Development of two quantitative real-time PCR diagnostic kits for HPV isolates from Korea.
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.
Department of Microbiology, Pukyong National University, Busan 608-737, Korea.
Subbiah
S
Jeeva
Nam-Il
NI
Kim
In-Kwon
IK
Jang
Tae-Jin
TJ
Choi
D000818:Animals; D001483:Base Sequence; D052160:Benzothiazoles; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000302:isolation & purification*; D003959:Diamines; D008969:Molecular Sequence Data; D009930:Organic Chemicals / Q000737:chemistry; D010322:Parvoviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D017992:Parvovirus / Q000235:genetics / Q000302:isolation & purification*; D033561:Penaeidae / Q000821:virology; D011804:Quinolines; D011933:Reagent Kits, Diagnostic / Q000592:standards / Q000662:veterinary*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards / Q000662:veterinary*; D015203:Reproducibility of Results; D056910:Republic of Korea; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013997:Time Factors
D052160:Benzothiazoles; D017931:DNA Primers; D004279:DNA, Viral; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D011933:Reagent Kits, Diagnostic; C098022:SYBR Green I