A one-step reverse transcription loop-mediated isothermal amplification for detection and discrimination of infectious bursal disease virus

Abstract

BackgroundInfectious bursal disease (IBD) is a highly contagious immunosuppressive disease in young chickens caused by infectious bursal disease virus (IBDV). It causes huge economic losses to the poultry industry. The objective of this study is to develop a loop-mediated isothermal amplification (LAMP) method for the detection and discrimination of IBDV.ResultsIn this study, we applied reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect IBDV in one simple step and further identified the very virulent strain from non-vvIBDVs with a simply post-amplification restriction enzyme analysis. Based on sequence analysis, a set of two inner, two outer and two loop primers were designed to target the VP5 gene and they showed great specificity with no cross reaction to the other common avian pathogens. The detection limit determined by both color change inspection and agarose gel electrophoresis was 28 copies viral RNA, which was almost as sensitive as a real-time RT-PCR previous developed in our laboratory. We also identified a unique Tfi I restriction site located exclusively in non-vvIBDVs, so very virulent strain could be distinguished from current vaccine strains. By screening a panel of clinical specimens, results showed that this method is high feasible in clinical settings, and it obtained results 100% correlated with real-time RT-PCR.ConclusionRT-LAMP is a rapid, simple and sensitive assay. In combination with the Tfi I restriction analysis, this method holds great promises not only in laboratory detection and discrimination of IBDV but also in large scale field and clinical studies.

Simultaneous detection and differentiation of dengue virus serotypes 1-4, Japanese encephalitis virus, and West Nile virus by a combined reverse-transcription loop-mediated isothermal amplification assay

“…This result is not consistent with the published one that a 65ºC incubation for 60 min yielded the best result [19]. The reason is unknown.…”

Colorimetric biosensors for point-of-care virus detections

“…Its high efficiency is due to the use of a single-step test tube at around 60-65°C for approximately thirty minutes [11][12][13][14]. The single step LAMP is obtained by combining reverse transcripts of RNAs with LAMP [15]. The reaction time for LAMP is very fast at just ten minutes [11], which enables the total time required for the entire LAMP process to be from fifty [12] to sixty minutes [13].…”

“…The four primers are two forward inner primers and two backward inner primers for first and second stages of the process respectively [12]. These primers are designed to be specific to the virus detected [15][16][17], such as a particular protein gene on the virus coat [18]. However, one can use five or six primers as well [14].…”

“…However, one can use five or six primers as well [14]. In the event when five primers are used, the primers consist of two inner primers, two outer ones and one loop primer [15]. Additionally, six primers are frequently used for the virus detection to be even more selective and efficient [12,14,19].…”

Rapid Detection of Viruses Using Loop-Mediated Isothermal Amplification (LAMP): A Review

“…Kiatpathomchai et al ( 2007 ) detected RT-TSV RNA in P. vannamei (collected from shrimp farms) by LAMP assay, and the sensitivity of RT-LAMP appears to be ten times more than RT-PCR. Wang et al ( 2011 ) detected infectious bursal disease virus (IBDV) in one simple step by reverse transcription loopmediated isothermal amplifi cation (RT-LAMP) and further identifi ed the very virulent strain from non-vvIBDVs with a simply post-amplifi cation restriction enzyme analysis. A set of two inner, two outer, and two loop primers were designed on the basis of sequence analysis to target the VP5 gene and showed great specifi city with no cross-reaction to the other common avian pathogens.…”