Evaluation of semi-quantitative colorimetric assays based on loop-mediated isothermal amplification indicators by using image analysis.

Colorimetric assays are some of the most convenient detection methods, creating discoloration in solutions that is visible to the naked eye. However, colorimetric reactions have some limitations regarding the variability in the color perception of individuals caused by factors such as color blindness, experience, and gender. Semi-quantitative chromatic analysis has been used as an alternative method to differentiate between two colors and accurately interpret the results from a numerical value, with high confidence. Therefore, we developed and 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.

Visualization and development of colorimetric loop-mediated isothermal amplification for the rapid detection and diagnosis of paramphistome infection: colorimetric PAR-LAMP.

Colorimetric detection can be applied to differentiate between positive and negative conditions. It can be coupled with loop-mediated isothermal amplification to diagnose rumen fluke or paramphistome infection, also called colorimetric PAR-LAMP. This study conducted LAMP using three candidate indicator dyes, namely malachite green (MLG), methyl green (MTG), and neutral red (NTR), and the results were observed by the naked eye. The dye concentration was optimized to obtain the most pronounced positive-negative result discrimination. Subsequently, we conducted target sensitivity tests using the DNA of Fischoederius elongatus at different concentrations. To validate the detection accuracy, the result was confirmed by gel electrophoresis. The sensitivity test presented the lowest detectable DNA concentration or limit of detection (LOD), with 1 pg for MLG, 0.5 ng for MTG, and 50 pg for NTR. Different LODs revealed inhibition of LAMP reaction and reduced efficiency of result presentation for colorimetric-based detection, particularly NTR and MTG. For MLG-LAMP, we observed no cross-reaction of non-target DNA and improved reaction with the DNA of Fischoederius cobboldi and Calicophoron sp., with multi-detection. In addition, naked eye observation and agarose gel electrophoresis (AGE) evaluation of the MLG-LAMP results showed a moderate and strong agreement with LAMP-AGE and microscopic examinations. Based on our results, colorimetric PAR-LAMP is a rapid, comfortable, and point-of-care procedure for the diagnosis of paramphistome infection.

Preliminary data on Ascaridia galli infections in Gallus gallus domesticus and the development of a specific primer based on the NADH dehydrogenase subunit 4.

One major problem of chicken (Gallus gallus domesticus) farming was various parasitic infections, especially Ascaridia galli that can cause the Ascaridiosis and is commonly found worldwide. The purpose of this study was to investigate the epidemiological situation of gastrointestinal tract parasitic infections and to develop species-specific primer for A. galli detection. A total of 247 chicken gastrointestinal tract specimens from 5 fresh markets in Bangkok. The species-specific primers of A. galli were manually designed using the mitochondrial genome at the NADH dehydrogenase subunit 4 (MT-ND 4) gene. As a result, PCR assays were optimized for the specific PCR product approximately 198 bp with the optimal temperature of 51 °C. In addition, sensitivity tests provided the detection of adult and egg stages at the minimum concentrations of 156.3 ng and 2.8 ng (70 eggs), respectively. This research can be used as preliminary information regarding the epidemic situation of gastrointestinal tract infections in chickens and detection of A. galli infection in definitive hosts, which plans programs for the effective control and prevention of parasitic infections.