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Orf is a non-systemic, ubiquitous disease of sheep and goats caused by the orf virus (ORFV). ORFV occasionally causes cutaneous lesions in humans in contact with infected animals. In the present study, a real-time PCR method was established for detection of ORFV using the fluorescent chimeric dye SYBR Green I. Specific primers were designed to target a highly conserved region of the ORFV B2L gene. This method was able to detect a minimum of 20 copies of ORFV genomic DNA. The results showed no cross-reactions with other common DNA viruses. The time required for the test was approximately 1.5 h. Clinical test samples showed that this method was faster and had a higher sensitivity than traditional PCR. In conclusion, this novel, real-time PCR-based assay provides a rapid, sensitive, and specific method for ORFV detection. This test provides improved technical support for studies regarding the clinical diagnosis and epidemiology of ORFV.

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In this study, we develop a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid and easy detection of goose astroviruses (GAstVs) in clinical samples. The specific LAMP primer sets were designed targeting the ORF2 gene of GAstV. The conditions of LAMP amplification were optimized in terms of reaction time and temperature. The optimal conditions are 60 min in a 60 °C water bath. No cross-reactivity was noted with fowl adenovirus serotype 4 (FAdV-4), duck tembusu virus (DTMUV), goose parvovirus (GPV), avian infectious bronchitis virus (IBV), or chicken anemia virus (CAV). The proposed RT-LAMP method was compared with conventional RT polymerase chain reaction (RT-PCR) and with nested RT-PCR. The results showed that the sensitivity of the proposed method was comparable to that of nested RT-PCR and tenfold higher than that of the conventional RT-PCR. Clinical samples (N = 129) of the liver and kidney from sick geese collected from six commercial goose farms were tested. The positive rate was 39.5% (51/129), 38.8% (50/129), and 34.9% (45/129) using RT-LAMP, nested RT-PCR and conventional RT-PCR, respectively. The developed RT-LAMP diagnostic method is not only simple, rapid, and highly specific, but also portable for use on the field. It may be used in epidemiological investigation to detect GAstVs.