Identification of Acinetobacter baumannii and its carbapenem-resistant gene blaOXA-23-like by multiple cross displacement amplification combined with lateral flow biosensor

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 bla_OXA-23-like gene. The duplex MCDA assay, which targets the pgaD and bla_OXA-23-like genes, could identify the A. baumannii isolates and differentiate these isolates harboring bla_OXA-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 bla_OXA-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 bla_OXA-23-like gene for appropriate antibiotic therapy.

Figure 1

Detection and confirmation of pgaD- and bla_OXA-23-like-MCDA products. (a,c), Color change of pgaD- and bla_OXA-23-like -MCDA tubes; (b,d), LFB applied for visual detection of pgaD- and bla_OXA-23-like-MCDA products. Tube a1 (biosensor b1), positive amplification; tube a2 (biosensor b2), negative amplification (K. pneumoniae); tube a3 (biosensor b3), negative amplification (S. aureus); tube a4 (biosensor b4), negative control (DW); Tube c1 (biosensor d1), positive amplification; tube c2 (biosensor d2), negative amplification (K. pneumoniae); tube c3 (biosensor d3), negative amplification (S. aureus); tube c4 (biosensor d4), negative control (DW).

Figure 2

Optimal amplification temperature for pgaD- and bla_OXA-23-like-MCDA primer sets. The MCDA amplifications for detection of pgaD (a) and bla_OXA-23-like (b) were monitored by real-time measurement of turbidity and the corresponding curves of concentrations of templates were marked in the figures. The threshold value was 0.1 and the turbidity of >0.1 was considered as positive. Five kinetic graphs (1–5) were generated at various temperatures (61 °C-65 °C, 1 °C intervals) with target pathogens DNA. (a) graphs from 2 (62 °C) to 4 (64 °C) showed robust amplification; (b) graphs from 2 (62 °C) to 4 (64 °C) showed robust amplification.

Figure 3

Detection of a single target in a MCDA reaction. Two sets of MCDA primers targeting the pgaD (a1,b1,c1,d1) and bla_OXA-23-like (a2,b2,c2,d2) genes were used in different reactions and the serial dilutions (10 ng, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg) of target templates were subjected to MCDA reactions. (a1) and (a2), real-time turbidity applied for analysis of pgaD- and bla_OXA-23-like-MCDA products. (b1) and (b2), MG applied for analysis of pgaD- and bla_OXA-23-like-MCDA products. (c1) and (c2), LFB applied for analysis of pgaD- and bla_OXA-23-like-MCDA products. (d1) and (d2), gel electrophoresis applied for analysis of pgaD- and bla_OXA-23-like-MCDA products. Signals/Tubes/Biosensors/Lanes 1–6: A. baumannii (SG-AB001) genomic templates (10 ng-1fg); Signal/Tube/Biosensor/Lane 7: negative control (K. pneumoniae); Signal/Tube/Biosensor/Lane 8: blank control (DW). The d1 and d2 were cropped from different gels, the full-length gels can be found as Supplementary Fig. S1.

Figure 4

Detection of multiplex targets in a m-MCDA reaction. Two sets of MCDA primers targeting pgaD- and bla_OXA-23-like-MCDA genes were simultaneously added to a reaction tube and the LoD of m-MCDA for simultaneously detecting pgaD and bla_OXA-23-like genes was confirmed using LFB. Biosensors 1, 2, 3, 4, 5 and 6 represent DNA levels of 10 ng (A. baumannii SG-AB001), 10 pg (A. baumannii SG-AB001), 1 pg (A. baumannii SG-AB001), 100 fg (A. baumannii SG-AB001), 10 fg (A. baumannii SG-AB001) and 1 fg (A. baumannii SG-AB001); biosensor 7, negative control (K. pneumoniae); biosensor 8, blank control (DW). The LoD of m-MCDA assay for pgaD and bla_OXA-23-like detection was 100 fg per vessel.

Figure 5

Specificity of duplex MCDA-LFB assay using different bacterial strains. The m-MCDA amplifications were carried out using different genomic DNA templates and the results were indicated using LFB. Biosensors 1–9, A. baumannii strains with bla_OXA-23-like gene; Biosensors 10–12, A. baumannii strains without bla_OXA-23-like gene; biosensors 13–27, Listeria monocytogenes, Bacillus cereus, Citrobacter braakii, Citrobacter freundii, Corynebacterium ammoniagenes, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Providencia rettgeri, Pseudomonas aeruginosa, Serratia marcescens, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus salivarius.

Figure 6

Nucleotide sequence and location of pgaD and bla_OXA-23-like genes used to design MCDA-LFB primers. The nucleotide sequences of the sense strands of pgaD (a) and bla_OXA-23-like (b) are showed. The sites of primer sequences were underlined. Left arrows and right arrows showed complementary and sense sequences that are used.