Development and Clinical Application of a Recombinase Polymerase Amplification-Lateral Flow Strip Assay for Detection of Carbapenem-Resistant Acinetobacter baumannii

Abstract

Acinetobacter baumannii is a worldwide, primary cause of respiratory tract infections, septicemia, urinary apparatus infections, and secondary meningitis. It can be fatal. Rapid and accurate detection methods are needed to control the spread of carbapenem-resistant A. baumannii (CRAB). Current molecular diagnostic methods are limited and not suitable for on-site detection. In this study, an isothermal detection method using recombinase polymerase amplification (RPA) combined with a lateral flow strip (LFS) was developed to target the bla_OXA-51 and bla_OXA-23 genes of A. baumannii. The reaction was completed in about 40 min at 37°C. This method can also effectively distinguish A. baumannii and CRAB. The limit of detection of 10⁰-10¹ CFU/reaction was equal to that of other detection methods. The detection accuracy was equal to that of the qPCR method with the use of clinical samples. The RPA-LFS assay is portable, rapid, and accurate and could replace existing detection methods for on-site detection of A. baumannii and CRAB.

Keywords: blaOXA-23 gene; blaOXA-51 gene; carbapenem-resistant Acinetobacter baumannii; lateral flow strip; recombinase polymerase amplification.

Figure 1

Screening of the primer-probe sets. (A) The RPA results of the four different primer sets targeting bla _OXA-51 and bla _OXA-23. The name of each set is shown at the top of each lane. A NTC was used in the reactions. All reactions were performed at 37°C for 30 min. The image represents the results of three independent experiments. (B, C) Pairing analysis and sequence modifications of the primer-probe sets for detection of bla _OXA-51 and bla _OXA-23 with Primer Premier 5 software. Relevant DNA bases of the probes and primers were excluded. The DNA strands are shown as horizontal lines and matching bases are indicated with vertical lines. Molecular markers are listed under the (C). (D) Effectiveness of the primer-probe sets for the RPA-LFS assay. The name of each set is shown at the top of each lane. A NTC was used in the reactions. The positions of the test and control lines are indicated on the right. All reactions were performed at 37°C for 30 min. The images represent the results of three independent experiments.

Figure 2

Applicability of the primer-probe sets. (A) The image shows the detection results of the RPA-LFS assay for 10 A. baumannii (bla _{OXA-51 without OXA-23}) isolates using the primer-probe sets bla _OXA-51-F3/R2B/P (#1-1, #2-1, #3-1, #4-1, #5-1, #6-1, #7-1, #8-1, #9-1, #10-1) and bla _OXA-23-F3/R2B/P (#1-2, #2-2, #3-2, #4-2, #5-2, #6-2, #7-2, #8-2, #9-2, #10-2). (B) The image shows the detection results of the RPA-LFS assay for 10 A. baumannii (bla _{OXA-51 and OXA-23}) isolates using the primer-probe sets bla _OXA-51-F3/R2B/P (#1-1, #2-1, #3-1, #4-1, #5-1, #6-1, #7-1, #8-1, #9-1, #10-1) and bla _OXA-23-F3/R2B/P (#1-2, #2-2, #3-2, #4-2, #5-2, #6-2, #7-2, #8-2, #9-2, #10-2). NTC-1, no template control with the primer-probe set bla _OXA-51-F3/R2B/P. NTC-2, no template control with the primer-probe set bla _OXA-23-F3/R2B/P. The name of each set is shown at the top of each lane. The positions of the test and control lines are indicated on the right. All reactions were performed at 37°C for 30 min. The images represent the results of three independent experiments.

Figure 3

Specificity of the primer-probe sets. The image shows the detection results of the RPA-LFS assay with different bacterial templates using the primer-probe set bla _OXA-51-F3/R2B/P. The name of the bacterium used for each reaction is shown at the top of each lane. NTC, no template control. The positions of the test and control lines are indicated on the right. All reactions were performed at 37°C for 30 min. The image represents the results of three independent experiments.

Figure 4

LOD of the RPA-LFS assay. (A) The image shows the detection results of the RPA-LFS assay with different CFUs of A. baumannii (bla _OXA-51) using the primer-probe set bla _OXA-51-F3/R2B/P. (B) The image shows the detection results of the RPA-LFS assay with different CFUs of A. baumannii (bla _OXA-51) and 10⁷ CFU of E. coli O157 using the primer-probe set bla _OXA-51-F3/R2B/P. (C) The image shows the detection results of the RPA-LFS assay with different CFUs of A. baumannii (bla _OXA-23) using the primer-probe set bla _OXA-23-F3/R2B/P. (D) The image shows the detection results of the RPA-LFS assay with different CFUs of A. baumannii (bla _OXA-23) and 10⁷ CFU of E. coli O157 using the primer-probe set bla _OXA-23-F3/R2B/P. NTC, no template control. All reactions were performed at 37°C for 30 min. The CFUs are indicated at the top of the strips. The positions of the test and control lines are indicated on the right. The images represent the results of three independent experiments.