Setting up a real-time Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method for detection of Enterovirus B species

Abstract

Enteroviruses (EV) are small, non-enveloped, single stranded RNA viruses that are highly abundant in nature. They are a common cause of both acute and chronic diseases in humans but can also spread undetected in population without showing any symptoms. Currently, there are no treatments or prevention methods for enterovirus infections except for polio and EV71 vaccines and the detection methods used are laborious and time consuming. This highlights a need for faster and more accurate diagnostic methods that could be utilised in basic research and clinical setting to detect these viruses. A nucleic acid-based detection method called loop-mediated isothermal amplification (LAMP) is based on auto-cycling strand displacement synthesis by DNA polymerase. The reaction happens in a constant temperature and results in different lengths of DNA structures. To date, it has been used to detect various microorganisms and even utilized in few clinical applications. However, what hinders their further utilization is the risk of false positives and difficulty of optimization. The aim of this thesis is to design a LAMP protocol, design primers and optimize an assay that can detect enterovirus B species.

In this project, a real-time fluorescent detection-based reverse transcription LAMP (RT-LAMP) assay was developed and optimized for the detection of type B enteroviruses. For the detection, one primer set from article and two self-designed primer sets were used that target the 5’UTR and VP1 area of enterovirus genome. Optimization consisted of testing different reagent combinations and changing the reaction conditions by adjusting temperature and salt concentration and incorporating additives into the mix. Additionally, sensitivity of the assay was investigated with a dilution series consisting of different amounts of virus particles. Lastly, electrophoresis and Sanger sequencing were used to examine the amplification products.

The results showed that primer design successfully created a primer set (CVB1-10796 5’UTR) that recognised CVB1 virus template and amplified the target in the RT-LAMP assay. Successful results came with reaction mix consisting of Bst 3.0 DNA polymerase, SuperScript IV reverse transcriptase and EvaGreen dye. The optimal temperature was found to be 65°C and the addition of DMSO did not improve sensitivity or specificity of the assay. Optimum concentration of magnesium was 8 mM but results showed variation. Additionally, results confirmed that RNA extraction is not needed before LAMP reaction. Sensitivity of the reaction needs to be verified in future experiments.

In conclusion, this project determined optimum conditions for a LAMP assay used to detect type B enteroviruses. Additionally, it showed the difference between literature and experimental work. The method is not as robust as pictured in literature and the problems with non-specific amplification and contamination are similar to PCR. However, this assay does have the potential to be used in wider spectrum but for this to happen more research needs to be done to study the underlying mechanism of LAMP and what affects it to create assays that could be utilized in other applications with minimal optimization.