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Focusing on the research and development and application of isothermal nucleic acid amplification technology

RAA Combined with CRISPR-Cas13a Enables Rapid Detection of Four Diarrheal Pathogens—An Bolin

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Objective: To establish a rapid, isothermal detection method for pathogenic bacteria based on recombinase-mediated amplification (RAA) combined with molecular beacons. Methods: The methodology was developed by designing specific primers and molecular beacon probes targeting the gene encoding Staphylococcus aureus protein A (SPA). The optimal primer concentration ratio was determined through continuous adjustment of primer concentrations to achieve asymmetric amplification. Asymmetric RAA was then performed, followed by hybridization with molecular beacon probes. The results were analyzed by agarose gel electrophoresis and fluorescence detection. The sensitivity of the method was evaluated by serially diluting a positive plasmid at 10-fold increments. The specificity of the RAA hybridization assay was tested by analyzing 72 strains—collected in December 2016 and preserved in the Microbiology Laboratory of the Department of Laboratory Medicine at Daping Hospital—including strains of Staphylococcus aureus and other species within the genus Staphylococcus. Based on the specificity study, 39 additional strains preserved in our laboratory from December 2016 were included to perform Kappa consistency analysis and evaluate clinical diagnostic performance. Results: When the concentration ratio of restrictive primers to non-restrictive primers was 1:20, the efficiency of single-stranded DNA (sDNA) generation was highest. The efficiency of asymmetric RAA amplification followed by hybridization with molecular beacon probes was significantly higher than that of symmetric amplification. The sensitivity of this method was 20 copies per reaction. The RAA hybridization assay could distinguish Staphylococcus aureus from other species within the genus Staphylococcus. Compared with the traditional gold standard, the Kappa value was 0.860, indicating good agreement. Among 111 strains tested, the sensitivity of this method was 92.5% (37/40), the specificity was 97.2% (69/71), the positive predictive value was 94.9% (37/39), the negative predictive value was 95.8% (69/72), the positive likelihood ratio was 33.04, the negative likelihood ratio was 0.077, and the Youden index was 0.897. Compared with the traditional gold standard, the Kappa value was 0.902, demonstrating excellent agreement between the two methods. Conclusion: By optimizing the conditions for asymmetric RAA amplification and combining it with molecular beacon probes, we have established a novel RAA hybridization technique for detecting bacterial DNA, laying a foundation for the application of isothermal amplification in clinical settings. (Chinese Journal of Clinical Laboratory Medicine, 2017, 40:309-313)

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Development of a reverse transcription recombinase-aided amplification assay for the detection of coxsackievirus A10 and coxsackievirus A6 RNA (Coxsackievirus)

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