Multiplex RT-PCR assay for simultaneous detection of six viruses of penaeid shrimp

Khawsak, Paisarn; Deesukon, Warin; Chaivisuthangkura, Parin; Sukhumsirichart, Wasana

doi:10.1016/j.mcp.2007.12.005

Cited by 31 publications

(37 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Several methods for single, nested, and multiplex PCR are now available for the detection of IHHNV (Anonymous, 2006;Flegel, 2006;Khawsak, Deesukon, Chaivisuthangkura, & Sukhumsirichart, 2008;Lightner & Redman, 1998;Mrotzek et al, 2010;Nunan, Poulos, & Lightner, 2000;Tang et al, 2000;Tang & Lightner, 2006;Yang et al, 2006). These methods are applicable for surveillance and virus screening of broodstock and populations in the hatchery, documenting the specific-pathogen-free (SPF) status of animals, and monitoring the prevalence of the virus in commercial ponds and in the wild.…”

Section: Histopathology and Virus Detectionmentioning

confidence: 99%

“…PCR methods have now been developed to detect multiple viral pathogens of shrimp, including IHHNV, simultaneously (Khawsak et al, 2008;Tan et al, 2009;Xie et al, 2007;Yang et al, 2006). PCR methods have now been developed to detect multiple viral pathogens of shrimp, including IHHNV, simultaneously (Khawsak et al, 2008;Tan et al, 2009;Xie et al, 2007;Yang et al, 2006).…”

Section: Histopathology and Virus Detectionmentioning

confidence: 99%

“…Multiplex RT-PCR methods are now available to simultaneously detect up to six viruses of penaeid shrimp including both DNA (IHHNV, HPV, monodon baculovirus, MBV, and WSSV) and RNA viruses (TSV and yellow head virus (YHV)) (Khawsak et al, 2008). Multiplex RT-PCR methods are now available to simultaneously detect up to six viruses of penaeid shrimp including both DNA (IHHNV, HPV, monodon baculovirus, MBV, and WSSV) and RNA viruses (TSV and yellow head virus (YHV)) (Khawsak et al, 2008).…”

Section: Histopathology and Virus Detectionmentioning

confidence: 99%

See 2 more Smart Citations

Biology, Genome Organization, and Evolution of Parvoviruses in Marine Shrimp

Dhar

Robles‐Sikisaka

Saksmerprome

et al. 2014

Advances in Virus Research

View full text Add to dashboard Cite

Section: Histopathology and Virus Detectionmentioning

confidence: 99%

Section: Histopathology and Virus Detectionmentioning

confidence: 99%

Section: Histopathology and Virus Detectionmentioning

confidence: 99%

See 1 more Smart Citation

Biology, Genome Organization, and Evolution of Parvoviruses in Marine Shrimp

Dhar

Robles‐Sikisaka

Saksmerprome

et al. 2014

Advances in Virus Research

View full text Add to dashboard Cite

“…However, this duplex qPCR assay detected 10- and 627-fold lower amounts of PstDV1 DNA than the previously described conventional duplex and multiplex PCR methods [21,22]. In addition, the WSSV detection limit for duplex qPCR assay was 1-fold lower than that obtained using previously described multiplex PCR methods [21,22], and 10 copies lower than that obtained using a previously described multiplex qPCR method [9]. These data demonstrate the superior performance of standardized duplex qPCR method, compared with other conventional and real-time PCR-based methods.…”

Section: Discussionmentioning

confidence: 82%

Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp

et al. 2014

View full text Add to dashboard Cite

BackgroundThe White spot syndrome virus (WSSV) and Penaeus stylirostris penstyldensovirus 1 (previously named Infectious hypodermal and hematopoietic necrosis virus-IHHNV) are two of the most important viral pathogens of penaeid shrimp. Different methods have been applied for diagnosis of these viruses, including Real-time PCR (qPCR) assays. A duplex qPCR method allows the simultaneous detection of two viruses in the same sample, which is more cost-effective than assaying for each virus separately. Currently, an assay for the simultaneous detection of the WSSV and the PstDV1 in shrimp is unavailable. The aim of this study was to develop and standardize a duplex qPCR assay for the simultaneous detection of the WSSV and the PstDV1 in clinical samples of diseased L. vannamei. In addition, to evaluate the performance of two qPCR master mixes with regard to the clinical sensitivity of the qPCR assay, as well as, different methods for qPCR results evaluation.ResultsThe duplex qPCR assay for detecting WSSV and PstDV1 in clinical samples was successfully standardized. No difference in the amplification of the standard curves was observed between the duplex and singleplex assays. Specificities and sensitivities similar to those of the singleplex assays were obtained using the optimized duplex qPCR. The analytical sensitivities of duplex qPCR were two copies of WSSV control plasmid and 20 copies of PstDV1 control plasmid. The standardized duplex qPCR confirmed the presence of viral DNA in 28 from 43 samples tested. There was no difference for WSSV detection using the two kits and the distinct methods for qPCR results evaluation. High clinical sensitivity for PstDV1 was obtained with TaqMan Universal Master Mix associated with relative threshold evaluation. Three cases of simultaneous infection by the WSSV and the PstDV1 were identified with duplex qPCR.ConclusionThe standardized duplex qPCR was shown to be a robust, highly sensitive, and feasible diagnostic tool for the simultaneous detection of the WSSV and the PstDV1 in whiteleg shrimp. The use of the TaqMan Universal Master Mix and the relative threshold method of data analysis in our duplex qPCR method provided optimal levels of sensitivity and specificity.

show abstract

“…A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was also developed for detection of six different viruses in penaeid shrimp including IHHNV [36]. Yang et al [95] developed a single-step multiplex PCR for simultaneous detection of WSSV and IHHNV in penaeid shrimp.…”

Section: Normal Cellmentioning

confidence: 99%

Genomics, Molecular Epidemiology and Diagnostics of Infectious hypodermal and hematopoietic necrosis virus

et al. 2012

View full text Add to dashboard Cite

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the major viral pathogens of penaeid shrimps worldwide, which has resulted in severe mortalities of up to 90 % in cultured Penaeus (Litopenaeus) stylirostris from Hawaii and hence designated Penaeus stylirostris densovirus (PstDNV). IHHNV is distributed in shrimp culture facilities worldwide. It causes large economic loss to the shrimp farming industry. Our knowledge about the natural reservoirs of IHHNV is still scarce. Recent studies suggest that there is sufficient sequence variation among the isolates from different locations in Asia, suggesting multiple geographical strains of the virus. Four complete genomes and several partial sequences of the virus are available in the GenBank. Complete genome information would be useful for assessing the specificity of diagnostics for viruses from different geographical areas. Comparisons of complete genome sequences will help us gain insights into point mutations that can affect virulence of the virus. In addition, because of unavailability of shrimp cell lines for culturing IHHNV in vitro, quantification of virus is difficult. The recent progress in research regarding clinical signs, geographical distribution, complete genome sequence and genetic variation, transmission has made it possible to obtain information on IHHNV. A comprehensive understanding of IHHNV infection process, pathogenesis, structural proteins and replication is essential for developing prevention measures. To date, no effective prophylactic measure for IHHNV infection is available for shrimp to reduce its impact. This review provides an overview of key issues regarding IHHNV infection and disease in commercially important shrimp species.

show abstract

Multiplex RT-PCR assay for simultaneous detection of six viruses of penaeid shrimp

Cited by 31 publications

References 32 publications

Biology, Genome Organization, and Evolution of Parvoviruses in Marine Shrimp

Biology, Genome Organization, and Evolution of Parvoviruses in Marine Shrimp

Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp

Genomics, Molecular Epidemiology and Diagnostics of Infectious hypodermal and hematopoietic necrosis virus

Contact Info

Product

Resources

About