title: Development of a rapid method for identifying carryover contamination of positive control DNA, using a chimeric positive control and restriction enzyme for the diagnosis of white spot syndrome virus by nested PCR.
abstract: Chimeric positive plasmids have been developed to minimize false-positive reactions caused by polymerase chain reaction (PCR) contamination. Here, we developed a rapid method for identifying false-positive results while detecting white spot syndrome virus (WSSV) by nested PCR, using chimeric positive plasmids. The results of PCRs using WSSV diagnostic primer sets showed PCR products of a similar size (WSSV 1st PCR product, 1,447 bp; WSSV 2nd PCR product, 941 bp) using WSSV chimeric plasmids or DNA from shrimp infected with WSSV. The PCR products were digested with DraI for 1 h at 37 °C. The digested chimeric DNA separated into two DNA bands; however, the WSSV-infected shrimp DNA did not separate. Thus, chimeric plasmid DNA may be used as positive control DNA instead of DNA from WSSV-infected shrimp, in order to prevent PCR contamination. Thus, the use of restriction enzyme digestion allowed us to rapidly distinguish between WSSV DNA and WSSV chimeric plasmid DNA.
authors.affiliation: National Fishery Products Quality Management Services, Head Office, Goyang-si, 410-315 Gyeonggi-do South Korea.
authors.forename: Hyoung Jun
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Department of Aquatic Life Medical Sciences, Sunmoon University, Asan-si, 336-708 Chungnam South Korea.
authors.forename: Se Ryun
authors.initials: SR
authors.lastname: Kwon
keywords: Chimeric positive control; Identifying carryover contamination; PCR; Restriction enzyme; White spot syndrome virus