This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dhar, A. K.
Right arrow Articles by Klimpel, K. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dhar, A. K.
Right arrow Articles by Klimpel, K. R.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, August 2001, p. 2835-2845, Vol. 39, No. 8
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.8.2835-2845.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Detection and Quantification of Infectious Hypodermal and Hematopoietic Necrosis Virus and White Spot Virus in Shrimp Using Real-Time Quantitative PCR and SYBR Green Chemistry

Arun K. Dhar, Michelle M. Roux, and Kurt R. Klimpel*

Super Shrimp, Inc., National City, California, California 91950

Received 14 December 2000/Returned for modification 10 May 2001/Accepted 26 May 2001

A rapid and highly sensitive real-time PCR detection and quantification method for infectious hypodermal and hematopoietic necrosis virus (IHHNV), a single-stranded DNA virus, and white spot virus (WSV), a double-stranded DNA (dsDNA) virus infecting penaeid shrimp (Penaeus sp.), was developed using the GeneAmp 5700 sequence detection system coupled with SYBR Green chemistry. The PCR mixture contains a fluorescence dye, SYBR Green, which upon binding to dsDNA exhibits fluorescence enhancement. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. A linear relationship was observed between the amount of input plasmid DNA and cycle threshold (CT) values over a range of 1 to 105 copies of the viral genome. To control the variation in sampling and processing among samples, the shrimp beta -actin gene was amplified in parallel with the viral DNA. The CT values of IHHNV- and WSV-infected samples were used to determine absolute viral copy numbers from the standard CT curves of these viruses. For each virus and its beta -actin control, the specificity of amplification was monitored by using the dissociation curve of the amplified product. Using genomic DNA as a template, SYBR Green PCR was found to be 100- to 2000-fold more sensitive than conventional PCR, depending on the virus, for the samples tested. The results demonstrate that SYBR Green PCR can be used as a rapid and highly sensitive detection and quantification method for shrimp viruses and that it is amenable to high-throughout assay.

* Corresponding author. Mailing address: Super Shrimp, Inc., 1545 Tidelands Ave., Suite J, National City, CA 91950. Phone: (619) 477-5394. Fax: (619) 477-5396. E-mail: klimpel{at}supershrimp.com.

Journal of Clinical Microbiology, August 2001, p. 2835-2845, Vol. 39, No. 8
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.8.2835-2845.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Wang, S., Liu, N., Chen, A.-J., Zhao, X.-F., Wang, J.-X. (2009). TRBP Homolog Interacts with Eukaryotic Initiation Factor 6 (eIF6) in Fenneropenaeus chinensis. J. Immunol. 182: 5250-5258 [Abstract] [Full Text]  
  • Ionescu, M., Belkin, S. (2009). Overproduction of Exopolysaccharides by an Escherichia coli K-12 rpoS Mutant in Response to Osmotic Stress. Appl. Environ. Microbiol. 75: 483-492 [Abstract] [Full Text]  
  • Schuurman, T., van Breda, A., de Boer, R., Kooistra-Smid, M., Beld, M., Savelkoul, P., Boom, R. (2005). Reduced PCR Sensitivity Due to Impaired DNA Recovery with the MagNA Pure LC Total Nucleic Acid Isolation Kit. J. Clin. Microbiol. 43: 4616-4622 [Abstract] [Full Text]  
  • Pillitteri, L. J., Lovatt, C. J., Walling, L. L. (2004). Isolation and Characterization of a TERMINAL FLOWER Homolog and Its Correlation with Juvenility in Citrus. Plant Physiol. 135: 1540-1551 [Abstract] [Full Text]  
  • Endoh, H., Tomida, S., Yatabe, Y., Konishi, H., Osada, H., Tajima, K., Kuwano, H., Takahashi, T., Mitsudomi, T. (2004). Prognostic Model of Pulmonary Adenocarcinoma by Expression Profiling of Eight Genes As Determined by Quantitative Real-Time Reverse Transcriptase Polymerase Chain Reaction. JCO 22: 811-819 [Abstract] [Full Text]  
  • Plikus, M., Wang, W. P., Liu, J., Wang, X., Jiang, T.-X., Chuong, C.-M. (2004). Morpho-Regulation of Ectodermal Organs: Integument Pathology and Phenotypic Variations in K14-Noggin Engineered Mice through Modulation of Bone Morphogenic Protein Pathway. Am. J. Pathol. 164: 1099-1114 [Abstract] [Full Text]  
  • Coulet, F., Nadaud, S., Agrapart, M., Soubrier, F. (2003). Identification of Hypoxia-response Element in the Human Endothelial Nitric-oxide Synthase Gene Promoter. J. Biol. Chem. 278: 46230-46240 [Abstract] [Full Text]  
  • Zhu, Y., Sun, Y., Xie, L., Jin, K., Sheibani, N., Greenberg, D. A. (2003). Hypoxic Induction of Endoglin via Mitogen-Activated Protein Kinases in Mouse Brain Microvascular Endothelial Cells. Stroke 34: 2483-2488 [Abstract] [Full Text]  
  • Roux, M. M., Pain, A., Klimpel, K. R., Dhar, A. K. (2002). The Lipopolysaccharide and {beta}-1,3-Glucan Binding Protein Gene Is Upregulated in White Spot Virus-Infected Shrimp (Penaeus stylirostris). J. Virol. 76: 7140-7149 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»