Development of quantitative polymerase chain reaction assay for detection of scale drop disease virus (SDDV) in Asian sea bass
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Issued Date
2021
Copyright Date
2021
Resource Type
Language
eng
File Type
application/pdf
No. of Pages/File Size
x, 64 leaves : ill.
Access Rights
open access
Rights
ผลงานนี้เป็นลิขสิทธิ์ของมหาวิทยาลัยมหิดล ขอสงวนไว้สำหรับเพื่อการศึกษาเท่านั้น ต้องอ้างอิงแหล่งที่มา ห้ามดัดแปลงเนื้อหา และห้ามนำไปใช้เพื่อการค้า
Rights Holder(s)
Mahidol University
Bibliographic Citation
Thesis (M.Sc. (Biotechnology))--Mahidol University, 2021
Suggested Citation
Sukhontip Sriisan Development of quantitative polymerase chain reaction assay for detection of scale drop disease virus (SDDV) in Asian sea bass. Thesis (M.Sc. (Biotechnology))--Mahidol University, 2021. Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/114216
Title
Development of quantitative polymerase chain reaction assay for detection of scale drop disease virus (SDDV) in Asian sea bass
Author(s)
Abstract
Scale drop disease virus (SDDV) is a novel Megalocytivirus causing scale drop disease (SDD) in Asian sea bass in Southeast Asia. In order to support disease diagnosis and surveillance, the present study developed a highly sensitive and specific SYBR Green qPCR assay for rapid detection of SDDV. Specific primers targeting a 135-bp fragment of ATPase coding gene and a 121-bp fragment of major capsid protein (MCP) coding gene of the SDDV genome were newly designed. The optimized qPCR conditions using either ATPase or MCP primers showed no cross amplification with DNA extracted from 12 viruses and bacteria commonly found in aquatic animals. However, the former protocol had a detection limit of 2 viral copies per reaction while the latter had 20 copies. Thus, the SDDV ATPase qPCR method was subsequently validated with field samples (n=86). The results revealed that all clinically sick fish (n=34) from 5 affected farms revealed positive results. Interestingly, 30/52 samples of apparently healthy fish from 8 unaffected farms which previously tested negative for SDDV by semi-nested PCR assay were positive by the newly developed qPCR method. This suggested that qPCR method is highly sensitive and suitable for early screening of SDDV from clinically healthy fish and for disease confirmation of sick fish. Investigation of tissue tropism and viral load of SDDV revealed systemic viral infection in all 8 tested organs with viral loads ranging from 8×10 2 to 6.8×10 4 copies per 200 ng of DNA template. The newly developed qPCR method in this study delivered an accurate and reliable method for rapid detection of SDDV that may facilitate active surveillance in Asian sea bass farming industry.
Degree Name
Master of Science
Degree Level
Master's degree
Degree Department
Faculty of Science
Degree Discipline
Biotechnology
Degree Grantor(s)
Mahidol University