Fast short-fragment PCR for rapid and sensitive detection of shrimp viruses.
This article describes a fast short-fragment PCR method for the detection of white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and monodon baculovirus (MBV). Fast two-temperature (95 degrees C denaturation and 60 degrees C annealing/extension) PCRs were performed in 5-10 microl volume samples in miniaturized microplates using a fast Peltier thermal cycler. 40 cycles were completed in 25-30 min. Rapid high-resolution agarose gel electrophoresis of 70-150 bp PCR fragments was performed in 10 min. High sensitivity of PCR product detection (50-100 pg) was obtained using ultra sensitive dyes such as GelStar and a gel documentation system equipped with a blue-light transilluminator. This novel method is faster and more sensitive than its TaqMan real-time PCR counterparts.
Mrotzek
Grit
G
Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute, Jena, Germany.
Haryanti
Koesharyani
Isti
I
Tretyakov
Alexander N
AN
Sugama
Ketut
K
Saluz
Hans P
HP
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000302:isolation & purification*; D019223:Densovirinae / Q000235:genetics / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D033561:Penaeidae / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D013696:Temperature; D013997:Time Factors; D014773:Virology / Q000379:methods*; D046848:White spot syndrome virus 1 / Q000235:genetics / Q000302:isolation & purification*