Abstract
Acinetobacter baumannii is a frequent cause of the nosocomial infections. Herein, a novel isothermal amplification technique, multiple cross displacement amplification (MCDA) is employed for detecting all A. baumannii strains and identifying the strains harboring blaOXA-23-like gene. The duplex MCDA assay, which targets the pgaD and blaOXA-23-like genes, could identify the A. baumannii isolates and differentiate these isolates harboring blaOXA-23-like gene. The disposable lateral flow biosensors (LFB) were used for analyzing the MCDA products. A total of sixty-eight isolates, include fifty-three A. baumannii strains and fifteen non-A. baumannii strains, were employed to optimize MCDA methods and determine the sensitivity, specificity and feasibility. The optimal reaction condition is found to be 63 °C within 1 h, with limit of detection at 100 fg templates per tube for pgaD and blaOXA-23-like genes in pure cultures. The specificity of this assay is 100%. Moreover, the practical application of the duplex MCDA-LFB assay was evaluated using clinical samples, and the results obtained from duplex MCDA-LFB method were consistent with conventional culture-based technique. In sum, the duplex MCDA-LFB assay appears to be a reliable, rapid and specific technique to detect all A. baumannii strains and identify these strains harboring blaOXA-23-like gene for appropriate antibiotic therapy.
Highlights
IntroductionA novel isothermal amplification technique, multiple cross displacement amplification (MCDA) is employed for detecting all A. baumannii strains and identifying the strains harboring blaOXA-23-like gene
Acinetobacter baumannii is a frequent cause of the nosocomial infections
Our data verified that the pgaD- and blaOXA-23-like-multiple cross displacement amplification (MCDA) primer sets were valuable candidates for development of the pgaD-MCDA-lateral flow biosensor (LFB), blaOXA-23-like-MCDA-LFB and duplex MCDA-LFB assays for target gene detection
Summary
A novel isothermal amplification technique, multiple cross displacement amplification (MCDA) is employed for detecting all A. baumannii strains and identifying the strains harboring blaOXA-23-like gene. The duplex MCDA-LFB assay appears to be a reliable, rapid and specific technique to detect all A. baumannii strains and identify these strains harboring blaOXA-23-like gene for appropriate antibiotic therapy. Specific and rapid identification of A. baumannii and the strains harboring blaOXA-23-like gene, will offer referential information on the therapeutic and control precautions for the nosocomial infections owing to the carbapenem-resistant A. baumannii. A total of ten primers were employed to recognize ten distinct regions on the target gene Given that this technique eliminated the use of a thermocycler, and did not require sophisticated training, MCDA showed the potential as a valuable diagnostic tool for field testing and point-of-care diagnosis[13]. Wang et al reported a new LFB technique of single labeling which can generate the double labeled amplicons as well and successfully eliminated the hybridization interaction of labeled primers[14,15]
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