Abstract:
Objective To establish a simple, rapid, and reliable diagnosis assay of loop-mediated isothermal amplification combined with nanoparticles-based lateral flow biosensor (LAMP-LFB), for the detection of Burkholderia pseudomallei.
Methods A set of six LAMP primers was designed to establish the LAMP assay for the specific amplifying one of type Ⅲ secretion system (TTS1) genes of B. pseudomallei. Nanoparticles-based lateral flow biosensor was used to indicating the results and clinical samples were used to evaluate the LAMP-LFB assay.
Results The optimal amplification conditions of LAMP-LFB assay were 67 ℃ for 40 mins. The sensitivity of the assay was 100 fg of genomic DNA from pure bacterial cultures, and the analytical specificity was 100% by the examination of 258 strains, including 227 strains of B. pseudomallei and 31strains of non-B. pseudomallei. To further demonstrate the feasibility, the LAMP-LFB assay exhibited a diagnostic accuracy of 100% in 46 clinical samples, which is comparable with the culture-biotechnical method. Particularly, the whole detection procedure of LAMP-LFB assay was completed within 1 h, including 15 mins for genomic DNA preparation, 40 mins for loop-mediated isothermal amplification, and 2 mins for the results interpretation visually by biosensor.
Conclusion B. pseudomallei-LAMP-LFB assay is a rapid, sensitive and specific method for the detection of B. pseudomallei, which can be developed as a potential tool to diagnose melioidosis either in field or clinical laboratory.
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