Rapid, sensitive and reliable detection of Klebsiella pneumoniae by label-free multiple cross displacement amplification coupled with nanoparticles-based biosensor.

Klebsiella pneumoniae (K. pneumoniae), as an important hospital-acquired bacterium, is responsible for severe morbidity and mortality among the elderly, newborn and immune-compromised people. We established a rcsA gene-based label-free multiple cross displacement amplification (MCDA) assay for rapid, simple and sensitive detection of K. pneumoniae by using lateral flow biosensor (LFB). MCDA reaction was conducted at a fixed temperature (65 °C) for only 30 min, and amplification results were directly indicated using LFB. The results showed that reaction products were detectable from as little as 100 fg and 4.8 CFU of pure K. pneumoniae templates, and from approximately 480 CFU in 1 mL of spiked clinical samples. All K. pneumoniae strains examined were positive for label-free MCDA-LFB analysis, and all non-K. pneumoniae strains used in the report were negative for label-free MCDA-LFB assay, indicating the high selectivity of the label free MCDA-LFB assay. Furthermore, to remove false-positive results, the label-free MCDA-LFB assay was supplemented with antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) to eliminate the carryover contamination. Thus, label-free MCDA-LFB assay complemented with AUDG enzyme was a rapid, simple, sensitive and reliable technique for detection of target pathogen, which has the ability to effectively avoid carryover contamination, and can be a valuable tool for "on-site" detection, clinical diagnosis, and primary quarantine purposes.