Multiplex real-time PCR and high-resolution melting analysis for detection of white spot syndrome virus, yellow-head virus, and Penaeus monodon densovirus in penaeid shrimp

Development of a multiplex PCR system for the simultaneous detection of white spot syndrome virus and hepatopancreatic parvovirus infection

“…(You, Nadala, Yang & Loh ). Therefore, PCR primers derived from the vp28 (Panichareon, Khawsak, Deesukon & Sukhumsirichart ) and vp26 (Tan et al . ) gene have been used.…”

“…Multiplex PCR for simultaneous detection of HPV and WSSV was attempted as part of multiple detection of three (Panichareon et al . ) or six viruses (Khawsak et al . ).…”

“…Conservation of the nucleotide sequence of these five major WSSV structural proteins among isolates collected from different shrimp species and/or geographical areas was also confirmed by You et al (You, Nadala, Yang & Loh 2004). Therefore, PCR primers derived from the vp28 (Panichareon, Khawsak, Deesukon & Sukhumsirichart 2011) and vp26 (Tan et al 2009) gene have been used. In our study, we compared five vp28 gene sequences and selected a 604-bp DNA fragment that showed 100% nucleotide sequence identity, corresponding to nucleotides 244, 252-244,855 of the AF332093 genome sequence.…”

“…A coinfection rate of 3.8% was observed in P. monodon from commercial ponds in Thailand (Flegel, Nielsen, Thamavit, Kongtim & Pasharawipas 2004), but the PCR for each virus was performed separately. Multiplex PCR for simultaneous detection of HPV and WSSV was attempted as part of multiple detection of three (Panichareon et al 2011) or six viruses (Khawsak et al 2008). Although the multiplex PCR exhibited high sensitivity and specificity, coinfection with WSSV and HPV was not detected.…”

Nidoviruses of Fish and Crustaceans

“…The inclusion of a nPCR step can improve YHV/GAV RNA detection efficiency up to 100-fold or more compared with one-step PCR (Cowley et al, 2000b(Cowley et al, , 2004b. Various real-time RT-PCR tests that provide viral RNA detection sensitivities further improved compared with nPCR as well as improved throughput capacity and linear dynamic range over which viral RNA loads and thus infection severity can be quantified accurately, have been described for detecting YHV, GAV or YHV genotypes 1-6 that utilize either SYBR-Green chemistry (Dhar et al, 2002;Mouillesseaux et al, 2003) or a TaqMan probe to generate fluorescence (de la Vega et al, 2004;Aranguren et al, 2012;Panichareon et al, 2011;Wijegoonawardane et al, 2010). The detection limits of these real-time PCR tests can approach a single RNA copy.…”