Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye

Abstract

A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid visual detection of turkey coronavirus (TCoV) infection. The reaction is performed in one step in a single tube at 65 °C for 45 min, with hydroxynaphthol blue (HNB) dye added prior to amplification. The detection limit of the RT-LAMP assay was approximately 10(2) EID(50/50 μl) TCoV genome, and no cross-reaction with other avian viruses was observed. The assay was evaluated further in tissue suspensions prepared from the ileum and ileum-caecal junctions of infected turkey embryos; 100% of these samples were positive in the RT-LAMP assay. All individual feces samples collected in the field were considered positive by both conventional RT-PCR and RT-LAMP. In conclusion, RT-LAMP with HNB dye was shown to be a sensitive, simple assay for the rapid diagnosis of TCoV infection, either directly from feces or in association with virus isolation methods.

Fig. 1

Sensitivity (A) and specificity (B) of RT-LAMP assay evaluated by eye after the addition of hydroxynaphthol blue dye. A) Lanes 1–6: cDNA from the TCoV (TCoV/BR/2007) at different titers: 10⁴ EID₅₀/_50μl (1), 10³ EID₅₀/_50μl (2), 10² EID₅₀/_50μl (3), 10¹ EID₅₀/_50μl (4), 10⁰ EID₅₀/_50μl (5), 10⁻¹ EID₅₀/_50μl (6); lane 7 corresponded to water instead of viral cDNA; B) Lanes 1–6: RNA from TCoV (TCoV/BR/2007) (1), NDV (2), TAstV-1 (3), IBV-M41 (4), IBV-H120 (5), AMPV (6); C) Agarose gel electrophoresis of RT-LAMP products from 10² EID₅₀/_50μl (line 2), 10³ EID₅₀/_50μl (line 3) dilution of TCoV; line 1, DNA marker 2 kb plus; D) Sensitivity and specificity of conventional RT-PCR; Lanes (1) corresponded to water instead of viral RNA, (2) TAstV-1, (3) IBV-41, (4) IBV-H120; Lanes 5–10: RT-PCR products from TCoV at different titers: 10⁻¹ EID₅₀/_50μl (5), 10⁰ EID₅₀/_50μl (6), 10¹ EID₅₀/_50μl (7), 10² EID₅₀/_50μl (8), 10³ EID₅₀/_50μl (9), 10⁴ EID₅₀/_50μl (10); Lanes 11–13 tissue corresponded to RT-PCR amplification from RNA extracted from uninfected poult embryos.