Real-Time Fluorescence Loop Mediated Isothermal Amplification for the Detection of Acinetobacter baumannii

Abstract

BackgroundDetection 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 FindingsSpecies-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.ConclusionThe 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.

Rapid detection of Acinetobacter baumannii and molecular epidemiology of carbapenem-resistant A. baumannii in two comprehensive hospitals of Beijing, China

“…Five sets of different primers were initially tested for detection of the bla OXA-51-like gene, and the primers for pga D gene of A. baumannii were added as a control ( Wang et al, 2013 ). Four of the five primer sets enabled successful amplification ( Figure 1 ).…”

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

“…LAMP assays also appeared unaffected by the presence of nontarget DNA in the acquired samples (Kaneko et al 2007;Lin et al 2012) which resembles its high specificity characteristic. The LAMP assay is stable against some PCR inhibitors such as blood and detection can be performed without the template extraction step and with a nonprocessed sample Francois et al 2011;Lin et al 2012;Mori et al 2013;Wang et al 2013).…”

<p>Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections</p>

“…17 LAMP has found application in detecting resistance genes such as bla KPC and bla NDM-1 in E. coli, Klebsiella pneumonia and Acinetobacter baumannii 32 as well as identification of bacteria like E. coli, 33 Mycobacteria, 34 Vibrio parahemolyticus, Salmonella spp 35 and A. baumannii. 36 LAMP with a detection limit of 1ng has also been successfully used for rapid detection of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, both of which cause periodontitis in humans. 37 LAMP assays for detecting yeasts, 38 influenza viruses 39 and Plasmodium have been developed.…”