Detection of shrimp infectious myonecrosis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick.
Infectious myonecrosis virus (IMNV) has caused a slowly progressive disease with cumulative mortalities of up to 70% or more in cultured Penaeus (Litopenaeus) vannamei in Northeast Brazil and Indonesia. Rapid detection of viruses by loop-mediated isothermal amplification (LAMP) of genomic material with high specificity and sensitivity can be applied for diagnosis, monitoring and control of diseases in shrimp aquaculture. Using an IMNV template, successful detection was achieved after a 60-min RT-LAMP reaction using biotin-labeled primers followed by 5min hybridization with an FITC-labeled DNA probe and 5min assay using a chromatographic lateral flow dipstick (LFD). Thus, the combined system of RT-LAMP and LFD required a total assay interval of less than 75min, excluding the RNA extraction time. The sensitivity of detection was comparable to that of other commonly used methods for nested RT-PCR detection of IMNV. In addition to reducing amplicon detection time when compared to electrophoresis, LFD confirmed amplicon identity by hybridization and eliminated the need to handle carcinogenic ethidium bromide. The RT-LAMP-LFD method gave negative test results with nucleic acid extracts from normal shrimp and from shrimp infected with other viruses including infectious hypodermal hematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), a hepatopancreatic parvovirus from P. monodon (PmDNV), white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Macrobrachium rosenbergii nodavirus (MrNV) and gill associated virus (GAV).
Puthawibool
Teeranart
T
CENTEX Shrimp, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand.
Senapin
Saengchan
S
Kiatpathomchai
Wansika
W
Flegel
Timothy W
TW
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D005814:Genes, Viral; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D033561:Penaeidae / Q000821:virology*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis; D012680:Sensitivity and Specificity; D013045:Species Specificity
D017931:DNA Primers; D012367:RNA, Viral