Reliable detection of Burkholderia pseudomallei using multiple cross displacement amplification label-based biosensor

Abstract

Background: Burkholderia pseudomallei (B. pseudomallei), as a highly pathogenic organism, causes melioidosis, which is a disease of public health importance in many tropical developing countries. Here, we present and validate a novel detection technique, termed multiple cross displacement amplification combined with nanoparticles-based lateral flow biosensor (MCDA-NB), for identifying B. pseudomallei and diagnosing melioidosis. Results: B. pseudomallei-MCDA targets the TTS1 (Type III secretion system gene cluster 1) to specifically design ten MCDA primers. The nanoparticles-based biosensor (NB) can be combined with B. pseudomallei-MCDA for visually, objective, simply and rapidly reporting reaction results. The optimal amplification conditions of B. pseudomallei-MCDA were 66 °C for 30 min. Assay’s sensitivity was 100 fg of genomic DNA in the pure cultures, and the analytical specificity was 100% by the examination of 257 strains, including 228 B. pseudomallei and 29 non-B. pseudomallei. As a result, the whole detection procedure was completed within 50 min, including 15 min for genomic DNA preparation, 30 min for l MCDA reaction, and 2 min for the interpretation of the results visually by biosensor. Conclusions: B. pseudomallei-MCDA assay is a rapid, sensitive and specific method for the detection of B. pseudomallei, and can be used as a potential tool for melioidosis diagnose in basic, field and clinical laboratories.