Development of a Reagent Kit for the Detection of Yellow Fever Virus RNA by Isothermal Amplification Method

Yellow fever virus (YFV), transmitted by Aedes aegypti mosquitoes, poses a serious threat to public health in the countries of tropical and subtropical regions, as well as causes a concern in the countries with a temperate climate. The virus provokes severe symptoms, including fever, jaundice, and damage to internal organs, and the mortality rate from the infection reaches 20–60%. A live vaccine, created back in 1936 and actively used in endemic regions, is the only effective preventive measure. Clinical diagnosis of the disease is difficult due to a similarity of the symptoms with other infections; however, modern PCR methods are highly accurate and promising for a rapid detection and differential laboratory diagnosis of YFV. To date, isothermal methods for nucleic acid amplification are becoming increasingly important due to their simplicity and independence from complex temperature programs. Their use allows the use of less resource-intensive and accessible equipment, which makes these methods promising for the development of express tests suitable for diagnostics directly at the patient’s bedside. The aim of the work was to develop a reagent kit for the detection of yellow fever virus RNA using the LAMP method with a colorimetric detection. Material and Methods. RNA obtained in this work (YFV-RNA-K+), human DNA isolated from the blood serum, as well as blood serum samples artificially contaminated with RNA of heterologous viruses, were used as the analyzed samples. Bioinformatics methods: analysis of whole genome sequences for primer selection; molecular genetic methods: nucleic acid extraction, reverse transcription reaction, PCR, isothermal amplification, electrophoresis in agarose gel, and Sanger sequencing. Results. An experimental reagent kit was created for the detection of YFV RNA using the LAMP method and visual detection. The detection limit of this kit (104 copies/mL) and optimal reaction time for achieving it were determined. Its specificity to the genetic material of the yellow fever virus and absence of response to heterologous viruses such as dengue, Zika, and tick-borne encephalitis were also confirmed. Conclusions. The creation of a reagent kit for the detection of YFV RNA using the LAMP method will allow us to determine the presence of the virus at early stages of the disease in less than 1 h with a visual assessment of the results. The reagent kit can be useful for laboratory verification of the pathogen in sick individuals or the presence of YFV RNA in mosquito carriers to study a possible virus distribution in both endemic and non-endemic regions. A possibility to use the thermostat instead of expensive amplifier with real-time detection, short reaction time (about 35–40 min), and ease of interpretation of the analysis results are the advantage of using the colorimetric detection method.