Creation of Standard DNA for Detection and Quantification of White Spot Syndrome Virus in Shrimps by Real-time PCR (qPCR)
Main Article Content
Abstract
White spot syndrome virus (WSSV) is a double stranded DNA virus that causes WSS diseases for many crustaceans including the Penaeidae family shrimp. In this study, standard DNA for detection and quantification of WSSV by real-time polymerase chain reaction (qPCR) method was created. A specific gene fragment of 126 bp from WSSV genome was successfully amplified by PCR and cloned into pGEM-T vector. qPCR calibration curves using the created recombinant pGEM vector harboring 126 bp gene fragment as the standard DNA at concentrations of 3x102 to 3x108 copies/mL showed to have good linear regression with an efficiency of 99.1%, correlation coefficient R2 of 0.998 and the slope of -3.344. The recombinant vector was also used as a positive standard for detection and quantification of WSSV in a number of WSSV-infected shrimp samples and the values ranging from 3.69x103 copies/mL to 1.25x108 copies/mL WSSV were found in the collected samples.
References
[2] K. Takemura, J. Satoh, J. Boonyakida, S. Park, A. D. Chowdhury, E. Y. Park, Electrochemical Detection of White Spot Syndrome Virus with a Silicone Rubber Disposable Electrode Composed of Graphene Quantum Dots and Gold Nanoparticle-Embedded Polyaniline Nanowires, Vol. 18, No. 152, 2009, https://doi.org/10.1186/s12951-020-00712-4.
[3] T. M. H. Truong, T. V. Pham, T. B. Phan, T. M. L. Huynh, T. V. Phan, A Study on White Spot Syndrome Virus (WSSV) Infection on Crayfish (Macrobrachium nipponense) and Posibility of WSSV Transmission to White Leg Shrimp (Litopenaeus vannamei), Vietnam Journal of Science, Technology and Engineering, Vol. 19, No. 8, 2017, pp. 33-38 (in Vietnamese).
[4] L. M. Nunan, D. V. Lightner, Optimized PCR Assay for Detection of White Spot Syndrome Virus (WSSV), Journal of Virological Methods, Vol. 171, 2011, pp. 18-21.
[5] F. Zhu, H. Quan, A New Method for Quantifying White Spot Syndrome Virus: Experimental Challenge Dose Using TaqMan Real-time PCR Assay, Journal of Virological Methods, Vol. 188, 2012, pp. 121-124,
http://dx.doi.org/10.1016/j.jviromet.2012.05.026.
[6] F. M. Cano, A. S. Paz, Development and Validation of a Quantitative Real-time Polymerase Chain Assay for Universal Detection of the White Spot Syndrome Virus in Marine Crustaceans, Virology Journal, Vol. 10, No. 186, 2013, http://www.virologyj.com/content/10/1/186.
[7] Y. M. Hafez, N. Y. Moustafa, A. F. Magouz, N. F. A. Maria, Isolation of White Spot Syndrome Virus (WSSV) Egyptian Shrimp Using Conventional PCR and Real time PCR (qPCR) Techniques, Slovenian Veterinary Research,
Vol. 56, No. 22, 2019, pp. 457-64, https://doi.org/10.26873/SVR-783-2019.
[8] T. T. H. Tran, A Polymerase Chain Reaction Protocol for the Detection of White Spot Syndrome Virus (WSSV) and Decapod Genes Serving as Internal Control in Various Infected Hosts, Can Tho University of Science, Vol. 17a, 2011, pp. 1-8 (in Vietnamese).
[9] T. Y. Hoang, T. N. Nguyen, T. H. Pham, T. H. Nguyen, M. T. Tran, et al., Detection of White Spot Syndrome Virus in Shrimp by Using a Rapid Test, Vietnam Journal of Biotechnology, Vol. 10, No. 1, 2012, pp. 145-150 (in Vietnamese).
[10] W. Wu, L. Wang, X. Zhangb, Identification of White Spot Syndrome Virus (WSSV) Envelope Proteins Involved in Shrimp Infection, Virology, Vol. 332, 2005, pp. 578-583, https://doi.org/10.1016/j.virol.2004.12.011.
[11] P. H. Choua, Y. C. Lina, P. H. Tenga, C. L. Chenb, P. Y. Leec, Real-time Target-specific Detection of Loop-mediated Isothermal Amplification for White Spot Syndrome Virus Using Fluorescence Energy Transfer-based Probes, Journal of Virological Methods, Vol. 173, 2011, pp. 67-74, https://doi.org/10.1016/j.jviromet.2011.01.009.
[12] B. Sun, Z. Wang, F. Zhu, The Crustin-like Peptide Plays Opposite Role in Shrimp Immune Response to Vibrio alginolyticus and White Spot Syndrome Virus (WSSV) Infection, Fish & Shellfish Immunology, Vol. 66, 2017, pp. 487-496, http://dx.doi.org/10.1016/j.fsi.2017.05.055.