Development of a portable reverse transcription loop‐mediated isothermal amplification system to detect the E1 region of Chikungunya virus in a cost‐effective manner

Abstract

Chikungunya fever is a mosquito‐borne disease cause of persistent arthralgia. The current diagnosis of Chikungunya virus (CHIKV) relies on a conventional reverse transcription polymerase chain reaction assay. Reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) is a rapid and simple tool used for DNA‐based diagnosis of a variety of infectious diseases. In this study, we established an RT‐LAMP system to recognize CHIKV by targeting the envelope protein 1 (E1) gene that could also detect CHIKV at a concentration of 8 PFU without incorrectly detecting other mosquito‐borne viruses. The system also amplified the E1 genome in the serum of CHIKV‐infected mice with high sensitivity and specificity. Moreover, we established a dry RT‐LAMP system that can be transported without a cold chain, which detected the virus genome in CHIKV‐infected patient samples with high accuracy. Thus, the dry RT‐LAMP system has great potential to be applied as a novel CHIKV screening kit in endemic areas.

Development of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Detecting Feline Coronavirus

“…The amplification is carried out by a set of six primers recognizing eight distinct regions of the targeted gene [ 60 ], yielding a large amount of cauliflower-like DNA structures and insoluble pyrophosphate as a by-product [ 51 ]. LAMP amplicons can be detected by intercalating fluorescent dye [ 61 ], the turbidity of white Mg-pyrophosphate precipitate [ 62 ], agarose gel electrophoresis [ 63 ], and visualization using a metal-sensitive dye such as calcein [ 63 ] or hydroxynaphthol blue (HNB) [ 64 ]. It can also be observed using the color change of pH-sensitive dyes such as xylenol orange [ 65 ], phenol red [ 66 ], and neutral red (NR) [ 67 ], as a result of the generation of protons from the polymerization reaction [ 68 ].…”

“…When compared to the methods using intercalating fluorescent dye and metal-sensitive dye, the NR-based detection system has two important advantages, that is distinct contrast of color change of positive (pink color) and negative (yellow color) reaction. While violet changed to blue for HNB [ 55 , 64 ], dark blue changed to light blue for malachite green [ 69 ], and dark yellow changed to yellow for calcein [ 63 ], which were less discernable color changes based on their effects, disadvantage for these dyes [ 70 ]. Another advantage of the NR dye is its ability to easily visualize the color change of the reaction by naked eye without the risk of post-contamination from re-opening the tube [ 67 , 71 , 72 ].…”

Naked‐eye detection strategies coupled with isothermal nucleic acid amplification techniques for the detection of human pathogens

“…HNB can be implemented in a similar way as of calcein, overcoming the limitations of SG, but without the need to supplement the reaction with Mn 2+ ions. Its application has been extensively reported (Anupama et al., 2020; Chen et al., 2017; Dong et al., 2015; Ma et al., 2010; Saechue et al., 2020; Srisrattakarn et al., 2017) (see Figure 7).…”

Development of a Loop-Mediated Isothermal Amplification Assay Coupled With a Lateral Flow Dipstick Test for Detection of Myosin Binding Protein C3 A31P Mutation in Maine Coon Cats

“…However, this method uses complex procedures and requires specialized equipment. The other commonly used methods for LAMP product verification are the visualization of colorimetric and lateral flow dipstick (LFD) tests, which are simple and straightforward for carrying out POC tests in a small animal hospital or small laboratory unit ( 22 ). Nonetheless, the application of LAMP combined with LFD for the detection of MYBPC3-A31P mutations has not yet been reported.…”