Abstract
The traditional microbiological methods used for detecting Acinetobacter baumannii were usually time-consuming and labor-intensive. Thus, we sought to establish a novel rapid detecting method for target pathogen. A set of multiple cross displacement amplification (MCDA) primers was designed to recognize 10 different regions of the pgaD gene, which was conservative and specific for the bacterium. In the MCDA system, amplification primers D1 and R1 were 5′-labeled with FITC (fluorescein) and biotin, respectively. Numerous FITC- and biotin-attached duplex amplicons were formed during the amplification stage, which were detected by nanoparticles-based lateral flow biosensors (LFB) through immunoreactions (FITC on the duplex and anti-FITC on the LFB test line) and biotin/streptavidin interaction (biotin on the duplex and streptavidin on the nanoparticles). The results showed that the optimized reaction condition of MCDA-LFB method was 62 °C within 25 min. There was no cross reaction with non-A. baumannii species and the non-Acinetobacter genera, and the detection limit for DNA samples was 100 fg/reaction. For 135 sputum samples, the detection results showed that the detection ability of MCDA-LFB assay was superior to the culture methods and conventional PCR. Therefore, MCDA-LFB assay could be a potential tool for the rapid detection of A. baumannii in clinical samples and low resource areas.
Highlights
Acinetobacter baumannii (A. baumannii), a strict-aerobic, gram-negative, rod-shaped, opportunistic pathogen, extensively exists in natural and medical environments (Bergongne-Berezin and Towner 1996; Van Looveren and Goossens 2004)
The reference strain ATCC19606 DNA produced positive results as shown in Fig. 2, the color of reaction mixture turned bright green at the end of amplification, two red bands were visible on the control line and the test line of a lateral flow biosensors (LFB), and a ladder-like band occurred on 2% gel electrophoresis
In the present study, a new amplification technique multiple cross displacement amplification (MCDA) combined with the LFB assay was developed for rapid detection of A. baumanni
Summary
Acinetobacter baumannii (A. baumannii), a strict-aerobic, gram-negative, rod-shaped, opportunistic pathogen, extensively exists in natural and medical environments (Bergongne-Berezin and Towner 1996; Van Looveren and Goossens 2004). The gold standard used for diagnosis of A. baumannii infection is traditional culture-biochemical methods, Cheng et al AMB Expr (2019) 9:30 which usually takes 24–72 h of incubation to produce positive results, and is typically labor intensive and time consuming This technique could not satisfy early detection of causative pathogens, which is crucial for the proper use of antimicrobial agents. These molecular methods require specialized high-cost equipment, which are not readily available in low-resource settings and outbreak control Most of these genes could not effectively distinguish A. baumannii from Acinetobacter pittii (Acinetobacter genospecies 3), Acinetobacter nosocomialis (Acinetobacter genospecies 13TU) and Acinetobacter lwoffii, which have been recognized as important nosocomial pathogens recently (Dijkshoorn et al 2007; Higgins et al 2007; Turton et al 2010). Another rapid, simple and specific assay detecting the other target gene is required to replace current PCR assays
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