Real-time fluorescence loop mediated isothermal amplification for the detection of Acinetobacter baumannii

Abstract

Background: Detection of Acinetobacter baumannii has been relying primarily on bacterial culture that often fails to return useful results in time. Although DNA-based assays are more sensitive than bacterial culture in detecting the pathogen, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. In addition, these molecular tools require expensive laboratory instruments. Therefore, establishing molecular tools for field use require simpler molecular platforms. The loop-mediated isothermal amplification method is relatively simple and can be improved for better use in a routine clinical bacteriology laboratory. A simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in the same platform has been developed in recent years. This method is referred to as real-time loop-mediated isothermal amplification. In this study, we attempted to utilize this method for rapid detection of A. baumannii.

Methodology and significant findings: Species-specific primers were designed to test the utility of this method. Clinical samples of A. baumannii were used to determine the sensitivity and specificity of this system compared to bacterial culture and a polymerase chain reaction method. All positive samples isolated from sputum were confirmed to be the species of Acinetobacter by 16S rRNA gene sequencing. The RealAmp method was found to be simpler and allowed real-time detection of DNA amplification, and could distinguish A. baumannii from Acinetobacter calcoaceticus and Acinetobacter genomic species 3. DNA was extracted by simple boiling method. Compared to bacterial culture, the sensitivity and specificity of RealAmp in detecting A. baumannii was 98.9% and 75.0%, respectively.

Conclusion: The RealAmp assay only requires a single unit, and the assay positivity can be verified by visual inspection. Therefore, this assay has great potential of field use as a molecular tool for detection of A. baumannii.

Figure 1. The correlation between time to amplification and amount of target DNA.

The plot reported the fluorescence in millivolts (mV) on the Y-axis and time in minutes on the X-axis. 1, 100 ng/μL; 2, 10 ng/μL; 3, 1 ng/μL; 4, 100 pg/μL; 5, 10 pg/μL; 6, 1 pg/μL; PC, positive control; NC, negative control.

Figure 2.Comparative. sensitivity of the RealAmp and PCR assays for the detection of A. baumannii ATCC19606.

(A) The sensitivity of the RealAmp assay for the detection of A. baumannii ATCC19606. The plot reported the fluorescence in millivolts (mV) on the Y-axis and time in minutes on the X-axis. 1, 1×10⁷ CFU/mL; 2, 1×10⁶ CFU/mL; 3, 1×10⁵ CFU/mL; 4, 1×10⁴ CFU/mL; 5, 1×10³ CFU/mL; 6, 1×10² CFU/mL; 7, 1×10¹ CFU/mL; NC, negative control. (B) The sensitivity of the PCR assay for the detection of A. baumannii ATCC19606. 1, 1×10⁷ CFU/mL; 2, 1×10⁶ CFU/mL; 3, 1×10⁵ CFU/mL; 4, 1×10⁴ CFU/mL; 5, 1×10³ CFU/mL; 6, 1×10² CFU/mL; 7, 1×10¹ CFU/mL; NC, negative control. The number of colony forming units/mL (CFU/mL) of A. baumannii ATCC19606 are indicated.

Figure 3. The RealAmp assay for the detection of A. calcoaceticus–A. baumannii (Ac–Ab) complex.

The plot reported the fluorescence in millivolts (mV) on the Y-axis and time in minutes on the X-axis. 1, Acinetobacter baumannii ATCC19606; 2, Acinetobacter genomic species 13TU; 3, Acinetobacter genomic species 3; 4, Acinetobacter calcoaceticus; PC, positive control; NC, negative control.