Development of simple and rapid assay to detect viral RNA of tick-borne encephalitis virus by reverse transcription-loop-mediated isothermal amplification

Abstract

BackgroundTick-borne encephalitis virus (TBEV) is a causative agent of acute central nervous system disease in humans. It has three subtypes, far eastern (FE), Siberian (Sib) and European (Eu) subtypes, which are distributed over a wide area of Europe and Asia. The objective of this study was to develop a simple and rapid assay for the detection of TBEV RNA by using reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) method that can differentiate the three subtypes of TBEV and can be used for clinical diagnosis and epidemiological study.MethodsPrimers for TBEV-specific and subtype-specific RT-LAMP assay were designed to target the consensus sequence in NS1 of all subtypes and the consensus sequence in the E gene of each subtype, respectiveluy. In vitro transcribed RNA of Oshima strain that belongs to FE subtype was serially diluted and used to examine the sensitivity of the assay. Cross-reactivity of subtype-specific RT-LAMP assay was tested by using the RNA of Oshima and Sofjin (FE), IR-99 (Sib) and Hochosterwitz (Eu) strains. RNA extracted from the mixtures of TBEV and ticks, and of TBEV and human blood, and the mouse tissues infected with TBEV, were evaluated in the assay. Positive amplification was observed by real-time monitoring of turbidity and by visual detection of color change.ResultsThe sensitivity of TBEV-specific RT-LAMP assay was 102 copies of target RNA per reaction volume. FE-specific RT-LAMP assay amplified viral genes of Oshima and Sofjin strains but not of IR-99 and Hochosterwitz strains, and of Japanese encephalitis virus. RT-LAMP assay for Sib and for Eu specifically amplified viral genes of IR-99 and Hochosterwitz strains, respectively. We also showed that tick or human blood extract did not inhibit the amplification of viral gene during the assay. Furthermore, we confirmed that the TBEV RT-LAMP could detect virus RNA from peripheral and central nervous system tissues of laboratory mice infected with TBEV.ConclusionTBEV RT-LAMP assay offers a sensitive, specific, rapid and easy-to-handle method for the detection of TBEV RNA in tick samples and this may be applied in the clinical samples collected from TBE-suspected patients.

Comparison of Fluorescent Intercalating Dyes for Quantitative Loop-Mediated Isothermal Amplification (qLAMP)

“…The reactions contained 1 ng of lambda phage DNA (11), 1.6 ng of E. coli genomic DNA (23), and 10 4 copies of pCR-TBEV plasmid (24), unless otherwise indicated. Fluorescent dyes were purchased from Invitrogen (SYTO-9, SYTO-13, SYTO-82, SYBR Green I, SYBR Gold) or Biotium (Hayward, CA) (EvaGreen).…”

Pathologic Potential of Variant Clones of the Oshima Strain of Far-Eastern Subtype Tick-Borne Encephalitis Virus

“…The levels of viral RNA were measured by real time-PCR as demonstrated previously [27, 28]. The copy numbers were calculated as a ratio of the copy numbers in 100 ng of RNA.…”

Colorimetric biosensors for point-of-care virus detections

“…Additionally, six primers are frequently used for the virus detection to be even more selective and efficient [12,14,19]. The six primers will be two inner primers, two outer primers and two loop primers [19][20][21]. LAMP primers can be designed by software such as Primer Explore V4 [22].…”

“…The presence of virus is shown by an increase in turbidity due to pyrophosphate ion attained from LAMP that causes white magnesium pyrophosphate precipitate to form in the assay [10,21,[35][36][37][38]. The higher turbidity is caused by a larger amount of amplified products that leads to a larger amount of white magnesium pyrophosphate precipitate, hence forming a more cloudy solution with higher turbidity [12].…”