèi)完成。儀器整體尺寸為270 mm × 275 mm × 250mm,重量為6.5 kg�，F(xiàn)場操作時僅需將帶樣本的卡盒放入儀器中,并配置實驗參數(shù),則可自動完成整個檢測流程。3.現(xiàn)場病原體檢測系統(tǒng)的性能測試首先使用百日咳病毒質(zhì)粒DNA進行了核酸提取效率的對比實驗,系統(tǒng)和手工提取的效率分別為95.49%和84.33%;使用HBV質(zhì)粒DNA測試獲得了 104 copies/mL的提取限,說明系統(tǒng)的樣本處理能力在效率和靈敏度方面均可滿足下游PCR檢測的需求。同時,分別使用E.coli及HBV病毒的真實臨床樣本進行了核酸提取對比實驗,結(jié)果顯示采用本系統(tǒng)操作在產(chǎn)量上比手工操作分別高出6.6 ng/μL和0.93 ng/μL。通過平行實驗測得本系統(tǒng)的實時熒光PCR檢測孔間差異為0.023(CV值),檢測限為104copies/mL。最后,使用本系統(tǒng)連續(xù)運行20次陰性和陽性樣本的間隔實驗,測試結(jié)果與預(yù)期一致,表明系統(tǒng)沒有發(fā)生批間交叉污染的現(xiàn)象。4.現(xiàn)場傳染病檢測實驗在江蘇省疾病防控中心進行了腺病毒的檢測實驗,并采用羅氏LightCycler 2.0熒光定量PCR儀進行實驗對照,獲得未知樣本的Ct值分別為31.15和31.68,陽參的Ct值分別為27.52和28.01,從擴增曲線形態(tài)和Ct值的比較說明兩系統(tǒng)的檢測結(jié)果較為一致。最后,使用本卡盒系統(tǒng)對沙門氏菌及志賀氏菌進行聯(lián)合檢測,并設(shè)置卡盒內(nèi)采用懸滴加樣的方式進行PCR體系的配置,同時采用ABI SteponePlus實時熒光定量PCR系統(tǒng)進行實驗對照。兩系統(tǒng)的擴增曲線形態(tài)一致,兩路熒光檢測(FAMandTexasRed)的Ct值均值差值分別為0.71和0.98左右,僅相差0.29,結(jié)果表明卡盒系統(tǒng)在多重檢測方面與商用系統(tǒng)的檢測結(jié)果較為一致。
[Abstract]:In recent years, due to changes in the environment, natural disasters, changes in species diversity and other factors, resulting in various infectious diseases caused by virus outbreak and appear repeatedly, such as SARS H1N1, H7N9 virus, avian influenza virus, coronavirus mers and this year to talk about the Ebola virus discoloration, Zika virus and so on. With the accelerating process of globalization and the variability of the pathogen of infectious diseases spread, the outbreak of the disease is no longer a local region, it is rapidly popular in the global scope, bring great harm to human health and society. Therefore, the prevention and control of infectious diseases, diagnosis and treatment has become the focus in the world in the field of public health. However, most of the traditional infectious pathogen detection method is only applicable to the lab deployment environment, the detection process has dispersed, cumbersome, complex operation experiment, The disadvantage of such sensitive environment, and experimental skills of laboratory technician requirements higher. These disadvantages greatly limits the application of traditional detection techniques in the detection of infectious disease in the field. The pathogens of infectious diseases on-site rapid detection requirements has the advantages of simple operation, low cost, portable and easy to realize automation, therefore, the urgent need to find a new in order to meet the needs of the scene detection technology platform for infectious diseases. This paper first use of 3D printing technology, design and production of the closed box card detection, on the basis of the research and development of portable automatic nucleic acid detection system, and the instruments for the test of the performance test and the practical field of infectious diseases, the successful completion of the site infectious diseases from the sample input to the automatic detection of process output results. The specific contents include: 1. the design and manufacture of box card closed 3D printing technology based on Detection Based on The experimental process of pathogen nucleic acid detection clear design requirements of the enclosed card box, the closed type flexible vane cartridge structure and its internal modules. Based on the design and manufacturing process using 3D printing technology with high accuracy and medical grade PC-ABS material, has experienced rapid iterative development process in 5 batches. The design and optimization of the structure of 7 special parts of the card box, card box size assembled 140mm * 90 mm * 30 mm. the internal integrated pipetting module, sample processing and detection area, sample processing range is 20 ~ 1000 L, and 1 ~ 6 real time fluorescent PCR detection of liquid operation. The range is 2 ~ 200 L, measured at 10 L and 100 L of the pipette relative errors are 1.71% and 0.76%, the repeatability CV values were 2.3% and 0.77%, are better than the national standard. At the same time, the method of nucleic acid extraction and PCR magnetic card box preparation process Optimized. Finally, experiments show that the upstream nucleic acid extraction reagent volatilization on the downstream PCR detection did not cause inhibitory effect, explained in the enclosed card box for the integration of nucleic acid detection is feasible based on the.2. research site blocking pathogen detection system card box with the enclosed card box based automation and development the auxiliary drive system, the internal control by pipetting, magnetic separation heating, thermal cycling, fluorescence collection and auxiliary circuit modules. The maximum operating range shift hydraulic control module is 260 L, the precision is 0.118 L, containing a filter to prevent cross contamination; heating module by inclined magnetic separation sliding structure, capable of controlling the position of magnetic enrichment, and the precision of temperature - 0.5 DEG C, but to the target temperature in 2min; thermal cycle module can provide the PCR reaction conditions of 3 channels, the temperature rise speed Don't is 4.6 DEG /s and 4 DEG /s, the control precision is + 0.3 DEG C; the fluorescence collection module of dual color fluorescence channel (FAM/SYBR Green and Texas Red range of wavelength), using monochromatic LED as excitation source, volatility is less than 1 W, the single fluorescence signal acquisition was completed within 2.5 s. The size of the instrument was 270 mm * 275 mm * 250mm, the weight for the card box 6.5 kg. site operation will only be with the sample into the instrument configuration, and experimental parameters can automatically complete the performance testing of the experimental detection process.3. pathogen detection system of the first use of pertussis virus plasmid DNA was extracted efficiency nucleic acids, efficiency of the system and manual extraction were 95.49% and 84.33%; the use of HBV plasmid DNA was extracted from 104 copies/mL test limit, explains the sample processing ability of the system in terms of efficiency and sensitivity can meet the test needs of downstream PCR Pray. At the same time, the real clinical samples were studied using E.coli and HBV virus were nucleic acid extraction experiments, results show that the yield ratio of manual operation were higher than 6.6 ng/ L and 0.93 ng/ L. through parallel experiments between real-time PCR detection system of the hole is 0.023 by the operation of the system (CV value), the detection limit is 104copies/mL. finally, the continuous operation of the system 20 times of positive and negative sample interval test, the test results were consistent with the expected.4. field indicates the phenomenon of infectious disease detection system without cross contamination between batch experiments of adenovirus detection in Jiangsu Province Center for Disease Control and prevention, and the use of Roche LightCycler 2 fluorescence quantitative PCR instrument experiment obtained the unknown sample Ct values were 31.15 and 31.68, Yang joined the Ct values were 27.52 and 28.01, compared with that from the amplification curve shape and Ct value Two more consistent detection system results. Finally, the use of the card box system for joint detection of Salmonella and Shigella, and set the PCR card system using the hanging drop and the way in the box configuration, at the same time the control experiment by using real-time quantitative PCR system ABI. SteponePlus amplification curve form two the two fluorescence detection (FAMandTexasRed) of the Ct mean value of difference is about 0.71 and 0.98, a difference of only 0.29. The results show that more consistent detection card box system and commercial system in the multiple detection results.

【學位授予單位】：東南大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：TP274;R446

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