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