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, 39 (8), 2835-45

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

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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

A K Dhar et al. J Clin Microbiol.

Abstract

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 (C(T)) values over a range of 1 to 10(5) 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 C(T) values of IHHNV- and WSV-infected samples were used to determine absolute viral copy numbers from the standard C(T) 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.

Figures

FIG. 1
FIG. 1
Linear relationship between CT values and the dilutions of the plasmid DNAs of IHHNV (A) and WSV (B) samples. The exact numbers of copies of the IHHNV plasmid added to each reaction mixture (corresponding to the numbers on the linear curve) were as follows: 1.88 × 105 (1), 1.88 × 104 (2), 1.88 × 103 (3), 1.88 × 102 (4), 1.88 × 101 (5), 9.4 (6), 4.7 (7), 2.35 (8), and 1.18 (9). For WSV samples, the plasmid copy numbers were 2.37 × 105 (1), 2.37 × 104 (2), 2.37 × 103 (3), 2.37 × 102 (4), 2.37 × 101 (5), 11.85 (6), 5.93 (7), 2.96 (8), and 1.48 (9). The amplifications of IHHNV and WSV DNAs from the same plasmid dilutions by conventional PCR are shown in panels C and D, respectively. The lane numbers refer to the plasmid copy numbers mentioned above. Lane M, 50-bp DNA step ladder (Sigma); lane −ve, negative control.
FIG. 2
FIG. 2
(A and B) Detection limits for IHHNV (A) and WSV (B) DNAs by conventional PCR. The dilutions of genomic DNAs of IHHNV- and WSV-infected P. stylirostris samples were amplified using virus-specific primers. (C and D) β-Actin DNA was amplified from each sample in parallel. The sizes of the amplicons were 329, 306, and 339 bp for IHHNV, WSV, and the β-actin gene, respectively. Lanes 1 to 9 contain 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg, respectively. Lane M, 50-bp DNA step ladder (Sigma); lane −ve, negative control.
FIG. 3
FIG. 3
Detection limits of IHHNV (A) and WSV (B) DNAs by SYBR Green PCR. The β-actin gene was amplified in parallel for each dilution of the IHHNV and WSV samples. There were three to four replicates for each dilution of DNA, and the CT value of each dilution was used to plot the graph.
FIG. 4
FIG. 4
Amplification profiles (A and C) and dissociation curves (B and D) of IHHNV-challenged-and healthy-P. stylirostris samples. Genomic DNAs of IHHNV-challenged- and healthy-P. stylirostris samples were amplified using IHHNV-specific (A) and β-actin-specific (C) primers by SYBR Green PCR. The dissociation curves for IHHNV and β-actin products are shown in panels B and D, respectively. The Tm values of IHHNV and β-actin amplicons are indicated alongside their corresponding dissociation curves.
FIG. 5
FIG. 5
Amplification profiles (A and C) and corresponding dissociation curves (B and D) for WSV and the β-actin gene amplified using genomic DNAs of WSV-challenged- and healthy-P. stylirostris samples. The Tm values of WSV and β-actin amplicons are indicated next to the dissociation curve of the corresponding gene product.
FIG. 6
FIG. 6
PCR amplification of IHHNV (A)- and WSV (B)-specific DNA in laboratory-challenged-P. stylirostris samples. (C and D) β-Actin amplifications for the samples used for panels A and B. The sizes of the amplified DNAs of IHHNV, WSV, and the β-actin gene were 329, 306, and 339 bp-, respectively. In each panel, lanes 1 to 6 contain DNAs from virus-challenged samples and lanes 7 and 8 contain DNAs from healthy samples. Lane M, 50-bp DNA step ladder (Sigma).

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