A TaqMan-based real-time PCR assay for specific detection of novel duck reovirus in China
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METHODOLOGY ARTICLE
Open Access
A TaqMan-based real-time PCR assay for specific detection of novel duck reovirus in China Shuai Zhang1,2,3†, Weihua Li4†, Xiaodong Liu5, Xudong Li1,2,3, Bin Gao1,2,3, Youxiang Diao1,2,3*† and Yi Tang1,2,3*†
Abstract Background: In China, Newly emerging duck reovirus (NDRV) variants have been causing major disease problems in cherry valley ducks. NDRV has the potential to cause high morbidity and 5–50% mortality rates. Severe hemorrhagic-necrosis in the liver and spleen were commonly seen in NDRV affected ducks. The availability of upgraded methods for rapid diagnosis of newly emerging DRV variants is crucial for successful DRV infection control and prevention. Results: In this study, we present a TaqMan-based real-time PCR assay (RT-qPCR) for the detection of NDRV infection. Using the conserved regions within the NDRV genome, we designed the specific primers and probe. The lower limit of detection for NDRV infection was 10 copies/μL (Ct values: 38.3) after the optimization of the RT-qPCR conditions. By cross-checking with other duck viral pathogens, no crossreactivity was observed confirming the assay was highly specific for the detection of NDRV. Reproducibility of the RT-qPCR was confirmed by intra- and inter-assay variability was less than 2.91%(Intra-assay variability of Ct values: 0.07–1.48%; Interassay variability of Ct values: 0.49–2.91%). This RT-qPCR and conventional PCR (cPCR) detected one hundred and twenty samples of NDRV infection from different regions. The result shows that the positive rates were 94.17 and 84.17% respectively. The detection rate of RT-qPCR rapid detection assay was 10% higher than that of the cPCR method. Conclusion: This research developed a highly sensitive, specific, reproducible and versatile of RT-qPCR for quantitatively detecting NDRV. It can be used to study the pathogenesis and epidemiology investigation of NDRV. Keywords: Novel duck reovirus, σC gene, Real-time PCR assay, TaqMan-based probe, Detection method
* Correspondence: [email protected]; [email protected] † Shuai Zhang, Weihua Li, Youxiang Diao and Yi Tang contributed equally to this work. 1 College of Animal Science and Technology, Shandong Agricultural University, 61 Daizong Street, Tai’an 271018, Shandong Province, China Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the pe
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