Jichu yixue yu linchuang (Nov 2020)
Capture and ligation-based loop-mediated isothermal amplification technology
Abstract
Objective To establish a capture and ligation-based loop-mediated isothermal amplification technology (CLIP-LAMP) based on probe capture and ligation reaction to provide a new method for laboratory diagnosis and pathogen detection. Methods Capture probes and detection probes were designed with step-loop structure which can hybridize with the target DNA/RNA sequence. Whole blood or dry blood smears were lysed directly in the 96-well capture plate and target nucleic acid was fixed in the tube with capture probes without nucleic acid extraction. Detection probes which hybridized to target sequence were ligated to form a dumb-bell form DNA structure, which can serve as a template in the loop-mediated isothermal amplification. The specificity and sensitivity of this technique were evaluated by application to quantitative detection of 18S rRNA in plasmodium. Moreover, the technique was used to test some clinical samples. Results This method was successfully applied in plasmodium detection with high sensitivity and specificity. The method detected as low as 0.01 parasites/μL, which was more sensitive than RDT and traditional microscopy. Moreover, it accurately detected the 18S rRNA of parasite in infected samples. The method was able to test 96 samples in 4 hours. Conclusions A nucleic acid extraction-free, capture and ligation-based loop-mediated isothermal amplification is developed, which provide a more efficient and sensitive method for molecular diagnosis and screening analysis.
