Detection of Novel duck reovirus (NDRV) using visual reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Abstract

Here we present a visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting the gene encoding the σB major outer-capsid protein of novel duck reovirus (NDRV). A set of primers, composed of two outer primers, two inner primers and two loop primers, was designed based on the gene of interest. The LAMP reaction was conducted in a traditional laboratory water bath at 65 °C for 50 min. We compared the performance of calcein/Mn²⁺ and SYBR Green I dyes, as well as electrophoresis on agarose gel stained with GoldView nucleic acid dye to detect the RT-LAMP-amplified products and all assays could be employed to discriminate between positive and negative specimens in visible or UV light. Our data showed that there is no cross-reaction with other viruses and the RT-LAMP technique displayed high sensitivity for detecting NDRV with a minimal detection limit of 200 fg RNA input. This assay was more sensitive than conventional PCR in detecting NDRV both in natural and experimental infection. In conclusion, the RT-LAMP technique was remarkably sensitive, specific, rapid, simple and profitable for the identification of NDRV.

Figure 1

Location of the NDRV RT-LAMP primer in the gene encoding the σB major outer-capsid protein.

Figure 2

Procedures optimization of the NDRV RT-LAMP reaction. (A) The effect of temperature: M, 2000 bp DNA Marker; lanes 1–8 (66 °C, 65 °C, 64 °C, 63 °C, 62 °C, 61 °C, 60 °C and 59 °C, respectively) and lane 9: ddH₂O (negative control; NC). (B) The effect of time: lanes 1–6 (10, 20, 30, 40, 50 and 60 min, respectively); lane 7 NC.

Figure 3

Primer optimization of the NDRV RT-LAMP reaction. (A) The effect of the ratio of inner and outer primers: M, 2000 bp DNA Marker, lanes 1–6 (2:1, 4:1, 6:1, 8:1, 10:1 and 12:1, respectively) and lane 7 NC. (B) The effect of the ratio of loop (Loop primer Fc and Loop primer B) and outer primers (F3 and B3): M, 2000 bp DNA Marker, lanes 1–7 (0:0.2, 1:1, 2:1, 3:1, 4:1, 5:1 and 6:1, respectively); lane 8 NC.

Figure 4

Agarose gel electrophoresis results for NDRV RT-LAMP reaction products and their HhaI restriction enzyme digestion results. M, 2000 bp DNA Marker; lane 1(HhaI restriction enzyme digestion results); lane 2 ladder-like pattern bands; lane 3 NC.

Figure 5

Identification specificity of the NDRV RT-LAMP assay. Samples were resolved on 2.0% agarose gels. LAMP was carried out with the different sources of nucleic acids. Lane M, 2000 bp DNA ladder; Lane 1, recombinant plasmid; Lane 2 NDRV sample; Lanes 3–11, MDRV, ARV, DPV, DHAV, NDV, H9 AIV, H5 AIV, DTMUV and NC.

Figure 6

Macroscopic lesions of NDRV affected 1-day-old ducklings. (A) liver and spleen in physiological saline-injected ducklings; (B) hepatomegaly and pleural exudates with yellow discoloration 48 hpi; (C) hepatomegaly with brittle texture and patchy hemorrhagic necrosis 72 hpi; (D) red darken splenomegaly, enlarged heart and inflated intestine 72 hpi.