Real time PCR quantification of WSSV infection in specific pathogen free (SPF) Litopenaeus vannamei (Boone, 1931) exposed to antiviral nucleotide

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• 作者：MA Badhul Haq^a ; ^{drmabhaqcas@gmail.com} ; K Kalai Priya^a ; R Rajaram^b ; R Vignesh^a ; M Srinivasan^a
• 关键词：Aquaculture ; Pathogen ; Infection ; Shrimps ; Drugs ; PCR ; Genome
• 刊名：Asian Pacific Journal of Tropical Biomedicine
• 出版年：2012
• 出版时间：February, 2012
• 年：2012
• 卷：2
• 期：2_supp_S
• 页码：S1120-S1129
• 全文大小：1K

文摘

Objective

To investigate the level of WSSV transmission from the infected tiger prawn Penaeus monodon (P. monodon) to specific pathogen free Litopenaeus vannamei (L. vannamei) in laboratory captivity condition in relation to PCR detection, histopathological observation and viral genome sequence.

Methods

Viral DNA was isolated from purified virions by treatment with proteinase K (0.2 mg/mL) and Sarkosyl (1 % ). The purity and concentration of the DNA were determined by agarose gel electrophoresis. Moribund and dead shrimp were removed and processed for indirect immunofluorescence (IIF) analysis. Histological observation of infected L. vannamei shrimps were revealed by the degenerated cells which were characterized by intranuclear inclusions in the tissues of WSSV infected mid-gut gland, lymphoid organ, gill lamellae and gut epithelium. Total DNA was extracted, from shrimp hemolymph and tissues, with a High Puree PCR template preparation kit. WSSV-DNA was detected using a commercial 2-step PCR detection kit.

Results

The present study compares the real-time PCR results with SYBR Green I concentration ranging from 0.2 to 0.7¡Á. The positive standard was used in the range of 10², 10⁴ 10⁶, 10⁸ and 10¹⁰ copies/ng of DNA in general. The PCR analysis showed the appearance of a prominent band from the PCR amplified product of WSSV-DNA at internal control band of 848 bp. Moderate and severe levels were observed as 650 bp and 910 bp (200 & 2?000 copies) in various transmission routes. The WSSV content in moribund shrimp of all the experimental species (L. vannamei) approximately ranged in nucleotide application by quantification method from 0.000?001 WSSV copies/¦Ìg of total DNA. In whole moribund infection animal, approximately 0.02 WSSV copies/¦Ìg of DNA was detected in nucleotide applied animal.

Conclusion

These results indicate that wild brood stock and native culture shrimp P. monodon may be infected with WSSV and can get transferred into the SPF L. vannamei farming environment. Based on the studies, we made in captivity condition in different WSSV transmission route in dissimilar infection range with the use of nucleotide for antiviral drugs. There is an urgent need to address and develop antiviral drugs and molecular based viral genome technique for control measures to salt away the aquaculture environments.