ORIGINAL PAPER Microchim Acta DOI 10.1007/s00604-014-1354-y Microfluidic method for rapid turbidimetric detection of the DNA of Mycobacterium tuberculosis using loop-mediated isothermal amplification in capillary tubes Adele Rafati & Pooria Gill Received: 16 May 2014 / Accepted: 1 September 2014 # Springer-Verlag Wien 2014 Abstract We describe a microfluidic method for rapid isother- mal turbidimetric detection of the DNA of Mycobacterium tuberculosis. Loop-mediated isothermal amplification is accom- plished in capillary tubes for amplifying DNA in less than 15 min, and sensitivity and specificity were compared to con- ventional loop-mediated isothermal amplification (LAMP). The method can detect as little as 1 pg mL -1 DNA in a sample. Results obtained with clinical specimens indicated 90 % sensi- tivity and 95 % specificity for microfluidic LAMP in compari- son to culture methods. No interference occurred due to the presence of nonspecific DNAs. The findings demonstrate the power of the new microfluidic LAMP test for rapid molecular detection of microorganisms even when using bare eyes. Keywords Microfluidic-LAMP . M. tuberculosis . Capillary tube Introduction Nucleic acid isothermal amplification technologies are the powerful molecular methods for detection of microorganisms [1]. The most known nucleic acid isothermal amplification is loop-mediated isothermal amplification of DNA (LAMP) A. Rafati : P. Gill Department of Medical Biotechnology, Faculty of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan 4713818518, Iran P. Gill (*) Department of Physiology, Pharmacology, and NanoBioMedicine, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari 4847191971, Iran e-mail: pooriagill@yahoo.com P. Gill e-mail: p.gill@mazums.ac.ir technology [2]. LAMP has attracted wide considerable inter- ests for molecular diagnosis of nucleic acid targets because of its special properties including the significant specificity, sen- sitivity, rapidness, and its high clinical performance in the practice [3]. LAMP occurs with an incubation time at a temperature between 60 – 65 °C. Its main reaction included a thermophilic DNA polymerase, with the strand displace- ment activity, betaine, and the primers for targeting DNA template [4]. The amplification process are conducted under a constant thermal condition and a more efficiently amplifica- tion achieved at a single temperature [5]. LAMP result can be assessed by its turbidity or via an extrinsic fluorescence of the cauliflower-like DNAs stained with a fluorescent dye [6]. The isothermal amplification pro- cess conventionally performed in a microtube; however, microfluidic chambers could be improved rapidness of the amplification process. These characteristics made various technological approaches adapted with this amplification technology for several applications [7]. For instance, the con- ventional LAMP assays were reported for detection of known infectious agents via targeting a specific region of the desired gene [8]. Also, two-step reverses transcription (RT)-LAMP assays were developed for identifying RNA of the pathogen as the viability marker [9]. Recently, microfluidic LAMP assay was introduced as a rapid and portable identification test [10]; however, the microfluidic LAMP would be useful in the point-of-care for diagnostic purposes [11]. On the other hand, the molecular detection of M. tuberculosis in clinical specimens is important as a clinical diagnostic tool [12]. The assays for identifying mycobacterial pathogens require detection of the physical characteristics via the acid-fast staining and the microscopic studying, physio- logical specifications via the growth on defined media, or the biochemical analysis according to the membrane lipid com- position [13]. These analyses require highly concentrations of Mycobacteria in the specimen and they need a long time [14]. brought to you by CORE View metadata, citation and similar papers at core.ac.uk provided by Golestan University of Medical Sciences Repository