Sensitive and rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimps by loop-mediated isothermal amplification.
A method for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) is a novel, sensitive and rapid technique, which can be applied for disease diagnosis in aquaculture. Using the LAMP method, a highly specific and sensitive diagnostic system for infectious hypodermal and hematopoietic necrosis virus (IHHNV) detection was designed. A set of four primers was designed by targeting the IHHNV genome DNA. By the detection system, target DNA was amplified and visualized on agarose gel within 60min under isothermal condition at 64 degrees C. Without gel electrophoresis, the LAMP amplicon was visualized directly in the reaction tube by addition of SYBR Green I for a naked-eye inspection. The LAMP reaction was also assessed by the white turbidity of magnesium pyrophosphate (a by-product of LAMP) in the tube. The assay had a detection limit of 5-500 copies of DNA template with gel electrophoresis, SYBR Green I and white turbidity with naked-eye inspection. The detection sensitivity of LAMP was 100-fold higher than the PCR. A diagnostic procedure which is rapid and highly sensitive was developed for IHHNV detection.
Sun
Zhao-Feng
ZF
The Key Laboratory of Applied Marine Biology of Guangdong Province and Chinese Academy of Sciences, South China Sea Institute of Oceanology, 164 West Xingang Road, Guangzhou 510301, PR China.
Hu
Chao-Qun
CQ
Ren
Chun-Hua
CH
Shen
Qi
Q
D000818:Animals; D001483:Base Sequence; D052160:Benzothiazoles; D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral / Q000235:genetics*; D019223:Densovirinae / Q000235:genetics / Q000302:isolation & purification*; D003959:Diamines; D015180:Electrophoresis, Gel, Two-Dimensional; D005814:Genes, Viral / Q000235:genetics; D017616:Magnesium Compounds; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009930:Organic Chemicals; D033561:Penaeidae / Q000821:virology*; D010710:Phosphates; D011804:Quinolines; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral; D003959:Diamines; D017616:Magnesium Compounds; D009930:Organic Chemicals; D010710:Phosphates; D011804:Quinolines; C098022:SYBR Green I; C030781:magnesium phosphate