RESUMO
In view of given that the current development trend of nucleic acid molecular detection technology is gradually shifting from clinical laboratory to field detection, there is an urgent need to establish a new nucleic acid diagnostic tool with high sensitivity, high specificity, great efficiency and convenience portability to meet the needs of clinical point-of-care testing(POCT). The detection method based on clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein (Cas) is a promising new nucleic acid detection method. In this method, the Cas effector protein would recognize highly specific nucleic acid sequences, and it can be used for POCT in combination with a highly sensitive small biosensor. This method can successfully achieves the high-efficiency and convenient detection characteristics while satisfying high sensitivity and high specificity. This article reviews the developments progress in the field of miniaturized analytical sensors related to CRISPR/Cas technology in recent years, and discusses the research prospects and challenges of related POCT detection.
RESUMO
ObjectiveTo establish a droplet digital PCR (ddPCR) method for detecting hepatitis B virus (HBV) covalently closed circular DNA (cccDNA). MethodsHBV cccDNA standard substance was constructed, and HBV cccDNA primers and probes were designed based on the structural differences between HBV cccDNA and relaxed circular DNA (rcDNA). HBV plasmid was amplified to obtain HBV cccDNA standard substance, and a ddPCR detection method was established with the standard substance after gradient dilution as the template for HBV cccDNA detection; the limit of detection and repeatability of this method were analyzed. Liver tissue samples were collected from 20 patients who attended Beijing YouAn Hospital, Capital Medical University, from June 2017 to October 2020, all of whom were diagnosed with HBV infection, and DNA of the samples was extracted and digested with plasmid-safe ATP-dependent DNA enzyme to obtain HBV cccDNA template; the ddPCR detection method was evaluated in clinical samples and was compared with the quantitative real-time PCR (qPCR) detection method. The chi-square test was used for comparison of categorical data between the two groups. ResultsThe HBV cccDNA detection method based on ddPCR was established, which accurately detected HBV cccDNA in standard substance after gradient dilution, with a limit of detection of 1 copy/μl, and the coefficients of variation of 1×103, 1×102, and 1×101 copies/μl standard substances were 441%, 3.98%, and 5.09%, respectively. HBV cccDNA was detected in the samples of 20 patients with HBV infection; the ddPCR detection method detected HBV cccDNA in 17 patients, with a positive rate of 85%, while the qPCR detection method detected HBV cccDNA in 11 patients, with a positive rate of 55%, and there was a significant difference between the two methods (χ2=4.286, P=0038). ConclusionThe established ddPCR method for detecting HBV cccDNA has a low limit of detection and good repeatability, which provides an effective tool for further clinical detection.