Development of SYBR Green and TaqMan quantitative real-time PCR assays for hepatopancreatic parvovirus (HPV) infecting Penaeus monodon in India. Issue 6 (December 2015)

• Record Type: Journal Article
• Authors: Yadav, Reena; Paria, Anutosh; Mankame, Smruti; Makesh, M.; Chaudhari, Aparna; Rajendran, K.V.
• Is Part Of: Molecular and cellular probes. Volume 29:Issue 6(2015)
• Page Start: 442
• Page End: 448
• Publication Date: 2015-12
• Subjects: Real-time PCR -- Hepatopancreatic parvovirus (HPV) -- Penaeus monodon -- Shrimp virus; Molecular probes -- Diagnostic use -- Periodicals; Pathology, Cellular -- Technique -- Periodicals; Cell Biology -- Periodicals; Molecular Biology -- Periodicals; Sondes moléculaires -- Utilisation diagnostique -- Périodiques; Cytopathologie -- Technique -- Périodiques; 572
• DOI: 10.1016/j.mcp.2015.07.006
• Languages: English
• ISSNs: 0890-8508

Abstract

Abstract: Hepatopancreatic parvovirus (HPV) infects Penaeus monodon and causes mortality in the larval stages. Further, it has been implicated in the growth retardation in cultured P. monodon . Though different geographical isolates of HPV show large sequence variations, a sensitive PCR assay specific to Indian isolate has not yet been reported. Here, we developed a sensitive SYBR Green-based and TaqMan real-time PCR for the detection and quantification of the virus. A 441-bp PCR amplicon was cloned in pTZ57 R/T vector and the plasmid copy number was estimated. A 10-fold serial dilution of the plasmid DNA from 1 × 10 9 copies to 1 copy was prepared and used as the standard. The primers were tested initially using the standard on a conventional PCR format to determine the linearity of detection. The standards were further tested on real-time PCR format using SYBR Green and TaqMan chemistry and standard curves were generated based on the Ct values from three well replicates for each dilution. The assays were found to be sensitive, specific and reproducible with a wide dynamic range (1 × 10 9 to 10 copies) with coefficient of regression ( R 2 ) > 0.99, calculated average slope −3.196 for SYBR Green assay whereas, for TaqMan assay it was >0.99 and −3.367, respectively. The intra- and inter-assay variance of the Ct values ranged from 0.26% to 0.94% and 0.12% to 0.81%, respectively, for SYBR Green assay, and the inter-assay variance of the Ct values for TaqMan assay ranged from 0.07% to 1.93%. The specificity of the assays was proved by testing other DNA viruses of shrimp such as WSSV, IHHNV and MBV. Standardized assays were further tested to detect and quantify HPV in the post-larvae of P. monodon . The result was further compared with conventional PCR to test the reproducibility of the test. The assay was also used to screen Litopeneaus vannamei, Macrobrachium rosenbergii and Scylla serrata for HPV. Highlights: Two sensitive real-time PCR assays for detection and quantification of Indian isolate of HPV were developed. The assays were compared for their efficiency in detecting and quantifying HPV. Detection limits of these assays were found to be as low as 10 viral copies. The assays can be used in the screening of post-larvae and in the development of HPV-free stock of Penaeus monodon .