靶向lytA和cpsA的一步法LAMP-CRISPR/Cas12a平台用于肺炎链球菌的快速平行鉴定和毒力分层

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中文摘要

肺炎链球菌（S. pneumoniae，SPN）是引起社区获得性肺炎（CAP）、化脓性脑膜炎、败血症和急性中耳炎的主要病原体。准确的菌种鉴定和侵袭性菌株的快速区分对临床管理和感染控制至关重要。本研究基于环介导等温扩增（LAMP）和CRISPR/Cas12a技术，建立了一个靶向肺炎链球菌特异性lytA基因和荚膜多糖合成调控基因cpsA的平行双靶标检测平台。采用荧光检测（FD）和侧流试纸条（LFTS）相结合的双读出模式，以满足多样化的实验室检测需求。对于每个靶标特异性反应，LAMP扩增和CRISPR/Cas12a切割在单个封闭管中利用石蜡油分层依次进行，以抑制溶剂蒸发，有效防止气溶胶污染。范围摸索的系列稀释试验显示，在全部三个技术重复中产生阳性信号的最低gDNA浓度达到1 fg μL-1（FD）和10 fg μL-1（LFTS）。该检测能特异性地将肺炎链球菌与其他常见临床病原体区分开来，并初步鉴定出有荚膜的侵袭性菌株，其敏感性和特异性均为100%，与传统PCR一致。该LAMP-CRISPR/Cas12a平台采用封闭管设计，避免了污染，同时FD和LFTS双读出满足多样化的检测需求。其具有高灵敏度、优异特异性、简便和快速的特点，为肺炎链球菌的快速诊断提供了一种创新且实用的解决方案，具有很大的临床潜力。

英文摘要

Streptococcus pneumoniae (S. pneumoniae, SPN) is the primary pathogen causing community-acquired pneumonia (CAP), purulent meningitis, sepsis, and acute otitis media. Accurate species identification and rapid differentiation of invasive strains are critical for clinical management and infection control. Based on loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a technologies, this study established a parallel dual-target detection platform targeting the S. pneumoniae-specific lytA gene and the capsular polysaccharide synthesis regulatory gene cpsA. A dual-readout mode combining fluorescence detection (FD) and lateral flow test strip (LFTS) was adopted to meet diverse laboratory testing requirements. For each target-specific reaction, LAMP amplification and CRISPR/Cas12a cleavage were sequentially performed in a single closed tube using paraffin-oil stratification to suppress solvent evaporation, effectively preventing aerosol contamination. Range-finding serial-dilution assays revealed that the lowest gDNA concentrations producing positive signals in all three technical replicates reached 1 fg μL-1 (FD) and 10 fg μL-1 (LFTS). This assay specifically distinguished S. pneumoniae from other common clinical pathogens and preliminarily identified encapsulated invasive strains, with 100% sensitivity and specificity consistent with conventional PCR. This LAMP-CRISPR/Cas12a platform, with its closed-tube design, avoids contamination, while FD and LFTS dual readouts cater to diverse testing needs. Featuring high sensitivity, excellent specificity, simplicity, and speed, it provides an innovative and practical solution for the rapid diagnosis of S. pneumoniae and holds great clinical potential.