Graphene oxide based fluorescence resonance energy transfer and loop-mediated isothermal amplification for white spot syndrome virus detection.
Graphene oxide (GO) is attractived for biological or medical applications due to its unique electrical, physical, optical and biological properties. In particular, GO can adsorb DNA via π-π stacking or non-covalent interactions, leading to fluorescence quenching phenomenon applicable for bio-molecular detection. In this work, a new method for white spot syndrome virus (WSSV)-DNA detection is developed based on loop-mediated isothermal amplification (LAMP) combined with fluorescence resonance energy transfer (FRET) between GO and fluorescein isothiocyanate-labeled probe (FITC-probe). The fluorescence quenching efficiency of FITC-probe was found to increase with increasing GO concentration and reached 98.7% at a GO concentration of 50 μg/ml. The fluorescence intensity of FITC-probe was recovered after hybridization with WSSV LAMP product with an optimal hybridization time of 10 min and increased accordingly with increasing amount of LAMP products. The detection limit was estimated to be as low as 10 copies of WSSV plasmid DNA or 0.6 fg of the total DNA extracted from shrimp infected with WSSV. In addition, no cross reaction was observed with other common shrimp viral pathogens. Therefore, the GO-FRET-LAMP technique is promising for fast, sensitive and specific detection of DNAs.
Thailand Organic and Printed Electronics Innovation Center, National Electronics and Computer Technology Center (NECTEC), National Science and Technology Development Agency (NSTDA), 112 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Neung, Amphoe Khlong Luang, Pathum Thani 12120, Thailand.
U
U
Waiwijit
Nanoelectronics and MEMS Laboratory, National Electronics and Computer Technology Center (NECTEC), National Science and Technology Development Agency (NSTDA), 112 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Neung, Amphoe Khlong Luang, Pathum Thani 12120, Thailand.
D
D
Phokaratkul
Bioengineering and Sensing Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Neung, Amphoe Khlong Luang, Pathum Thani 12120, Thailand.
J
J
Kampeera
Thailand Organic and Printed Electronics Innovation Center, National Electronics and Computer Technology Center (NECTEC), National Science and Technology Development Agency (NSTDA), 112 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Neung, Amphoe Khlong Luang, Pathum Thani 12120, Thailand.
T
T
Lomas
Nanoelectronics and MEMS Laboratory, National Electronics and Computer Technology Center (NECTEC), National Science and Technology Development Agency (NSTDA), 112 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Neung, Amphoe Khlong Luang, Pathum Thani 12120, Thailand.
A
A
Wisitsoraat
Bioengineering and Sensing Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Neung, Amphoe Khlong Luang, Pathum Thani 12120, Thailand. Electronic address: wansika@biotec.or.th.
W
W
Kiatpathomchai
Thailand Organic and Printed Electronics Innovation Center, National Electronics and Computer Technology Center (NECTEC), National Science and Technology Development Agency (NSTDA), 112 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Neung, Amphoe Khlong Luang, Pathum Thani 12120, Thailand. Electronic address: adisorn.tuantranont@nectec.or.th.
A
A
Tuantranont
D000818:Animals; D003445:Crustacea / Q000821:virology; D004266:DNA Virus Infections / Q000821:virology; D004279:DNA, Viral / Q000032:analysis*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006108:Graphite / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010087:Oxides / Q000737:chemistry; D010957:Plasmids; D046848:White spot syndrome virus 1 / Q000235:genetics*
D004279:DNA, Viral; D005456:Fluorescent Dyes; D010087:Oxides; D006108:Graphite; D016650:Fluorescein-5-isothiocyanate
Fluorescence quenching; Fluorescence resonance energy transfer (FRET); Graphene oxide (GO); Loop-mediated isothermal amplification (LAMP); White spot syndrome virus (WSSV)