（1. 公安部第一研究所，北京 100048；2. 公安部鉴定中心，北京 100038；3. 西安交通大学生物证据研究院，
国家生物安全证据基地，西安 710049；4. 北京中盾安民分析技术有限公司，北京 102200）
摘　要：本文使用国产24道遗传分析仪搭配国产36 cm毛细管阵列，解决无胶筛分毛细管电泳技术为基础的遗传分析仪在测序中荧光信号校正、运行电压、迁移校正问题。以24道遗传分析仪原始光谱荧光信号为基础，建立光谱校正模型，获得基线噪声阈值。通过运行电压与峰间距值绘制标准曲线，利用测序分析软件计算清晰片段长度，确定最佳运行电压和峰间距，进而计算迁移偏移值，建立碱基大小与迁移时间的线性模型，实现碱基的准确识别。研究表明，在合适的光谱校正和基线噪声阈值限制下，当运行电压设置为10 kV，峰间距为13.05帧时，分析仪的最长清晰片段检验能力最强，为561 bp。通过迁移修正值的补偿，建立碱基大小与迁移时间的线性模型，碱基G、A、T、C的R2（标准曲线）值分别从0.992 7、0.992 7、0.994 5、0.987 9提高到0.999 6、0.999 8、0.999 6、0.999 7，且修正后，各个荧光标记的碱基均能得到正确标记，无漏标和错标，清晰片段长度延长到621 bp。本研究可以指导国产遗传分析仪测序模块的优化和设计，使分析仪的DNA 碱基识别功能更高效和准确。
关键词：国产遗传分析仪；无胶筛分毛细管电泳；碱基测序；运行电压；迁移校正
中图分类号：R394-33                          文献标志码：ADOI：10.3969/j.issn.1007-7146.2024.03.004

(1. First Research Institute of the Ministry of Public Security of PRC, Beijing 100048, China; 2. Institute of Forensic Science of China, Beijing 100038, China; 3. Bio-evidence Sciences Academy, Xi’an Jiaotong University & National Biosafety Evidence Foundation (NBEF), Xi’an 710049, China; 4. Beijing Zhongdun Anmin Analysis Technology CO. Ltd., Beijing 102200, China)
Abstract: A domestic 24 channels genetic analyzer combined with a domestic 36 cm capillary arrays were used to solve the problems of fluorescence signal correction, running voltage, and migration correction during sequencing on the genetic analyzer based on non-gel sieving capillary electrophoresis technology. Spectral calibration model and appropriate baseline noise values were obtained under the condition that the original spectral fluorescence signal of the analyzer was used as a regulation basis. A standard curve was plotted by combining an running voltage and a base spacing value, while a clear range length was also calculated by a sequencing analysis software, so as to determine the optimal running voltage and base spacing. In addition, a migration offset value was further calculated and a linear model was eventually established between the base size and the migration time, which achieved an accurate identification of bases. It was reported that with the appropriate spectral calibration model and baseline noise threshold limitation, the strongest ability to detect the longest clear range length, that is 561 bp, was found in the analyzer when the running voltage was set to 10 kV and the base spacing was 13.05 frames. By compensating for migration corrections, a linear model was established between the base size and the migration time. Among them, R2 values of bases G, A, T, and C increased from 0.992 7, 0.992 7, 0.994 5, and 0.987 9 to 0.999 6, 0.999 8, 0.999 6, and 0.999 7, respectively. After correction, all fluorescent bases were accurately labeled without any omissions or errors, clear range length extended to 621 bp. This study could better guide the optimization and design of a sequencing module of the domestic genetic analyzer, making the DNA base recognition function of the analyzer more efficient and accurate.
Key words: domestic genetic analyzer;  non-gel capillary electrophoresis; base  sequencing; run voltage;  migration correction
（Acta Laser Biology Sinica, 2024, 33(3): 217-226）