基于LAMP技术的等温PCR仪校准试剂研究

陈娴; 吴枭; 王志栋; 高运华

doi:10.12338/j.issn.2096-9015.2024.0203

基于LAMP技术的等温PCR仪校准试剂研究

doi: 10.12338/j.issn.2096-9015.2024.0203

中国计量科学研究院，北京 100029

基金项目: 国家质量基础设施体系专项（2023YFF0614603）；中国计量科学研究院基本科研业务费项目（AKYCX2305）。

详细信息

作者简介:
陈娴（1997-），中国计量科学研究院助理研究员，研究方向：生物计量，邮箱：chenxian@nim.ac.cn

通讯作者:
高运华（1972-），中国计量科学研究院研究员，研究方向：生物计量，邮箱：gaoyh@nim.ac.cn

计量
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出版历程
- 收稿日期: 2024-06-25
- 录用日期: 2024-08-13
- 修回日期: 2024-07-28
- 网络出版日期: 2024-08-29

Calibration Reagent Research of Isothermal PCR Analyzer Based on Loop-mediated Isothermal Amplification Technology

National Institute of Metrology, Beijing 100029, China

摘要

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。
- 计量学 /
- 等温聚合酶链式反应 /
- 环介导等温扩增技术 /
- 校准方法 /
- 校准试剂
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.
- metrology /
- isothermal polymerase chain reaction /
- LAMP /
- calibration method /
- calibration reagent

HTML全文

图 1 LAMP基础引物筛选实验结果

Figure 1. Results of screening test for regular primers

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图 2 LAMP环引物筛选实验结果

Figure 2. Results of screening test for loop primers

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图 3 LAMP温度筛选实验结果

Figure 3. Results of screening test for temperature

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图 4 LAMP浓度梯度测试实验结果

Figure 4. Results of concentration gradient testing

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图 5 市售等温PCR仪校准实验结果

Figure 5. Calibration results of commercially available isothermal PCR instrument

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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

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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

下载: 导出CSV

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