Comparison of white spot syndrome virus PCR-detection methods that use electrophoresis or antibody-mediated lateral flow chromatographic strips to visualize PCR amplicons.
White spot syndrome virus (WSSV) PCR-detection methods that used electrophoresis or lateral flow chromatographic strips (LFCS) were to compare and visualize PCR amplicons. Real-time PCR was used to prepare a stock template solution containing 2.85 x 10 (6) copies WSSV/microl from WSSV-infected shrimp. Serial stock dilutions were used as templates for PCR amplification of a WSSV-specific DNA fragment that was detected either in ethidium bromide stained agarose electrophoresis gels or on a chromatographic strip where it interacted with antibody to markers labeled on hybridization complex. PCR amplification employed both 1-step PCR and semi-nested PCR methods. By using 1-step PCR, the LFCS method (100 copies) gave 10 times higher sensitivity than gel electrophoresis (10(3) copies). A combination of a semi-nested PCR with LFCS gave a comparable sensitivity to those with commercial kits for nested PCR (20 copies). In addition, LFCS confirmed amplicon identity, avoided handling of carcinogenic ethidium bromide and could be completed in approximately 20-30 min post-PCR compared with 1h for gel electrophoresis. The costs for the two methods were comparable. In conclusion, semi-nested PCR followed by LFCS is a safe and rapid alternative method for detection of WSSV that provides sensitivity similar to that obtained by standard nested PCR methods.
Srisala
Jiraporn
J
National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Klong Luang, Patumthani 12120, Thailand.
Tacon
Philip
P
Flegel
Timothy W
TW
Sritunyalucksana
Kallaya
K