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

Qinqin Wang,Siqi Xu,Chao Zhuo,Kang Liao,Shaoli Li,Lixia Huang,Yanbin Zhou,Ling Yang,Shuang-Yong Xu

doi:10.1371/journal.pone.0066406

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.

Highlights

Acinetobacter baumannii, one of the important nosocomial pathogens, is often associated with epidemic outbreaks of infection
The real-time loop-mediated isothermal amplification (RealAmp) assay only requires a single unit, and the assay positivity can be verified by visual inspection
The results showed that the loop-mediated isothermal amplification (LAMP) method was more suitable than polymerase chain reaction (PCR) for rapid detection of A. baumannii in clinical samples, especially for infection control purposes

Summary

Introduction

Acinetobacter baumannii, one of the important nosocomial pathogens, is often associated with epidemic outbreaks of infection. The organism is frequently pandrug-resistant and capable of causing substantial morbidity and mortality in patients with severe underlying diseases, both in the hospital and in the community [1] Rapid identification of this pathogen is critical for the appropriate therapy and outbreak control. 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. These molecular tools require expensive laboratory instruments. We attempted to utilize this method for rapid detection of A. baumannii

Methods

Results

Discussion

Conclusion