Improvement in the specificity and sensitivity of detection for the Taura syndrome virus and yellow head virus of penaeid shrimp by increasing the amplicon size in SYBR Green real-time RT-PCR.
Real-time RT-PCR using SYBR Green chemistry uses a green fluorescence dye, SYBR Green I, that binds to double stranded DNA (dsDNA) and exhibits enhancement of fluorescence upon binding to the DNA. The indiscriminate binding ability of SYBR Green I dye to dsDNA often results in non-specific products. We have shown that increasing the amplicon size from approximately 50 to approximately 75-100 bp increases the specificity due to higher melting temperature of the amplicon and also enhances the sensitivity of detection of real-time RT-PCR using SYBR Green chemistry while detecting two RNA viruses in laboratory-challenged shrimp, the Taura syndrome virus (TSV), and yellow head virus (YHV). The increased sensitivity of the larger amplicon over the smaller amplicon varied from 1.6 to 6.82-fold (with a median value of 4-fold) for the TSV-infected samples, and 1.80-10.27-fold (with a median value of 4-fold) for the YHV-infected samples. The longer amplicon also has a higher Tm value compared with the shorter amplicon (75.6 vs. 72.0 degrees C for TSV, and 81.3 vs. 72.5 degrees C for YHV). The increased melting temperature of the longer amplicon compared with the shorter amplicon will enable easier discrimination of a specific product from a primer dimer or other non-specific products. The improved method for the detection of TSV and YHV will be applicable not only to the detection of other viral pathogens but also to the quantitative measurement of cellular gene expression by real-time SYBR Green RT-PCR.
Mouillesseaux
Kevin P
KP
Scripps Research Institute, 10466 North Torrey Pines Road, La Jolla, CA 92037, USA.
Klimpel
Kurt R
KR
Dhar
Arun K
AK
D000818:Animals; D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D028381:Nidovirales / Q000235:genetics / Q000302:isolation & purification*; D009930:Organic Chemicals; D033561:Penaeidae / Q000821:virology*; D011804:Quinolines; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I