1. Introduction
Nucleic acid amplification is the first and main step for most gene-based detection methods and development of diagnostics technologies in life sciences. Polymerase chain reaction (PCR)-based nucleic acid amplification was conceived by Kary Mullis in 1983 [1]. PCR provides DNA amplification from a specific region of a DNA strand using a thermal cycler. It consists of 20-40 repeated thermal changes with discrete temperature steps (50 ˚C – 98 ˚C). Mostly, PCR reactions are carried out within a few hours. Also, PCR amplification products are usually visualized by gel electrophoresis. Although the PCR method is the gold standard for nucleic acid detection and feasible for laboratory conditions, it is labor and time consuming, requiring bulky and expensive instruments and trained personnel. Therefore, it is not convenient for on-field deployment or resource-limited settings. Here, the main purpose is to make nucleic acid-based diagnosis at the point of sample collection, starting from nucleic acid isolation to detection, using an easy-to-use, portable, low-cost platform with a reliable, precise, and rapid method.
To achieve this goal, various methods have been developed since 1990 [2-8]. Among these methods, Notomi and co-workers’ Loop-mediated isothermal amplification (LAMP) technique succeeded in performing nucleic acid amplification and detection at the point of sample collection [2]. In comparison to PCR, LAMP is a rapid (~ 30 min) and robust technique uses a constant temperature (~ 65 ˚C) instead of a series of alternating temperature cycles. It does not require highly pure DNA isolates. Hence, LAMP methods decrease cost, time and the need of expensive instruments or trained personnel. It has a great potential for breaking through the walls of laboratories to fields [9,10,11], clinics [12,13,14], farms [15,16,17], crime scenes [18,19] or even excavation sites.
To perform reliable, rapid, and robust nucleic acid detection outside laboratories, LAMP methods have been optimized and user-friendly, portable, low-cost devices to provide stable LAMP reactions have been developed [10,20-25]. Most of these optimization studies focused on primer design to achieve high specificity [20-22] or visualization methodology to obtain sensitive readouts [10,23-25]. Readouts were mostly determined by spectroscopic detection of turbidity either by the naked eye or cameras. Although the LAMP reactions were conducted in a simple to use, low-cost, and widely available equipment such as water baths, heat blocks, or ovens, still these instruments are bulky, and their usage is not practical for the point of sample collection. Moreover, a trained person is often required for nucleic-acid extraction, preparation of LAMP reactions, and interpretation of readouts in some circumstances. To benefit from the rapid, reliable, robust, and easy-to-use features of the LAMP technique, portable, low-cost, user-friendly platforms which can combine nucleic-acid extraction, LAMP reaction, and readout of detection are still very limited.
The recent urgency for the detection and diagnosis of COVID-19 cases has accelerated the transition of diagnostic assays and technologies from laboratories to points of sample collection for rapid, reliable, and ready-to-use tests [23,26-28]. Papadakis and co-workers developed the real-time colorimetric LAMP system to be used for COVID-19 diagnosis from clinical biopsy samples [23]. However, this device was not convenient to be used in resource limited settings or by non-trained personnel, since it requires digital image analysis using the images acquired by smartphone cameras [23]. Likewise, development of fully automated, low-cost, rapid, and portable LAMP assays for detection of bacterial pathogens are still at their crawling phase. Yan’s group focused on food safety and implemented LAMP assay for bacterial culture and bacterial colonies of 116 strains of Escherichia coli(E. coli ), Salmonella typhimurium, Vibrio parahaemolyticus, Pseudomonas aeruginosa (P. aeruginosa ), and Listeria monocytogenes using laboratory equipment [14]. They achieved LAMP amplification of target DNA without any DNA extraction and purification from bacterial colonies or cultures. For laboratory-free LAMP reactions, Sayad and co-workers developed a centrifugal microfluidic platform to detect the genus Salmonellain spiked tomato samples [24]. They used a hot air gun to maintain constant temperature required for a LAMP reaction which might influence thermal stability of reactions and triggers non-repeatable results. Therefore, their method was not fully automated, rapid, reusable, or low-cost, in other words convenient for field studies. A comparable study was performed by Seo and co-workers. They used a colorimetric, high-throughput centrifugal LAMP microdevice for the multiplex pathogen detection using E. coli , Salmonella typhimurium , andVibrio parahaemolyticus samples [25]. Like Sayad’s development, fabrication, and operation of the centrifugal microdevice was very expensive and complex. Moreover, the entire genetic analysis on the microdevice was longer than 30 min (~ 1 h). Boyle and co-workers developed a portable LAMP microfluidic device to detectStreptococcus equi subsp. equi in guttural pouch lavage specimens from convalescent horses [29]. Although this assay shows potential for field deployment, still DNA extraction requires standard laboratory protocols and laboratory equipment. Several microfluidic chips capable of generating and trapping emulsion droplets for digital loop-mediated isothermal amplification analysis have been developed [30,31]. The underlying motivation for these technologies were their low detection limits, however their complexity, high cost, and need of high-technical equipment are among their drawbacks.
Most of the studies cited above showed that simple, portable, low-cost, robust, and field-deployable devices to perform LAMP-based diagnosis are still limited [32]. To overcome these limitations, we developed a low-cost (~ 32 Euro), portable (~ 70 g), remotely controlled (wireless), real-time, colorimetric LAMP device. We integrated a proportional-integral (PI) controller to maintain isothermal conditions. We showed that our device successfully conducts LAMP reactions without a prior DNA extraction step. It provides naked-eye detection of bacterial amplicons during the amplification reactions. We confirmed specificity of our method using P. aeruginosa colonies and its genomic DNA with yaiO2primer set. We characterized the sensitivity of our system usingE. coli and P. aeruginosa colonies in different sizes (1 mm – 2.5 mm) in the Colony LAMP and Colony PCR reactions. Moreover, we prepared serial dilutions of genomic DNA (1%, 10%, 100%) for E. coli and P. aeruginosa cells and performed conventional LAMP assays. In this study, the LAMP device is tailored for Colony-LAMP reactions, however, it has a great potential to be used for rapid, reliable, simple, and high-throughput diagnosis in versatile operational environments.
2. Experimental section