Development of SYBR Green based real time PCR assay for detection of monodon baculovirus in Penaeus monodon.
Shrimp farming is one of the most important aquaculture activities. Expansion and intensification of shrimp farming has been accompanied with the outbreak of diseases, which threaten the development and sustainability of the industry. Viral diseases are the major challenges faced by shrimp farming industries. The prevention/control of such diseases have become critical in determining the viability of the shrimp farming. The disease caused by monodon baculovirus (MBV) is the major limiting factor especially in shrimp hatchery. There are no therapeutic measures to control the viral diseases. So the detection of the disease is crucial in the prevention and spread of the disease. Hence, in this study, SYBR Green based real time polymerase chain reaction (PCR) assay was developed for the detection of MBV. The sensitivity of the real time PCR was determined using 10-fold dilutions of purified plasmid DNA with the concentration in the range of 10(1)-10(5) copies per reaction. The assay could detect as low as 12 copies indicating that the assay was sensitive and could be effectively used for the quantification of MBV. The real time PCR assay was found to be specific and did not show any amplification with P. monodon infected with infectious hypodermal and hematopoietic necrosis virus (IHHNV), white spot syndrome virus (WSSV) and hepatopancreatic parvo-like virus (HPV). The novelty of this assay is that it could be employed for diagnosis of low level MBV infection in broodstock using fecal matter of shrimp, a non-invasive diagnostic tool.
Central Institute of Brackishwater Aquaculture, Indian Council of Agricultural Research, 75, Santhome High Road, Raja Annamalai Puram, Chennai 600028, India.
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Ramesh Kumar
Central Institute of Brackishwater Aquaculture, Indian Council of Agricultural Research, 75, Santhome High Road, Raja Annamalai Puram, Chennai 600028, India.
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Sanjuktha
Central Institute of Brackishwater Aquaculture, Indian Council of Agricultural Research, 75, Santhome High Road, Raja Annamalai Puram, Chennai 600028, India.
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Rajan
Central Institute of Brackishwater Aquaculture, Indian Council of Agricultural Research, 75, Santhome High Road, Raja Annamalai Puram, Chennai 600028, India.
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Ananda Bharathi
Central Institute of Brackishwater Aquaculture, Indian Council of Agricultural Research, 75, Santhome High Road, Raja Annamalai Puram, Chennai 600028, India.
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Santiago
Central Institute of Brackishwater Aquaculture, Indian Council of Agricultural Research, 75, Santhome High Road, Raja Annamalai Puram, Chennai 600028, India.
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Alavandi
Central Institute of Brackishwater Aquaculture, Indian Council of Agricultural Research, 75, Santhome High Road, Raja Annamalai Puram, Chennai 600028, India. Electronic address: poornima_vet@yahoo.com.
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Poornima
D000818:Animals; D017756:Aquaculture; D004279:DNA, Viral / Q000032:analysis; D005243:Feces / Q000821:virology; D005456:Fluorescent Dyes; D043143:Hepatopancreas / Q000821:virology; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000302:isolation & purification*; D033561:Penaeidae / Q000821:virology*; D010957:Plasmids / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D019562:Viral Load
D004279:DNA, Viral; D005456:Fluorescent Dyes
Monodon baculovirus; Penaeus monodon; SYBR Green real time PCR; Specific pathogen free (SPF)