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Asian Journal of Animal and Veterinary Advances

Year: 2016 | Volume: 11 | Issue: 7 | Page No.: 405-410
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Authors

Jianchang Wang

Jinfeng Wang

Libing Liu

Ruiying Chai

Wanzhe Yuan

Keywords

Research Article

Rapid and Sensitive Detection of Schmallenberg Virus by a One-step Reverse Transcription Loop-mediated Isothermal Amplification Method

Jianchang Wang, Jinfeng Wang, Libing Liu, Ruiying Chai and Wanzhe Yuan
Background: Schmallenberg virus (SBV), a novel orthobunyavirus was first identified in Germany in 2011. It primarily affects ruminants, e.g., cattle, sheep and goat, etc., causing schmallenberg disease. So far multiple diagnostic methods such as antibody testing and real time reverse transcription polymerase chain reaction (RT-PCR) have been developed for SBV detection. While antibody testing can be used for blood samples, it is not suitable for the test of other samples such as semen, cerebrum and spinal cord. Real time RT-PCR involves complex techniques and requires an expensive thermocycler. Loop-mediated isothermal amplification (LAMP), a novel isothermal gene amplification technique has been explored for the detection of diverse pathogens. The LAMP has several advantages over PCR, including the use of a water bath and being less sensitive to inhibitors that often appear in test samples. In this study, RT-LAMP was explored for SBV detection. Materials and Methods: Viral genes including SBV genomic RNA were used as template. A set of primers targeting the S gene were designed. The RT-LAMP reactions were performed at 61°C in a water bath. Sensitivity of the RT-LAMP assay was determined and compared with that of real time RT-PCR by using different copy numbers of SBV genomic RNA as template. Specificity was evaluated by using different vial genes as template. The RT-LAMP assay was further validated with SBV-spiked animal blood samples. Results: The detection limit of RT-LAMP was 10 copies of SBV genomic RNA, comparable to real time RT-PCR. Specificity analysis revealed that RT-LAMP detected SBV but did not amplify other viruses commonly found in ruminants. Like some real time RT-PCR assays, RT-LAMP cross-detected shamonda virus and Australian douglas virus, members of the Bunyaviridae family, which required further DNA sequencing for differentiation. Validation results showed that RT-LAMP had a complete agreement with real time RT-PCR in detecting SBV in spiked blood samples. Conclusion: A one-step RT-LAMP method was successfully developed. The RT-LAMP assay is comparable to real time RT-PCR in term of assay sensitivity and specificity but it is simpler and more cost-effective. The RT-LAMP method might be potentially applied to replace real time RT-PCR for SBV detection in resource-limited settings.
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