Development of Two Quantitative Real-Time PCR Diagnostic Kits for HPV Isolates from Korea

Abstract

Viral pathogens, alongside other pathogens, have major effects on crustacean aquaculture. Hepatopancreatic parvovirus (HPV) is an emerging virus in the shrimp industry and has been detected in shrimp farms worldwide. The HPV genome has greater diversity than other shrimp viruses owing to its wide host range and geographical distribution. Therefore, developing diagnostic tools is essential to detect even small copy numbers from the target region of native HPV isolates. We have developed two easy to use quantitative real-time PCR kits, called Green Star and Dual Star, which contain all of the necessary components for real-time PCR, including HPV primers, using the primers obtained from the sequences of HPV isolates from Korea, and analyzed their specificity, efficiency, and reproducibility. These two kits could detect from 1 to 1 × 10 9 copies of cloned HPV DNA. The minimum detection limits obtained from HPV-infected shrimp were 7.74 × 10 1 and 9.06 × 10 1 copies in the Green Star and Dual Star assay kits, respectively. These kits can be used for rapid, sensitive, and efficient screening for HPV isolates from Korea before the introduction of postlarval stages into culture ponds, thereby decreasing the incidence of early development of the disease.

Artificial infection of the native Korean freshwater prawn Macrobrachium nipponense (De Haan, 1849) (Decapoda, Palaemonidae) with white spot syndrome virus (WSSV)

“…A controlled bioassay system for WSSV infection by intramuscular injection was tested, but the experiment was carried out using serial dilutions of filtrate from infected shrimp (Prior et al, 2003). In our injection trials, we determined the viral titres using real-time PCR, as described by Jeeva et al (2012). Although differences can exist between the viral copy number and infectivity of the inoculum, we showed a clear relationship between viral titer and the progress of the viral infection.…”

“…The genetic background of M. nipponense was confirmed by sequencing the 18s rDNA using primers 143F (5'-TGCCTTATCAGC TNTC GATTGTAG-3') and 145R (5'-TCAGNTTTGCAACCATACTTCCC-3') (Ayub et al, 2008). The presence of WSSV in the captured prawn was tested by PCR using the two primers described above (Jeeva et al, 2012).…”

“…A plasmid containing a partial region of 604 bp of the vp28 gene from WSSV was used as the positive control (Jeeva et al, 2012). The DNA concen tration was then assessed using a spectrophotometer (VICTOR3, PerkinElmer, Waltham, MA, U.S.A.).…”

“…The DNA concen tration was then assessed using a spectrophotometer (VICTOR3, PerkinElmer, Waltham, MA, U.S.A.). The primers WS-155F (5'-AGGGAACATTCAAGGTGT GG-30 and WS-155R (5'-GGTGCCAACTTCATCCTCAT-3') (Jeeva et al, 2012) were used in real-time PCR using the AccuPower® GreenStar qPCR PreMix (Bioneer, Daejeon, South Korea), producing a 155-bp product. In each reaction of 20 Ail, a dilution series of the WSSV plasmid standard (1 x 103 -1 x 109) was used to produce a standard curve, and 80 ng of viral DNA extracted from purified virus was used to determine the copy number of the viral inoculum.…”

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

“…Real-time PCRs have 241 been previously developed for HPV and tested using TaqMan chemistry on isolates from 242 Australia [14], Thailand [34, 39], China, Taiwan, Madagascar, New Caledonia, Tanzania [34, 243 36], Malaysia, Indonesia [39] and Korea using TaqMan as well as SYBR Green chemistry[35]. 244 However, this is the first report of a detection and quantification assay for HPV infecting P. 245Through various research [23, 29, 31, 32] using varied primers, it is proven that PCR 247 technique detects not less than 1fg purified HPV DNA (approximately 340 viral particles).Our 248 analysis with a range of serial dilutions has yielded result that is similar to the previously 249 published data.…”