摘要

Background: The SNP rs671 of Human aldehyde dehydrogenase (ALDH) is G-A transition at 1510th nucleotides, which is an important clinical indicator of alcoholic liver disease, digestive tract cancer and some drug efficiency. The commonly used genotyping assay of this polymorphism is relatively time-consuming and costly. Finding: This study develops a rapid and accurate one-step CRISPR/Cas12b assay to distinguish the G1510A polymorphism of human ALDH2 free of DNA amplification. The method we established requires only one step of adding 1 μl genomic DNA sample to premixed system, and waiting for the acquisition of fluorescent signal, taking approximate 30 min. Conclusions: This method provides a potential tool for more accurate and reliable nucleic acid detection with a single base difference and supports the relevant disease diagnosis and personalized medicine.

A novel method using vitrified CRISPR/Cas12b reagent is developed for rapid, amplification-free genotyping of two single nucleotide polymorphisms (SNPs) in human SLCO1B1 genes. With optimized vitrification, the reagents can be stored at room temperature.

Background: Heat-labile uracil-DNA glycosylase (HL-UDG) is commonly employed to eliminate carry-over contamination in DNA amplifications. However, the prevailing HL-UDG is markedly inactivated at 50°C, rendering it unsuitable for specific one-step RT-qPCR protocols utilizing reverse transcriptase at an optimal temperature of 42°C. Objective: This study aimed to explore novel HL-UDG with lower inactivation temperature and for recombinant expression. Methods: The gene encoding an HL-UDG was cloned from the cold-water fish rainbow trout (Oncorhynchus mykiss) and expressed in Escherichia coli with high yield. The thermostability of this enzyme and other enzymatic characteristics were thoroughly examined. The novel HL-UDG was then applied for controlling carry-over contamination in one-step RT-qPCR. Results: This recombinantly expressed truncated HL-UDG of rainbow trout (OmUDG) exhibited high amino acids similarity (84.1% identity) to recombinant Atlantic cod UDG (rcUDG) and was easily denatured at 40°C. The optimal pH of OmUDG was 8.0, and the optimal concentrations of both Na+ and K+ were 10 mM. Since its inactivation temperature was lower than that of rcUDG, the OmUDG could be used to eliminate carry-over contamination in one-step RT-qPCR with moderate reverse transcription temperature. Conclusion: We successfully identified and recombinantly expressed a novel HL-UDG with an inactivation temperature of 40°C. It is suitable for eliminating carry-over contamination in one-step RT-qPCR.