Accurate estimation and rapid temperature control methods of PCR temperature in tube
ZHANG Yueye, YAO Jia, ZHANG Zhiqi, LI Jinze, ZHOU Lianqun
1. School of Mechanical and Electrical Engineering, Changchun University of Science and Technology, Changchun 130022, China;
2. Key Laboratory of Bio-medical Diagnosis, Suzhou Institude of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, China
Abstract: The polymerase chain reaction (PCR) analyzer takes the sample block temperature as the control object to control the sample temperature, resulting in thermal hysteresis. To reduce the thermal hysteresis and shorten the temperature change time of sample, this paper is based on the establishment of single-hole sample thermal conduction model by the finite element analysis technique, a three-parameter model that fused the initial temperature-target temperature-equivalent thermal resistance is constructed to predicted the sample temperature in tube and achieve real-time temperature tracking in the PCR process. According to the predicted temperature in the tube, the temperature of the sample block is accurately and quickly controlled, which greatly shorten the heating and cooling time and reduce the heat transfer hysteresis of the sample in the tube. The experimental results show that compared with the actual temperature in the tube, the error of the method is less than ±1.5 ℃. The temperature hysteresis time in tube is reduced by more than 27% after optimizing the target curve of sample block temperature. The optimization method of accurate control of the temperature overshoot in this paper significantly shorted the overall PCR thermal cycle time on the basis of maintaining the sample temperature in the tube, which was conducive to achieving faster and more accurate nucleic acid quantitative detection results.
Keywords: polymerase chain reaction;finite element analysis;temperature prediction;temperature optimization
2023, 49(11):150-156  收稿日期: 2023-02-11;收到修改稿日期: 2023-04-07
基金项目: 国家重点研发计划资助项目（2022YFC2409300）；江苏省社会发展重点研究开发项目（BE2020768）；中国科学院生物医学检验技术重点实验室开放课题资助项目(A2023F001)
作者简介: 张月业(1997-),男,河南台前县人,硕士研究生,专业方向为PCR温度控制。
参考文献
[1] ZHU H L, ZHANG H Q, XU Y, et al. PCR past, present and future[J]. Biotechniques, 2020, 69(4): 317-325.
[2] CODONY F, DINH-THANH M, AGUSTI G. Key factors for removing bias in viability PCR-based methods: a review[J]. Current Microbiology, 2020, 77(4): 682-687.
[3] CHEN H, SUN C, WANG Y, et al. Rapid detection of SARS-CoV-2 using duplex reverse transcription-multienzyme isothermal rapid amplification in a point-of-care testing[J]. Frontiers in Cellular and Infection Microbiology, 2021, 11: 678706.
[4] LU Y, LI M C, LIU H C, et al. Detecting mycobacterium tuberculosis complex and rifampicin resistance via a new rapid multienzyme isothermal point mutation assay[J]. Analytical Biochemistry, 2021, 630: 114341.
[5] 明焱, 冯汝鹏, 朴永杰. 基于新冠病毒荧光检测的RT-qPCR温控系统[J]. 电子测量技术, 2022, 45(6): 18-23.
[6] 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.
[7] LAMIEN-MEDA A, FUEHRER H-P, LEITSCH D, et al. A powerful qPCR-high resolution melting assay with taqman probe in plasmodium species differentiation[J]. Malaria Journal, 2021, 20(1): 121.
[8] KREITLOW A, BECKER A, SCHOTTE U, et al. Establishment and validation of a loop-mediated isothermal amplification (LAMP) assay targeting the ttrRSBCA locus for rapid detection of Salmonella spp. in food[J]. Food Control, 2021, 126: 107973.
[9] 罗媛媛, 姚佳, 李东书, 等. 实时数字PCR扩增曲线的分类[J]. 光学精密工程, 2021, 29(9): 2178-2188.
[10] 李姗姗, 王子超, 于成壮, 等. 数字液滴PCR图像的荧光信息提取[J]. 光学精密工程, 2022, 30(7): 821-829.
[11] 李树力, 李金泽, 郭振, 等. 蜂窝状数字PCR微阵列荧光图像的信息提取[J]. 光学精密工程, 2020, 28(12): 2745-2755.
[12] JIANG Y, LI B, WU W. Application of automatic feedback photographing by portable smartphone in PCR[J]. Sensors and Actuators B-Chemical, 2019, 298: 126782.
[13] 陈刘平. 面向芯片级核酸扩增的微流体温度控制方法研究[D]. 天津: 河北工业大学, 2022.
[14] 尚君鹏, 贺晓伟, 方秋雨, 等. 智能数字聚合酶链式反应系统的开发与应用验证[J]. 分析测试技术与仪器, 2022, 28(1): 18-23.
[15] SANFORD L N, WITTWER C T. Monitoring temperature with fluorescence during real-time PCR and melting analysis[J]. Analytical Biochemistry, 2013, 434(1): 26-33.
[16] 许秀峰, 陆敏恂, 周爱国, 等. 基于Smith 预估器的PCR仪时滞温控系统[J]. 同济大学学报(自然科学版), 2015, 43(2): 293-298.
[17] HUANG J, CHEN Z, YAO Y, et al. Simulation and experimental study of temprature lag time for polymerase chain reaction instrument[C]//2010 3rd International Conference on Biomedical Engineering and Informatics (BMEI 2010), 2010.
[18] BU M, PERCH-NIELSEN I R, SORENSEN K S, et al. A temperature control method for shortening thermal cycling time to achieve rapid polymerase chain reaction (PCR) in a disposable polymer microfluidic device[J]. Journal of Micromechanics and Microengineering, 2013, 23(7): 074002.
[19] 张辰, 孙继贤, 梁晓会, 等. PCR仪温度过冲特性有限元仿真研究[J]. 中国测试, 2022, 48(4): 117-122. DOI: 10.11857/j.issn. 1674-5124.2021030032.