Neisseria meningitidis is a Gram-negative bacterium that causes severe meningitis and sepsis. These diseases require fast and accurate diagnostics to indicate proper antimicrobial therapies [1, 2]. So far, together with blood culture, polymerase chain reaction (PCR) is recommended as a routine technique for the diagnostic confirmation [3, 4]. PCR requires laboratories with sophisticated infrastructures and well-trained personnel, therefore making challenges for deploying in limited-resource areas. Loop-mediated isothermal amplification (LAMP) based approaches have been used to detect pathogens . LAMP-based assays are faster and require no sophisticated instruments or/and skilled personnel, therefore having the advantage to use as on-site diagnostic device .
LAMP can achieve PCR’s sensitivity without complicated thermocycling, some LAMP assays can be completed within 30 min. However, LAMP detection step acquires non-specific indicators (such as Mg2+, intercalating dyes, labelled primers) that cannot distinguish spurious amplicons [7,8,9]. We documented several phenomena that real-time PCR protocols [4, 10] could not recapitulate positive results gained by LAMP reactions and some LAMP positive cases lacked meningitidis specific clinical symptoms .
We suspected that LAMP assay might embed risks of generating false positive [7,8,9]. Whereas, with abilities to accurately recognize specific sequences, the CRISPR-Cas system holds promising potentials to tackle the above-mentioned problem: In this system, the DNAse cas12a forms a complex with their cognate CRISPR RNAs to induce the cleavage of pathogen’s RNA or DNA in a sequence-specific manner. Afterwards, the collateral transcleavage activity is also triggered to unbiasedly cut the nearby off-target fragments. If relevant fluorescent-quencher-nucleic probes are present in the reaction, the non-target cleavage of probes will release fluorescent signals and establish diagnostics read-outs [11,12,13]. Thus, in this study, we have established an effective method combining the LAMP assay and the CRISPR-Cas12a system for the diagnosis of patients infected with Neisseria meningitidis.