作者：李鑫^1,2, 尚林东^1,2, 陈福原^1,2, 包晓栋^1,2, 邵帅^1,2, 李备^1,2,3

作者单位：1. 中国科学院长春光学精密机械与物理研究所, 吉林 长春 130028;
2. 中国科学院大学, 北京 100049;
3. 长春长光辰英生物科学仪器有限公司, 吉林 长春 130033

关键词：微流控;LAMP;Covid-19;实时检测;聚类分析

摘要：

肆虐全球近三年的新型冠状病毒肺炎对人类健康构成全球威胁。为更加快速准确地检测鉴定新型冠状病毒肺炎（corona virus disease 2019，Covid-19），该文提出一种基于离心式微流控芯片和比色环介导等温扩增（loop-mediated isothermal amplification，LAMP）的集成化智能检测系统。检测系统主要包括微流芯片、一步上样管、红外温控系统和光电探测器，可以完成对检测样本的定量进样、等温扩增和实时吸光度检测等操作。该文以Covid-19流行病毒RNA模板为实验对象，对该检测系统进行实际表征和优化，使用LDA、SVM聚类分析算法对结果进行实时判定。最终实验结果表明：该检测系统的检测限为200 copies/mL，完全满足现阶段新冠病毒实际检测的现实要求。

A real-time isothermal nucleic acid detection system based on microfluidic technology
LI Xin, SHANG Lindong, CHEN Fuyuan, BAO Xiaodong, SHAO Shuai, LI Bei
1. Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun 130028, China;
2. University of Chinese Academy of Sciences, Beijing 100049, China;
3. Changchun Hooke Instrument Co., Ltd., Changchun 130033, China
Abstract: Corona Virus Disease 2019，which has ravaged the world for nearly three years, poses a global threat to human health. To identify Covid-19 with a faster and more accurate test, an integrated intelligent detection system based on centrifugal microfluidic chips and colorimetric loop-mediated isothermal amplification was presented in this paper. The detection system mainly includes microfluidic chip, one-step sample tube, infrared temperature control system and photodetector, which can complete quantitative sampling, isothermal amplification and real-time absorbance detection of the detected samples. In this paper, the Covid-19 pandemic virus RNA template was used as the experimental object, the actual characterization and optimization of the detection system were carried out, and LDA and SVM clustering analysis algorithms were used for real-time judgment of the results. The final experimental results show that the detection limit of this detection system is 200 copies/mL, which fully meets the practical requirements of Covid-19 detection at the present stage.
Keywords: microfluidic;LAMP;Covid-19;real-time detection;cluster analysis
2023, 49(11):7-15  收稿日期: 2022-10-24;收到修改稿日期: 2022-12-28
基金项目:
作者简介: 李鑫(1996-),男,内蒙古鄂尔多斯市人,硕士,研究方向为微流控疾病检测。
参考文献
[1] 陈秋竹, 吴思娴, 余洋, 等. 新型冠状病毒肺炎COVID-19的实验室诊断技术及相关疾病的鉴别诊断[J]. 中国测试, 2020, 46(3): 64-72.
[2] ZHANG Z, MA P, AHMED R, et al. Advanced point-of-care testing technologies for human acute respiratory virus detection[J]. Adv Mater, 2022, 34(1): e2103646.
[3] TAUBENBERGER J K, MORENS D M. 1918 influenza: the mother of all pandemics[J]. Emerging Infectious Diseases, 2006, 12(1): 15-22.
[4] LU R, WU X, WAN Z, et al. A novel reverse transcription loop-mediated isothermal amplification method for rapid detection of SARS-CoV-2[J]. Int J Mol Sci, 2020, 21(8): 2826.
[5] ROHAIM M A, CLAYTON E, SAHIN I, et al. Artificial intelligence-assisted loop mediated isothermal amplification (AI-LAMP) for rapid detection of SARS-CoV-2[J]. Viruses, 2020, 12(9): 972.
[6] CHEN Q, HE Z, MAO F, et al. Diagnostic technologies for COVID-19: a review[J]. RSC Adv, 2020, 10(58): 35257-35264.
[7] ZHOU C, FANG Z, ZHAO C, et al. Sample-to-answer robotic ELISA[J]. Anal Chem, 2021, 93(33): 11424-11432.
[8] RASTON N H A, NGUYEN V T, GU M B. A new lateral flow strip assay (LFSA) using a pair of aptamers for the detection of Vaspin[J]. Biosensors & Bioelectronics, 2017, 93: 21-25.
[9] 常芮, 蔡伊娜, 王周平, 等. 基于适配体胶体金侧向层析试纸快速检测卡那霉素的方法研究[J]. 分析测试学报, 2021, 40(12): 1728-1735.
[10] ERILL I, CAMPOY S, RUS J, et al. Development of a CMOS-compatible PCR chip: comparison of design and system strategies[J]. Journal of Micromechanics and Microengineering, 2004, 14(11): 1558-1568.
[11] AUGUSTINE R, HASAN A, DAS S, et al. Loop-mediated isothermal amplification (LAMP): a rapid, sensitive, specific, and cost-effective point-of-care test for coronaviruses in the context of COVID-19 pandemic[J]. Biology (Basel), 2020, 9(8): 182.
[12] GONZALEZ-GONZALEZ E, LARA-MAYORGA I M, RODRIGUEZ-SANCHEZ I P, et al. Colorimetric loop-mediated isothermal amplification (LAMP) for cost-effective and quantitative detection of SARS-CoV-2: the change in color in LAMP-based assays quantitatively correlates with viral copy number[J]. Anal Methods, 2021, 13(2): 169-178.
[13] LI Y, FAN P, ZHOU S, et al. Loop-mediated isothermal amplification (LAMP): A novel rapid detection platform for pathogens[J]. Microb Pathog, 2017, 107: 54-61.
[14] MAUTNER L, BAILLIE C K, HEROLD H M, et al. Rapid point-of-care detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP)[J]. Virol J, 2020, 17(1): 160.
[15] NAWATTANAPAIBOON K, PASOMSUB E, PROMBUN P, et al. Colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a visual diagnostic platform for the detection of the emerging coronavirus SARS-CoV-2[J]. Analyst, 2021, 146(2): 471-477.
[16] VAN NGUYEN H, SEO T S. High-throughput human DNA purification on a centrifugal microfluidic device for rapid forensic sex-typing[J]. Biosens Bioelectron, 2021, 181: 113161.
[17] SANGER K, ZÓR K, BILLE JENDRESEN C, et al. Lab-on-a-disc platform for screening of genetically modified E. coli cells via cell-free electrochemical detection of p-Coumaric acid[J]. Sensors and Actuators B: Chemical, 2017, 253: 999-1005.
[18] MADADI M, FATHIPOUR M, GHASEMI J B. Separation of human granulocytes and mononuclear cells from whole blood using percoll on a centrifugal microfluidic disc[J]. Microchemical Journal, 2021, 167: 106316.
[19] 李志刚, 吴晓松, 汪磊, 等. 用于血液病毒核酸检测的微流控PCR温度控制技术研究[J]. 电子测量技术, 2020, 43(13): 152-156.
[20] 李凌梅, 胡建华, 路瑞军, 等. 杠杆表检定数据处理和快速定级的Excel模板[J]. 国外电子测量技术, 2016, 35(10): 21-24.
[21] MAES L, CAROLUS T, DE PRETER V, et al. Technical and clinical validation of three commercial real-time PCR kits for the diagnosis of neuroborreliosis in cerebrospinal fluid on three different real-time PCR platforms[J]. Eur J Clin Microbiol Infect Dis, 2017, 36(2): 273-279.
[22] PARK J E, KIM J Y, YUN S A, et al. Performance evaluation of the real-q cytomegalovirus (CMV) quantification kit using two real-time PCR systems for quantifying CMV DNA in whole blood[J]. Ann Lab Med, 2016, 36(6): 603-606.
[23] SIMOES DUTRA CORREA H, BRESCIA G, CORTELLINI V, et al. DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratories[J]. Electrophoresis, 2020, 41(9): 714-719.
[24] STARR C R. Optimizing the roche LightCycler(R) single-tube multiplexed RT-PCR assays[J]. Journal of Clinical and Diagnostic Research, 2015, 9(1): DM01-DM03.
[25] HATANO B, MAKI T, OBARA T, et al. LAMP using a disposable pocket warmer for anthrax detection, a highly mobile and reliable method for anti-bioterrorism[J]. Japanese Journal of Infectious Diseases, 2010, 63(1): 36-40.
[26] YEH E C, FU C C, HU L, et al. Self-powered integrated microfluidic point-of-care low-cost enabling (SIMPLE) chip[J]. Science Advances, 2017, 3(3): e1501645.
[27] ALAWSI T, MATTIA G P, AL-BAWI Z, et al. Smartphone-based colorimetric sensor application for measuring biochemical material concentration[J]. Sensing and Bio-Sensing Research, 2021, 32: 100404.
[28] 许文艳, 盛涛, 刘升, 等. 基于多波长光谱传感器的水质检测系统[J]. 国外电子测量技术, 2022, 41(6): 112-116.
[29] GERHARDT N, SCHWOLOW S, ROHN S, et al. Quality assessment of olive oils based on temperature-ramped HS-GC-IMS and sensory evaluation: Comparison of different processing approaches by LDA, kNN, and SVM[J]. Food Chemistry, 2019, 278: 720-728.
[30] MAHMODI K, MOSTAFAEI M, MIRZAEE-GHALEH E. Detection and classification of diesel-biodiesel blends by LDA, QDA and SVM approaches using an electronic nose[J]. Fuel, 2019, 258: 116114.1-116114.10.
[31] SAWETWONG P, CHAIRAM S, JARUJAMRUS P, et al. Enhanced selectivity and sensitivity for colorimetric determination of glyphosate using Mn-ZnS quantum dot embedded molecularly imprinted polymers combined with a 3D-microfluidic paper-based analytical device[J]. Talanta, 2021, 225: 122077.
[32] SHAH J H, SHARIF M, YASMIN M, et al. Facial expressions classification and false label reduction using LDA and threefold SVM[J]. Pattern Recognition Letters, 2020, 139: 166-173.
[33] 王瑞, 龙华, 邵玉斌, 等. 基于Labeled-LDA模型的文本特征提取方法[J]. 电子测量技术, 2020, 43(1): 141-146.
[34] SHAMBULINGA M, SADASHIVAPPA G. Supervised hyperspectral image classification using svm and linear discriminant analysis[J]. International Journal of Advanced Computer Science and Applications, 2020, 11(10): 403-409.
[35] 李楠, 邓威, 王晨, 等. 基于K-means聚类与概率神经网络的模拟电路故障诊断方法[J]. 中国测试, 2021, 47(3): 98-103.