Author:
Trung Ngo Tat,Son Le Huu Phuc,Hien Trinh Xuan,Quyen Dao Thanh,Bang Mai Hong,Song Le Huu
Abstract
Abstract
Background
Loop isothermal amplification (LAMP) has recently been proposed as a point-of-care diagnostic tool to detect acute infectious pathogens; however, this technique embeds risk of generating false-positive results. Whereas, with abilities to accurately recognize specific sequence, the CRISPR/Cas12a can forms complexes with cognate RNA sensors and cleave pathogen’s DNA targets complimerntary to its cognate RNA, afterward acquiring the collateral activity to unbiasedly cut nearby off-target fragments. Therefore, if relevant fluorescent-quencher-nucleic probes are present in the reaction, the non-specific cleavage of probes releases fluorescences and establish diagnostic read-outs.
Methods
The MetA gene of N. meningitidis was selected as target to optimize the LAMP reaction, whereas pseudo-dilution series of N. meningitidis gemonics DNA was used to establish the detection limit of LAMP/Cas12a combination assay. The diagnostic performance of established LAMP/Cas12a combination assay was validated in comparation with standard real-time PCR on 51 CSF samples (14 N. meningitidis confirmed patients and 37 control subjects).
Results
In relevant biochemical conditions, CRISPR-Cas12a and LAMP can work synchronously to accurately identify genetics materials of Nesseria menitigistis at the level 40 copies/reaction less than 2 h.
Conclusions
In properly optimized conditions, the CRISPR-Cas12a system helps to alleviate false positive result hence enhancing the specificity of the LAMP assays.
Funder
Vietnam National Foundation for Science and Technology Development
The Vietnamese Ministry of National Defence
Publisher
Springer Science and Business Media LLC
Reference14 articles.
1. Rosenstein NE, Perkins BA, Stephens DS, Popovic T, Hughes JM. Meningococcal disease. N Engl J Med. 2001;344(18):1378–88.
2. Stephens DS, Greenwood B, Brandtzaeg P. Epidemic meningitis, meningococcaemia, and Neisseria meningitidis. Lancet. 2007;369(9580):2196–210.
3. Laboratory Manual for the Diagnosis of Meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae http://www.who.int/emc.
4. Taha MK, Alonso JM, Cafferkey M, Caugant DA, Clarke SC, Diggle MA, Fox A, Frosch M, Gray SJ, Guiver M, et al. Interlaboratory comparison of PCR-based identification and genogrouping of Neisseria meningitidis. J Clin Microbiol. 2005;43(1):144–9.
5. Wong YP, Othman S, Lau YL, Radu S, Chee HY. Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms. J Appl Microbiol. 2018;124(3):626–43.
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