Calibration Reagent Research of Isothermal PCR Analyzer Based on Loop-mediated Isothermal Amplification Technology
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摘要: 针对市场上对等温聚合酶链式反应分析仪(PCR仪)校准的迫切需求,研发基于环介导等温扩增(LAMP)技术的等温PCR仪校准试剂和标准体系,以荧光定量PCR仪校准用标准物质(GBW(E)091100-091108)特异性量值和特异性序列为标准,设计LAMP基础引物(F3、B3、FIP、BIP)和环引物(LF、LB),优化筛选最佳基础引物和环引物,确定最佳反应温度,考察试剂盒对应标准物质浓度梯度范围、灵敏度和重复性等,并在市售等温PCR仪上进行校准试剂性能验证。结果显示,LAMP校准试剂盒的最佳反应温度为65℃,标准物质范围覆盖7个数量级(100~106 copies/μL),灵敏度可达100 copies/μL,重复性好。建立的基于LAMP技术的等温PCR仪校准试剂适用于等温PCR仪的计量校准测试,支撑等温PCR仪的性能评价及市场监管,为国家等温PCR仪相关计量校准规范的制定和实施奠定基础。Abstract: To meet the urgent calibration requirements of the isothermal polymerase chain reaction analyzer (PCR instrument) in the market, this paper developed the calibration reagent and standard system for isothermal PCR instruments based on loop-mediated isothermal amplification (LAMP) technology. The specific values and characteristic sequences of the fluorescence quantitative PCR instrument standard material (GBW(E)091100-091108) was utilized as the standard, and LAMP regular primers (F3, B3, FIP, BIP) and loop primers (LF, LB) were designed. The screening tests for optimum regular primers, loop primers and temperature testing were performed; besides, the performance of the calibration kit was investigated by examining its corresponding standard material concentration gradient range, sensitivity, and reproducibility, and then validated based on a commercial isothermal PCR instrument. The results showed that the optimum temperature of LAMP calibration kit is 65℃, the calibration concentration range covers 7 orders of magnitude (100~106 copies/μL), and the minimum detectable concentration can reach 100 copies/μL, demonstrating the advantage of good repeatability. In conclusion, the developed isothermal PCR instruments calibration reagents based on the LAMP technology are suitable for the calibration test of isothermal PCR instruments, support the performance evaluation and market supervision of isothermal PCR instruments, and lay a foundation for the formulation and implementation of national calibration and verification standards for isothermal PCR instruments.
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Key words:
- metrology /
- isothermal polymerase chain reaction /
- LAMP /
- calibration method /
- calibration reagent
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表 1 LAMP基础引物设计
Table 1. LAMP regular primer design
基础引物名 序列 1号-F3 CTGGGTGAAAGGAGACTGT 1号-B3 AGACAGGGCTGTGTTGGC 1号-FIP TGAGAGGGAAATGATTTCAGGACATGAGGGTTACCCCTCGG 1号-BIP GGCATGGAGTCCTCACCCATCGTACAGGTCTTTGCGGATGT 2号-F3 ATGAGGGTTACCCCTCGG 2号-B3 TGCATCCTGTCGGCAATG 2号-FIP GCCACAGGACTCCATGCCTGGCTGTGCTGTGGAAGCTAAG 2号-BIP TGAAGTGTGACGTGGACATCCGCAGGGTACATGGTGGTGC 3号-F3 ACTCTTCCAGCCTTCCTTCC 3号-B3 GCGTACAGGTCTTTGCGG 3号-FIP TAGCTTCCACAGCACAGCCCTGGGTGAGTGGAGACTGTC 3号-BIP TTCCCTCTCAGGCATGGAGTCCTGTCCACGTCACACTTCATG 表 2 LAMP环引物设计
Table 2. LAMP loop primer design
环引物名 序列 1号-LF TAGCTTAATCAGGAGAGCC 1号-LB ACCTTCTTCTCCATCATGAAGTG 2号-LF TTAGCTTCCACAGCACAGCC 2号-LB TTCAACTCCATCATGAAGTGTGAC 3号-LB CTCCATCATGAAGTGTGACGTG -
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