One-Pot Ligation LAMP Assay of miRNA for Pancreatic Cancer Screening

Abstract

Pancreatic cancer is one of the deadliest cancers due to its late diagnosis rates and low survivability. Typical diagnosis methods such as endoscopic ultrasound and magnetic resonance imaging are not very accessible or affordable. Instead, we can use miRNAs as a cancer biomarker to provide an affordable, time-efficient, and non-invasive cancer screening tool. miRNAs such as miR-21, miR-196a, and miR-221 are upregulated in the blood of patients with pancreatic cancer. In our study, we focused on using ligation-loop-mediated amplification (ligation-LAMP) to detect these biomarkers. To reduce the cost and nonspecific amplification associated with commercial mastermix, we created a buffer for LAMP by varying the amounts of polymerase, H1, H2, and filtered phenol red. Our 2x buffer successfully detected target concentrations of 1 nm, changing color during colorimetric ligation LAMP at around 50 minutes. We also included the One-Pot ligation LAMP system designed to isolate target miRNAs using magnetic beads and TRAP wax layers. Initially, the system failed due to the ineffective isothermal amplification buffer. To improve the One-Pot system, we added an isothermal amplification buffer containing 0.1% Tween 20, but this modification was also unsuccessful. Our buffer will make ligation-LAMP more affordable in future efforts to refine the One-Pot system.