title: Evaluation of real-time loop-mediated isothermal amplification (RealAmp) for rapid detection of Mycobacterium tuberculosis from sputum samples.
abstract: Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) leads to serious health problems as a chronic respiratory infectious disease. Here we established a real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) using a portable ESE Quant tube scanner as a convenient rapid detection method for MTB. The method efficacy from sputum samples was further investigated, and the reaction time was only 20min with the detection limit low to 10(2)CFU/ml concentration of MTB. We assessed a total of 1067 samples by the RealAmp assay, comparing the results with smear microscopy and conventional culture methods. To examine whether the failure to detect TB by culturing is due to low sensitivity or true absence, we examined the culture negative samples by commercial real time PCR MTB detection kit, and the results were compared with RealAmp. The data showed that RealAmp assay had a higher positive rate than that of sputum smear and culture methods. RealAmp had a sensitivity of 96.70% and a specificity of 91.55% when compared with culture. In addition, its sensitivity and specificity were 95.29% and 86.88% respectively compared with examination of smear samples using light microscopy. The sensitivity of RealAmp in comparison to real time PCR was 98.25% and specificity was 99.11% in validation of culture negative samples. The present study revealed the newly established RealAmp assay as a convenient, efficient, sensitive and specific method that could be an alternative for rapid detection of MTB and a tool to validate culture and smear negative samples. Furthermore, the portability of the ESE Quant tube scanner also contributed to the promising application for grassroots and field detection of MTB.
authors: College of Light Industry and Food Sciences, South China University of Technology, 510640 Guangzhou, PR China.
authors: Yiming
authors: Y
authors: Li
authors: College of Light Industry and Food Sciences, South China University of Technology, 510640 Guangzhou, PR China.
authors: Lei
authors: L
authors: Shi
authors: College of Light Industry and Food Sciences, South China University of Technology, 510640 Guangzhou, PR China.
authors: Anqi
authors: A
authors: Pan
authors: Guangzhou DE AOU Biotechnology Technology Co., Ltd., Innovation Building, Science Road, Guangzhou Science City, 510663 Guangzhou, PR China.
authors: Weiwei
authors: W
authors: Cao
authors: Guangzhou DE AOU Biotechnology Technology Co., Ltd., Innovation Building, Science Road, Guangzhou Science City, 510663 Guangzhou, PR China.
authors: Xun
authors: X
authors: Chen
authors: College of Light Industry and Food Sciences, South China University of Technology, 510640 Guangzhou, PR China.
authors: Hecheng
authors: H
authors: Meng
authors: College of Light Industry and Food Sciences, South China University of Technology, 510640 Guangzhou, PR China.
authors: He
authors: H
authors: Yan
authors: Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.
authors: Shin-ichi
authors: S
authors: Miyoshi
authors: College of Light Industry and Food Sciences, South China University of Technology, 510640 Guangzhou, PR China. Electronic address: yelei176003871@sina.com.
authors: Lei
authors: L
authors: Ye
mesh_terms: D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology*; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis / Q000382:microbiology*
keywords: ESE Quant tube scanner; Mycobacterium tuberculosis; RealAmp; Sputum