Analytical Biochemistry 688 (2024) 115481

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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

Evaluation of semi-quantitative colorimetric assays based on loop-mediated

isothermal amplification indicators by using image analysis
Wasin Panich a, Sirapat Nak-on a, Metawee Sabaijai a, Awika Raksaman a,
Chokchai Puttharugsa c, Thanawan Tejangkura a, b, Thapana Chontananarth a, b, *
a
Applied Parasitology Research Laboratory, Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110, Thailand
b
Research and Innovation Unit for Diagnosis of Medical and Veterinary Important Parasites, Faculty of Science, Srinakharinwirot University, Bangkok, 10110, Thailand
c
Department of Physics, Faculty of Science, Srinakharinwirot University, Bangkok, 10110, Thailand

A R T I C L E I N F O A B S T R A C T

Keywords: Colorimetric assays are some of the most convenient detection methods, creating discoloration in solutions that is
Semi-quantitative colorimetric analysis visible to the naked eye. However, colorimetric reactions have some limitations regarding the variability in the
CIELab color space color perception of individuals caused by factors such as color blindness, experience, and gender. Semi-
Loop-mediated isothermal amplification
quantitative chromatic analysis has been used as an alternative method to differentiate between two colors
Malachite green
and accurately interpret the results from a numerical value, with high confidence. Therefore, we developed and
Phenol red
Ascaridia galli determined the optimal model between Red-Green-Blue (RGB) and Commission Internationale de l’Eclairage
(CIE) Lab color spaces to establish a semi-quantitative colorimetric assay via image analysis by the ImageJ
program for loop-mediated isothermal amplification (LAMP), using the dyes malachite green and phenol red. The
semi-quantitative colorimetric assays using the color distance values of the CIELab color space (ΔEab) were more
suitable than those using the RGB color space (ΔERGB) for chromatic differentiation between positive and
negative reactions in both indicator dyes, demonstrating the feasibility of this assay to be applied in the detection
of a wide range of pathogens and infectious diseases.

1. Introduction
Generally, the diagnosis of gastrointestinal helminths requires
morphological examination of the adult stage or eggs in feces under a
stereomicroscope, which relies on the specific knowledge and training of
the personnel and is time-consuming [1–3]. Recently, DNA-based mo­
lecular methods have been successfully developed to diagnose the
infection based on a small amount of DNA from eggs or tissue in the
feces, without post-mortem investigation. Such methods include con­
ventional polymerase chain reaction (PCR) [4], multiplex PCR [5],
droplet digital PCR [6], and loop-mediated isothermal amplification
(LAMP) [7]. However, the analysis still requires opening the reaction
tube to perform post-amplification analysis, such as gel electrophoresis
[8], SYBR Green I fluorescence examination [9], and lateral flow
dipstick assay (LFD) [10], which increases the risk of self-contamination
[11,12].
Colorimetric readout assays using an indicator dye coupled with
LAMP are the simplest, rapidity and cost-effective to generate visible
color changes in the solution for monitoring the results [13]. Moreover,
the dye can be added into the mixture before start a reaction without
interfering with the reactions [11,14]. The indicator dyes can be divided
into two categories according to their discoloration properties, as
described by Scott et al. [15]: (1) direct methods rely on the insertion
between double-stranded DNA products, such as malachite green,
methyl green, and leuco crystal violet [16–18]; (2) indirect methods
depend on the detection of a byproduct of DNA amplification reactions
(e.g., proton and Mg2+ free), including phenol red, cresol red, and
hydroxynaphthol blue [19–22]. Nevertheless, visual observation has an
important limitation: it provides only qualitative results (detected/not
detected) depending on the color perception of an individual, which
requires trained and experienced personnel to discriminate between
positive and negative results [23].
To quantitatively distinguish colors, a color space coupled with a
chromatic analysis program via image acquisition was used to numeri­
cally measure the discoloration in solution and compute the standard
threshold value for result interpretation. Previously, several studies

* Corresponding author. Parasitology Research Laboratory, Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110, Thailand.
E-mail address: thapana@g.swu.ac.th (T. Chontananarth).

https://doi.org/10.1016/j.ab.2024.115481
Received 13 November 2023; Received in revised form 29 January 2024; Accepted 6 February 2024
Available online 13 February 2024
0003-2697/© 2024 Elsevier Inc. All rights reserved.
W. Panich et al.
Fig. 1. Graphical color spaces of RGB (A) and CIELab (B) systems.
reported the use of chromatic components in color space to interpret the
diagnosis results. For example, the Red-Green-Blue (RGB) color space
was applied using the three coordinates separately, such as the “Green
minus Blue formula”, which can discriminate between positive and
negative results of phenol red dye, whereas the hydroxy naphthol blue
indicator is the “Green minus Red formula”, and “Green divide Red
formula” provided in better differentiation [24,25]. In a similar way,
only hue degree in the Hue-Saturation-Intensity (HSI) color space is the
most popular scheme to differentiate between two colors [23,26,27]. In
case of Commission Internationale de l’Eclairage (CIE) Lab color space,
with L*, a*, and b* coordinates, the perception is uniform, meaning that
a change in the any coordinates will produce an equal change in the
other coordinates; thus, the three coordinates were used to compute the
sum value demonstrating the color distance between positive and
negative outcomes obtained with the use of phenol red and hydroxy
naphthol blue [28,29].
Hence, we propose semi-quantitative colorimetric assays using color
spaces, including RGB and CIELab, for the analysis of malachite green
and phenol red integrated with LAMP via imaging. The reaction tube
images were imported and semi-quantitative analyzed using ImageJ
software, which extracted chromatic components Red, Green, Blue and
L*, a*, b* in the RGB and CIELab color spaces, respectively. Finally, the
color distances (ΔE) of each color spaces were computed between pos­
itive reactions and negative controls to generate a numerical threshold
value for determining between positive and negative results. This
developed assay can also be applied in a wider range of analyses, such as
the diagnosis of pathogenic bacteria, viruses, fungi, and parasites.
2. Materials and methods
2.1. Sample preparation and DNA extraction
Adult stages of Ascaridia galli were obtained from the intestines of
chickens (Gallus gallus domesticus) from Maha Sarakham province,
Thailand, and identified to species level prior to DNA extraction. The
total genomic of A. galli was extracted using a GF-1 Tissue DNA
Extraction Kit (Vivantis, Malaysia) according to the manufacturer’s
recommendations. The DNA was diluted to a concentration of 5 ng/μL
with deionized water before being used in LAMP processes.
2.2. LAMP reaction and optimal indicator dye concentration
The LAMP primers targeting the internal transcribed spacer 2 (ITS2)
region of A. galli were as described in Panich et al. [30]. Table S1 shows
2
Analytical Biochemistry 688 (2024) 115481
the details of the primers. To determine the optimal concentration of
each indicator dye, various dye concentrations were added to the re­
action mixture to obtain final concentrations of 100, 200, and 400 μM
for malachite green and 50, 100, and 200 μM for phenol red. The LAMP
reagents from a commercial kit, a Bst 2.0 DNA Polymerase kit (New
England Biolabs), can immediately be used with the malachite green.
The discoloration of phenol red depends on the pH of the reaction
mixture, which was newly prepared; the reaction was conducted in
weakly buffered solution without Tris-HCl. Briefly, the working LAMP
solution of 12.5 μL contained 1x buffer of the New England Biolabs
commercial kit (with 20 mM Tris-HCl) at pH 8.8 for malachite green and
1x buffer (comprised of 10 mM (NH4)2SO4, 50 mM KCl, 6 mM MgSO4,
0.1% v/v Tween 20, without Tris-HCl) at pH 8.0–8.5, manually prepared
at 25 ◦ C for phenol red, 0.4 μM each of F3 and B3, 1.6 μM each of FIP and
BIP, 1.4 mM of each dNTP, 1.0 M betaine (Sigma-Aldrich), 1 μL of
template DNA (5 ng/μL), 1 μL of indicator dye, and 4U Bst DNA poly­
merase (New England Biolabs). Deionized water was used as a template
in the negative control. The LAMP reaction was performed at 66 ◦ C for
60 min in a thermocycler (Analytik Jena AG, Germany), and the results
were analyzed via 1.0% agarose gel electrophoresis.
2.3. Image acquisition and semi-quantitative color analyses
At the end of the amplification, the reaction tubes were placed on a
white background with a static white light, and images were captured
immediately to prevent color-fading, using the camera of an iPhone 13
(f = 1.6 mm; auto white balance). To control the ambient conditions,
each replicate LAMP reaction was photographed in the same frame for
controlling the ISO, speed shutter, and ambient lighting. The acquired
images were imported and semi-quantitatively analyzed using the
ImageJ software, which measured the chromatic components in the RGB
and CIELab color spaces. The RGB system is a device-dependent color
model and fundamental color space that was developed into other color
spaces, such as CIE 1931, HSI, and CIELab, relying on the coordinates of
Red (0–255), Green (0–255), and Blue (0–255). Regarding the CIELab
color space, each color component is shown as a uniform perception in
three dimensions, recommended for device-independent color presen­
tation in digital image processing. The CIELab color space covers the
entire human color perception, defined by the L* coordinate is the
luminance (which ranges from 0 to 100). The a* coordinate is the green-
red axis (which ranges from -50–50), and the b* coordinate is the blue-
yellow axis (which ranges from -50–50), as shown in Fig. 1 [29,31,32].
Hence, the uniformity of the CIELab system is feasible to measure the
degree of differences between two colors. The selected region of interest
W. Panich et al.
Fig. 2. Comparative validation of malachite green (A) and phenol red (B) at different final concentrations analyzed by the color distance of RGB (ΔERGB) and CIELab
(ΔEab) systems (P: positive reactions; N: negative reactions; squares: regions of interest that were used to extract chromatic components).
(ROI) was 30 × 30 pixels, using rectangular in the toolbar to define and
measure the solution color (Fig. 2). Briefly, Red, Green, and Blue com­
ponents were measured using ROI selection > Plugins > Analyze > RGB
Measure, whereas L*, a*, and b* components were determined using
Image > Type > Lab Stack > ROI selection > Analyze > Measure in each
channel. The color distances of RGB (ΔERGB) and CIELab (ΔEab) were
computed between sample reactions and negative controls in each
replicate, using the Excel software (Microsoft Office). Graphs were
generated using GraphPad Prism. The ΔERGB (Equation (1)) and ΔEab
(Equation (2)) were calculated as follows:
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
ΔERGB = ΔR2 + ΔG2 + ΔB2 (1)
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
ΔEab = ΔL∗2 + Δa∗2 + Δb∗2 , (2)
where Δ is the different value of the color components from the sample
reaction minus the negative control; R, G, B, and L*, a*, b* values are the
color components of the RGB and CIELab color spaces, respectively.
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Analytical Biochemistry 688 (2024) 115481
2.4. Chromatic aberration and threshold setup
The colorimetric LAMP assays using malachite green and phenol red
were performed with initial 5 ng of A. galli DNA, which was diluted 10-
fold with serial dilutions (5–5 × 10− 6 ng/μL) to determine the range of
color distance between positive and negative results, with three repli­
cates per dye. Threshold values of each color distance system were
calculated based on mean negative results plus three-fold standard de­
viation (SD) of the negative result (threshold value = mean negative
results + 3SD); in this sense, a reactive above the threshold value was
interpreted a positive result. The color space suitable for each indicator
dye was decided upon based on the highest proportion of color distance
between positive and negative results (mean color distance of the pos­
itive divided by the mean color distance of the negative result).
2.5. Color variability evaluation
The indicator dyes can be displayed in shades depending on the
amplification performance of the DNA quantity. To further confirm the
efficiency of selected color spaces in each dye, colorimetric LAMP
W. Panich et al. Analytical Biochemistry 688 (2024) 115481

Table 1 u., respectively (Fig. 2B). Therefore, the optimal final concentrations of
The ΔERGB and ΔEab values between positive and negative colors of malachite malachite green and phenol red were 400 and 100 μM, respectively
green and phenol red in different final concentrations. (Table 1), which were used to estimate the threshold values of the semi-
Colorimetric LAMP assay quantitative colorimetric assays in the following section.
Dye (positive - negative colors) Malachite green Phenol red (yellow -
(turquoise - colorless) red)
3.2. Threshold value evaluation for semi-quantitative colorimetric assays
Color distance values (d.u.) ΔERGB ΔEab ΔERGB ΔEab

Final concentration The colorimetric LAMP assays were tested with initial DNA con­
50 μM – – 93.94 54.40 centrations from 5 to 5 × 10− 6 ng/μL (dilution 1–7). Positive results
100 μM 18.78 6.19 111.11 65.71
were obtained from three reactions from dilution 1–3 (5, 0.5, 0.05 ng/
200 μM 51.05 14.97 84.52 31.73
400 μM 59.03 16.39 – – μL) for both colorimetric LAMP assays, which was consistent with the
color interpretation using the naked eye (Fig. 3). After that, the negative
results (dilution 4–7) were calculated to estimate the threshold values of
coupled with semi-quantitative chromatic analysis was performed with ΔERGB and ΔEab. Fig. 3 indicates the color distances and threshold values
additional specimens of A. galli (n = 30) from other populations in of ΔERGB and ΔEab in malachite green and phenol red calculated from the
Thailand (Nakhon Pathom, Chumphon, and Bangkok provinces) for dilution 4–7. As a result, the threshold values of ΔERGB and ΔEab of
different DNA concentrations, ranging from 4.4 to 460.9 ng/μL malachite green were 34.93 and 7.09 d.u. (Fig. 3A), whereas those of
(Table S2). phenol red were 53.62 and 12.78 d.u. (Fig. 3B), respectively. However,
the best color spaces for semi-quantitative analysis in each dye were
3. Results
Table 2
3.1. Validations of indicator dye concentration for the colorimetric LAMP Result summaries of this study.
assays
Result summaries Colorimetric LAMP assay

To validate and optimize the colorimetric LAMP assays, various final Malachite green (400 Phenol red (100
μM) μM)
concentrations of indicator dyes were pre-added to the LAMP mixture
before the reaction was carried out in a thermal cycler. A total of 5 ng/μL Type intercalated with pH indicator
dsDNA
of A. galli DNA and deionized water were used as positive and negative
controls, respectively. The ΔERGB and ΔEab values were computed Threshold value of ΔERGB 34.93 d.u. 53.62 d.u.
separately in each concentration and compared to determine the ΔERGB Ratio (mean positive/mean 3.86 5.46
negative)
optimal concentration that provided the most distance values for both Threshold value of ΔEab 7.09 d.u. 12.78 d.u.
indicator dyes. For malachite green, the optimal concentration was 400 ΔEab Ratio (mean positive/mean 5.82 11.78
μM per reaction, which resulted in the maximum color distance values of negative)
59.03 and 16.39 distance unit (d.u.) for ΔERGB and ΔEab, respectively Color variability assessment
- naked-eye observation 30/30 29/30
(Fig. 2A). Regarding phenol red, the final optimal concentration of 100
- semi-quantitative assay 30/30 30/30
μM provided the highest ΔERGB and ΔEab values of 111.11 and 65.71 d.

Fig. 3. Evaluations of a range of color values to set thresholds using various DNA concentrations for three replicates in malachite green (A) and phenol red (B) (1–7:
10-fold serial dilution of A. galli DNA ranging from 5 to 5 × 10− 6 ng/μL; N: negative controls; (i-iii): number of replicates in each dye; ΔERGB: color distance of RGB;
ΔEab: color distance of CIELab). A suggested positive and negative threshold value for both color distance systems is shown with the lines.

4
W. Panich et al. Analytical Biochemistry 688 (2024) 115481

Fig. 4. Assessment of the semi-quantitative colorimetric assays with malachite green and phenol red LAMP detecting 30 samples of A. galli from Nakhon Pathom
(NP), Chumphon (CP), and Bangkok (BK) provinces (1–30: number of A. galli samples; N: negative controls; ΔEab: the CIELab color distance). A suggested positive and
negative threshold value for both color distance systems is shown with the lines.

decided upon based on the proportion of mean positive ΔE divided by

Table 3
mean negative ΔE. Hence, the CIELab color space was more suitable to
Comparison of indicator dye coupled with color space analysis for colorimetric
quantitatively distinguish between positive and negative colors in mal­
LAMP assay.
achite green and phenol red, with a ratio of 5.82 and 11.78 (Fig. 3),
Indicator dye Positive - Negative Color space (chromatic Reference
respectively (see Table 2 for details).
colors value)

Hydroxy naphthol Blue - Violet RGB (Red) [15]

3.3. Semi-quantitative chromatic analysis
blue
Phenol red Yellow - Red HSB (Hue)
A total of 30 samples of A. galli were further used for validating the Leuco crystal violet Crystal violet - HSB (Saturation)
efficacy of semi-quantitative chromatic analysis. Based on the semi- Colorless
quantitative results, malachite green LAMP clearly facilitated the dif­ Malachite green Turquoise - HSB (Saturation)
Colorless
ferentiation between positive and negative samples (Fig. 4; Table S2).
Calcein Green - Orange RGB (Green)
On the contrary, phenol red LAMP led to ambiguous (shaded) results for Hydroxy naphthol Blue - Violet HSI (Hue) [23]
some samples by visual observation (which was most pronounced for blue
sample 26), whereas the semi-quantitative chromatic analysis was more Eriochrome black Blue - Violet RGB (Green/Red) [24,42]
T
accurate (Fig. 4; Table S2). Therefore, the semi-quantitative chromatic
Phenol red Yellow - Red RGB (Green-Blue) [25]
analysis based on the CIELab color distance was successfully developed, Hydroxy naphthol Blue - Violet RGB (Green-Red)
and the confidence regarding result interpretation was increased for the blue
colorimetric detections of malachite green and phenol red. The results Eriochrome black Blue - Violet HSI (Hue) [26]
for each dye are summarized in Table 2. T
Phenol red Yellow - Red HSI (Hue) [27]
Hydroxy naphthol Blue - Violet CIELab (ΔEab) [28]
4. Discussion blue
Phenol red Yellow - Red CIELab (ΔEab) [29]
In terms of user and environmental safety, although chemical re­ Phenol red Yellow - Red HSB (Hue) [39]
Malachite green Turquoise - RGB (ΔERGB), CIELab This study
agents play an important role in several diagnostic assays, including
Colorless (ΔEab)
LAMP reaction, but the reagents can affect user’s health. To preliminary Phenol red Yellow - Red RGB (ΔERGB), CIELab
enhance health safety, colorimetric LAMP assays can be performed in a (ΔEab)
single-step closed tube without the post-amplification processing, lead
to reducing the risk of chemical contamination for user and environment
[33,34]. Malachite green and phenol red are generally used in colori­ individual color perception is not universal [39]. Moreover, a major
metric LAMP reactions to monitor the results via the color change of the issue arises when the indicator dye is presented in shades, which is
solution for the detection of pathogens such as Leishmania spp. [35], caused by the LAMP amplification itself or an insufficient amplification
Plasmodium spp. [36], Toxocara spp. [37], Aeromonas spp. [38], and time, leading to difficulties in result interpretation [38,40]. In this case,
Aspergillus spp. [20]. semi-quantitative chromatic analysis is a powerful method to overcome
Colorimetric assays are some of the simplest analytical methods, and these limitations.
the results can be assessed immediately with the naked eye without post- In this study, we successfully developed and validated a semi-
amplification handling, which decreases the risk of contamination. quantitative colorimetric assay using the CIELab color space via the
Nevertheless, visual interpretation of color-creating reactions by the sum of the color distances, which provided the distance value that could
naked eye could be prone to errors, depending on personal and envi­ distinguish between colors better than using either value alone [32].
ronmental factors, such as color vision deficiency, personal experience, However, the RGB color space is more convenient for image acquisition,
ambient lighting, and temperature (for pH-sensitive dyes); therefore, display, and direct measurement using a smartphone. Nevertheless,

5
W. Panich et al.
regardless of its simple use, this color space is sensitive to lighting, which
affects non-constant brightness in the same frame, thereby impeding its
use in digital image processing [41]. This weakness affects color vari­
ability, resulting in a low ratio of ΔERGB compared with ΔEab for both
indicator dyes (Fig. 3). However, the results indicated that the RGB and
CIELab color spaces can be combined and used with phenol red, whereas
malachite green can only be used with the CIELab system due to the
threshold value of RGB, which overlapped with a standard deviation of
positive results (Fig. 3A). A previous study reported that CIELab color
distance coupled with colorimetric LAMP was successfully used to
interpret the results by the discoloration of phenol red for SARS-CoV-2
diagnosis [29]. Malachite green LAMP analyzed with the CIELab color
distance has not been previously reported. Therefore, this study is the
first work that uses the CIELab system to discriminate between positive
and negative colors of malachite green LAMP (Table 3). As a result, this
color space is especially useful for color differentiation and can be
applied in other colorimetric detections.
We are aware that our analytical assay used the white background
and the static white light to capture the images, which are not available
to every laboratory, but the focus was on lighting, comprising the sta­
bility of the light and the camera conditions. We therefore suggest
solving this problem by capturing the samples and negative control
tubes in the same frame to make sure that they obtain an equal amount
of light and to eliminate the factors in regard to the different camera
conditions. Imaging has become an advantage in these assays regarding
data recording as the reaction tubes do not need to be stored, which
prevents fading, and the results can be analyzed wherever and whenever
necessary.
5. Conclusions
Our semi-quantitative colorimetric assays based on the color dis­
tance of the CIELab color space were successfully developed to accu­
rately differentiate the solution chromaticity between positive and
negative results of malachite green and phenol red coupled with LAMP
via images. The assays have the potential to be applied in diagnosis and
result confirmation for medicine, veterinary healthcare, and epidemio­
logical investigations. To our knowledge, the color distance of the CIE­
Lab color space may be applied with other indicator dyes to detect
pathogenic organisms.
Ethics approval
All experimental methods related to animals were proceeded by the
National Research Council of Thailand and approved by the Committee
for Animal Care and Use Committee Srinakharinwirot University (Li­
cense NO. SWU-A-025-2562).
Disclosure statement
We declare that all authors have no conflict of interest.
CRediT authorship contribution statement
Wasin Panich: Data curation, Formal analysis, Methodology, Vali­
dation, Visualization, Writing – original draft, Writing – review &
editing. Sirapat Nak-on: Formal analysis, Investigation, Methodology,
Visualization, Writing – original draft, Writing – review & editing.
Metawee Sabaijai: Formal analysis, Investigation. Awika Raksaman:
Investigation, Writing – original draft. Chokchai Puttharugsa: Meth­
odology. Thanawan Tejangkura: Methodology, Writing – original
draft, Writing – review & editing. Thapana Chontananarth: Concep­
tualization, Formal analysis, Funding acquisition, Methodology, Project
administration, Resources, Supervision, Writing – original draft, Writing
– review & editing.
6
Analytical Biochemistry 688 (2024) 115481
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
The authors do not have permission to share data.
Acknowledgements
We considerably acknowledge Srinakharinwirot University,
Thailand, National Research Council of Thailand (NRCT) the Science
Achievement Scholarship of Thailand (SAST) for providing research
funding.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ab.2024.115481.
References
[1] J.R. Martín-Pacho, M.N. Montoya, T. Arangüena, C. Toro, R. Morchón, C. Marcos-
Atxutegi, F. Simon, A coprological and serological survey for the prevalence of
Ascaridia spp. in laying hens, J. Vet. Med. B 52 (2005) 238–242, https://doi.org/
10.1111/j.1439-0450.2005.00853.x.
[2] A. Permin, J.P. Christensen, M. Bisgaard, Consequences of concurrent Ascaridia
galli and Escherichia coli infections in chickens, Acta Vet. Scand. 47 (2006) 43–54,
https://doi.org/10.1186/1751-0147-47-43.
[3] L. Zada, T. Rehman, S. Niaz, M.A. Zeb, B. Ruqia, K.M. Salma, A. Khan, Prevalence
of Ascaridia galli in some poultry farms of district Mardan, J. Adv. Parasitol. 2
(2015) 75–79, https://doi.org/10.14737/journal.jap/2015/2.4.75.79.
[4] C.D. Pauling, A.R. Oller, V. Jackson, Fecal parasite identification by microscopy
and PCR in scimitar-horned oryx, Oryx dammah, managed at two sites, Int. J.
Parasitol. Parasites Wildl. 5 (2016) 312–320, https://doi.org/10.1016/j.
ijppaw.2016.11.001.
[5] W. Panich, S. Nak-On, T. Chontananarth, High-performance triplex PCR detection
of three tapeworm species belonging to the genus Raillietina in infected poultry,
Acta Trop. 232 (2022) 106516, https://doi.org/10.1016/j.
actatropica.2022.106516.
[6] B. Tarbiat, N. Enweji, P. Baltrusis, P. Halvarsson, E. Osterman-Lind, D.S. Jansson,
J. Höglund, A novel duplex ddPCR assay for detection and differential diagnosis of
Ascaridia galli and Heterakis gallinarum eggs from chickens feces, Vet. Parasitol. 296
(2021) 109499, https://doi.org/10.1016/j.vetpar.2021.109499.
[7] S. Nak-on, T. Tejangkura, T. Chontananarth, Multi-detection for paramphistomes
using novel manually designed PAR-LAMP primers and its application in a lateral
flow dipstick (LAMP-LFD) system, Vet. Parasitol. 317 (2023) 109905, https://doi.
org/10.1016/j.vetpar.2023.109905.
[8] H. Iseki, A. Alhassan, N. Ohta, O.M. Thekisoe, N. Yokoyama, N. Inoue, et al.,
Development of a multiplex loop-mediated isothermal amplification (mLAMP)
method for the simultaneous detection of bovine Babesia parasites, J. Microbiol.
Methods 71 (2007) 281–287, https://doi.org/10.1016/j.mimet.2007.09.019.
[9] S.M. Tsai, K.W. Chan, W.L. Hsu, T.J. Chang, M.L. Wong, C.Y. Wang, Development
of a loop-mediated isothermal amplification for rapid detection of orf virus,
J. Virol. Methods 157 (2009) 200–204, https://doi.org/10.1016/j.
jviromet.2009.01.003.
[10] W. Panich, T. Tejangkura, T. Chontananarth, Novel high-performance detection of
Raillietina echinobothrida, Raillietina tetragona, and Raillietina cesticillus using loop-
mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-
LFD), Vet. Parasitol. 292 (2021) 109396, https://doi.org/10.1016/j.
vetpar.2021.109396.
[11] C. Sriworarat, A. Phumee, M. Mungthin, S. Leelayoova, P. Siriyasatien,
Development of loop-mediated isothermal amplification (LAMP) for simple
detection of Leishmania infection, Parasites Vectors 8 (2015) 1–8, https://doi.org/
10.1186/s13071-015-1202-x.
[12] Y. Wang, J. Feng, X. Tian, Application of loop-mediated isothermal amplification
(LAMP) for rapid detection of Atlantic cod (Gadus morhua), Pacific cod (Gadus
macrocephalus) and haddock (Melanogrammus aeglefinus), Mol. Cell. Probes 47
(2019) 101420, https://doi.org/10.1016/j.mcp.2019.07.003.
[13] S. Ahmadi, N. Rabiee, Y. Fatahi, S.E. Hooshmand, M. Bagherzadeh, M. Rabiee, et
al., Green chemistry and coronavirus, Sustain. Chem. Pharm. 21 (2021) 100415,
https://doi.org/10.1016/j.scp.2021.100415.
[14] F. Zhang, C. Gao, L. Bai, Y. Chen, S. Liang, X. Lv, et al., Dual-color blending based
visual LAMP for food allergen detection: a strategy with enlarged color variation
range and contrast, Food Chem. X 13 (2022) 100201, https://doi.org/10.1016/j.
fochx.2021.100201.
W. Panich et al.
[15] A.T. Scott, T.R. Layne, K.C. O’Connell, N.A. Tanner, J.P. Landers, Comparative
evaluation and quantitative analysis of loop-mediated isothermal amplification
indicators, Anal. Chem. 92 (2020) 13343–13353, https://doi.org/10.1021/acs.
analchem.0c02666.
[16] S. Miyamoto, S. Sano, K. Takahashi, T. Jikihara, Method for colorimetric detection
of double-stranded nucleic acid using leuco triphenylmethane dyes, Anal. Biochem.
473 (2015) 28–33, https://doi.org/10.1016/j.ab.2014.12.016.
[17] N.W. Lucchi, D. Ljolje, L. Silva-Flannery, V. Udhayakumar, Use of malachite green-
loop mediated isothermal amplification for detection of Plasmodium spp. parasites,
PLoS One 11 (2016) e0151437, https://doi.org/10.1371/journal.pone.0151437.
[18] J. Thapa, B. Maharjan, M. Malla, Y. Fukushima, A. Poudel, B.D. Pandey, et al.,
Direct detection of Mycobacterium tuberculosis in clinical samples by a dry methyl
green loop-mediated isothermal amplification (LAMP) method, Tuberculosis 117
(2019) 1–6, https://doi.org/10.1016/j.tube.2019.05.004.
[19] X.J. Ma, Y.L. Shu, K. Nie, M. Qin, D.Y. Wang, R.B. Gao, et al., Visual detection of
pandemic influenza A H1N1 Virus 2009 by reverse-transcription loop-mediated
isothermal amplification with hydroxynaphthol blue dye, J. Virol. Methods 167
(2010) 214–217, https://doi.org/10.1016/j.jviromet.2010.03.027.
[20] M. Ferrara, A.F. Logrieco, A. Moretti, A. Susca, A loop-mediated isothermal
amplification (LAMP) assay for rapid detection of fumonisin producing Aspergillus
species, Food Microbiol. 90 (2020) 103469, https://doi.org/10.1016/j.
fm.2020.103469.
[21] H. Gou, Z. Bian, R. Cai, Z. Jiang, S. Song, Y. Li, et al., The colorimetric isothermal
multiple-self-matching-initiated amplification using cresol red for rapid and
sensitive detection of porcine circovirus 3, Front. Vet. Sci. 7 (2020) 1–7, https://
doi.org/10.3389/fvets.2020.00407.
[22] J. Xiong, B. Huang, J.S. Xu, W.S. Huang, A closed-tube loop-mediated isothermal
amplification assay for the visual detection of Staphylococcus aureus, Appl.
Biochem. Biotechnol. 191 (2020) 201–211, https://doi.org/10.1007/s12010-020-
03278-x.
[23] K. Yin, V. Pandian, K. Kadimisetty, C. Ruiz, K. Cooper, J. You, C. Liu,
Synergistically enhanced colorimetric molecular detection using smart cup: a case
for instrument-free HPV-associated cancer screening, Theranostics 9 (2019)
2637–2645, https://doi.org/10.7150/thno.32224.
[24] H.V. Nguyen, V.D. Nguyen, F. Liu, T.S. Seo, An integrated smartphone-based
genetic analyzer for qualitative and quantitative pathogen detection, ACS Omega 5
(2020) 22208–22214, https://doi.org/10.1021/acsomega.0c02317.
[25] G. Papadakis, A.K. Pantazis, N. Fikas, S. Chatziioannidou, V. Tsiakalou,
K. Michaelidou, et al., Portable real-time colorimetric LAMP-device for rapid
quantitative detection of nucleic acids in crude samples, Sci. Rep. 12 (2022) 3775,
https://doi.org/10.1038/s41598-022-06632-7.
[26] K. Yin, V. Pandian, K. Kadimisetty, X. Zhang, C. Ruiz, K. Cooper, C. Liu, Real-time
colorimetric quantitative molecular detection of infectious diseases on
smartphone-based diagnostic platform, Sci. Rep. 10 (2020) 9009, https://doi.org/
10.1038/s41598-020-65899-w.
[27] L. Bokelmann, O. Nickel, T. Maricic, S. Pääbo, M. Meyer, S. Borte, S. Riesenberg,
Point-of-care bulk testing for SARS-CoV-2 by combining hybridization capture with
improved colorimetric LAMP, Nat. Commun. 12 (2021) 1467, https://doi.org/
10.1038/s41467-021-21627-0.
[28] Y.D. Ma, Y.S. Chen, G.B. Lee, An integrated self-driven microfluidic device for
rapid detection of the influenza A (H1N1) virus by reverse transcription loop-
mediated isothermal amplification, Sensor. Actuator. B Chem. 296 (2019) 126647,
https://doi.org/10.1016/j.snb.2019.126647.
[29] E. González-González, I.M. Lara-Mayorga, I.P. Rodríguez-Sánchez, Y.S. Zhang, S.
O. Martínez-Chapa, G. Trujillo-de Santiago, M.M. Alvarez, Colorimetric loop-
mediated isothermal amplification (LAMP) for cost-effective and quantitative
Analytical Biochemistry 688 (2024) 115481
detection of SARS-CoV-2: the change in color in LAMP-based assays quantitatively
correlates with viral copy number, Anal. Methods 13 (2021) 169–178, https://doi.
org/10.1039/d0ay01658f.
[30] W. Panich, T. Tejangkura, T. Chontananarth, Feasibility of a DNA biosensor assay
based on loop-mediated isothermal amplification combined with a lateral flow
dipstick assay for the visual detection of Ascaridia galli eggs in faecal samples,
Avian Pathol. 52 (2023) 209–218, https://doi.org/10.1080/
03079457.2023.2196251.
[31] B. Hill, T. Roger, F.W. Vorhagen, Comparative analysis of the quantization of color
spaces on the basis of the CIELAB color-difference formula, ACM Trans. Graph. 16
(1997) 109–154, https://doi.org/10.1145/248210.248212.
[32] L.F. Capitán-Vallvey, N. Lopez-Ruiz, A. Martinez-Olmos, M.M. Erenas, A.J. Palma,
Recent developments in computer vision-based analytical chemistry: a tutorial
review, Anal. Chim. Acta 899 (2015) 23–56, https://doi.org/10.1016/j.
aca.2015.10.009.
[33] N. Rabiee, M.R. Dokmeci, A. Zarrabi, P. Makvandi, M.R. Saeb, H. Karimi-Maleh, et
al., Green Biomaterials: fundamental principles, Green Biom 1 (2023) 1–4, https://
doi.org/10.1080/29934168.2023.2268943.
[34] N. Rabiee, M. Atarod, M. Tavakolizadeh, S. Asgari, M. Rezaei, O. Akhavan, et al.,
Green metal-organic frameworks (MOFs) for biomedical applications, Microporous
Mesoporous Mater. 335 (2022) 111670, https://doi.org/10.1016/j.
micromeso.2021.111670.
[35] C.O. Nzelu, E.A. Gomez, A.G. Cáceres, T. Sakurai, L. Martini-Robles, H. Uezato, et
al., Development of a loop-mediated isothermal amplification method for rapid
mass-screening of sand flies for Leishmania infection, Acta Trop. 132 (2014) 1–6,
https://doi.org/10.1016/j.actatropica.2013.12.016.
[36] K.A. Barazorda, C.J. Salas, D.K. Bishop, N. Lucchi, H.O. Valdivia, Comparison of
real time and malachite-green based loop-mediated isothermal amplification
assays for the detection of Plasmodium vivax and P. falciparum, PLoS One 15 (2020)
e0234263, https://doi.org/10.1371/journal.pone.0234263.
[37] H.G. Avila, M.G. Risso, P. Ruybal, S.A. Repetto, M.J. Butti, M.D. Trangoni, et al.,
Development of a low-cost copro-LAMP assay for simultaneous copro-detection of
Toxocara canis and Toxocara cati, Parasitology 148 (2021) 819–826, https://doi.
org/10.1017/S0031182021000342.
[38] J. Xiong, S.G. Wu, Y. Liang, Y.L. Zou, X.M. Xie, W.S. Huang, Y.B. Zhang, Visual
closed-tube loop-mediated isothermal amplification (LAMP) assay targeting
aerolysin gene: a practical screening method for virulent Aeromonas species
affecting cultured eels in China, Aquacult. Int. 28 (2020) 2319–2332, https://doi.
org/10.1007/s10499-020-00588-z.
[39] T. Layne, K. Jackson, A. Scott, N.A. Tanner, A. Piland, D.M. Haverstick, J.
P. Landers, Optimization of novel loop-mediated isothermal amplification with
colorimetric image analysis for forensic body fluid identification, J. Forensic Sci.
66 (2021) 1033–1041, https://doi.org/10.1111/1556-4029.14682.
[40] W. Jaroenram, P. Cecere, P.P. Pompa, Xylenol orange-based loop-mediated DNA
isothermal amplification for sensitive naked-eye detection of Escherichia coli,
J. Microbiol. Methods 156 (2019) 9–14, https://doi.org/10.1016/j.
mimet.2018.11.020.
[41] L.S. Yu, S.Y. Chou, H.Y. Wu, Y.C. Chen, Y.H. Chen, Rapid and semi-quantitative
colorimetric loop-mediated isothermal amplification detection of ASFV via HSV
color model transformation, J. Microbiol. Immunol. Infect. 54 (2021) 963–970,
https://doi.org/10.1016/j.jmii.2020.08.003.
[42] J. Rodriguez-Manzano, M.A. Karymov, S. Begolo, D.A. Selck, D.V. Zhukov, E. Jue,
R.F. Ismagilov, Reading out single-molecule digital RNA and DNA isothermal
amplification in nanoliter volumes with unmodified camera phones, ACS Nano 10
(2016) 3102–3113, https://doi.org/10.1021/acsnano.5b07338.