Shanvi, San Diego, CA, USA.
Department of Pediatrics, University of California San Diego, 9500 Gilman Drive, MC 0703, Leichtag Building 132, La Jolla, CA, 92093-0703, USA.
Sci Rep. 2024 Jan 10;14(1):1013. doi: 10.1038/s41598-023-49651-8.
mRNA measurement is dominated by RT-PCR, which requires expensive laboratory equipment and personnel with advanced degrees. Loop-mediated isothermal amplification (LAMP) is a versatile technique for detecting target DNA and RNA. The sensitivity of LAMP in early reports has been below that of the standard RT-PCR tests. Here, we report the use of a fluorescence-based RT-LAMP protocol to measure CDX2 expression patterns, which match extremely well to the standards of sophisticated RT-PCR techniques (r = 0.99, p < 0.001). The assay works on diverse sample types such as cDNA, mRNA, and direct tissue sample testing in 25 min compared to more than 3 h for RT-PCR. We have developed a new protocol for designing RT-LAMP primers that reduce false positives due to self-amplification and improve quantification. A simple device with a 3D-printed box enables the measurement of mRNA expression at home, outdoors, and point-of-care setting.
mRNA 的测量主要依赖 RT-PCR，它需要昂贵的实验室设备和具有高等学位的人员。环介导等温扩增 (LAMP) 是一种用于检测靶 DNA 和 RNA 的多功能技术。在早期报告中，LAMP 的灵敏度低于标准 RT-PCR 检测。在这里，我们报告了使用基于荧光的 RT-LAMP 方案来测量 CDX2 表达模式，其与复杂 RT-PCR 技术的标准非常吻合（r = 0.99，p < 0.001）。该测定适用于多种样本类型，例如 cDNA、mRNA 以及直接组织样本测试，只需 25 分钟，而 RT-PCR 则需要 3 小时以上。我们已经开发了一种新的 RT-LAMP 引物设计方案，该方案可减少由于自我扩增引起的假阳性并提高定量准确性。一个带有 3D 打印盒的简单设备可实现在家庭、户外和护理点进行 mRNA 表达测量。
