Rapid and sensitive detection of Macrobrachium rosenbergii nodavirus in giant freshwater prawns by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick.
Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions. It can be combined with a chromatographic lateral flow dipstick (LFD) for much more efficient, field-friendly detection of MrNV. In this work, RT-LAMP was performed at 65 degrees C for 40 min, followed by 5 min for hybridization with an FITC-labeled DNA probe and 5 min for LFD resulted in visualization of DNA amplicons trapped at the LFD test line. Thus, total assay time, including 10 min for rapid RNA extraction was approximately 60 min. In addition to advantages of short assay time, confirmation of amplicon identity by hybridization and elimination of electrophoresis with carcinogenic ethidium bromide, the RT-LAMP-LFD was more sensitive than an existing RT-PCR method for detection of MrNV. The RT-LAMP-LFD method gave negative test results with nucleic acid extracts from normal shrimp and from shrimp infected with other viruses including DNA viruses [PstDNV (IHHNV), PemoNPV (MBV), PmDNV (HPV), WSSV] and RNA viruses (TSV, IMNV, YHV/GAV).
Puthawibool
Teeranart
T
Centex Shrimp, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand.
Senapin
Saengchan
S
Flegel
Timothy W
TW
Kiatpathomchai
Wansika
W
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005618:Fresh Water; D008969:Molecular Sequence Data; D030381:Nodaviridae / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D033541:Palaemonidae / Q000821:virology*; D012367:RNA, Viral / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013696:Temperature
D017931:DNA Primers; D012367:RNA, Viral