title: Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of yellow head virus in shrimp.
abstract: A real-time reverse transcription loop-mediated isothermal amplification (real-time RT-LAMP) method was applied for detecting the replicase polyprotein-encoding gene of yellow head virus (YHV) in shrimp, Penaeus monodon. It is a novel, gene-specific assay that amplifies nucleic acid with high specificity, sensitivity and rapidity under isothermal conditions using a set of six specially designed primers that recognize eight distinct sequences of the target gene. This method works with even low copies of DNA and is based on magnesium pyrophosphate turbidity detection by an inexpensive photometer for quantitative analysis. A user-friendly protocol was developed with optimal conditions standardized at 63 degrees C for 60 min. With this protocol, the assay sensitivity was 10 times higher than the widely used YHV nested RT-PCR system. Cross-reactivity analysis using other shrimp virus DNA/cDNA and YHV-negative shrimp demonstrated high specificity of the assay. The real-time RT-LAMP method was performed also for an internal control gene, EF-1alpha, to compare with the expressions of the YHV gene in different organs of infected shrimp, and the resulting standard curves showed high correlation coefficient values. These results suggest that this assay is applicable widely as a new quantitative detection method in the pursuit of YHV-free shrimp culture.
authors: Interdisciplinary Graduate School of Agriculture and Engineering, University of Miyazaki, 1-1 Gakuen Kibanadai-nishi, 889-2192 Miyazaki, Japan.
authors: Tohru
authors: T
authors: Mekata
authors: Raja
authors: R
authors: Sudhakaran
authors: Tomoya
authors: T
authors: Kono
authors: Kittichon
authors: K
authors: U-taynapun
authors: Kidchakan
authors: K
authors: Supamattaya
authors: Yoshihiro
authors: Y
authors: Suzuki
authors: Masahiro
authors: M
authors: Sakai
authors: Toshiaki
authors: T
authors: Itami
mesh_terms: D000818:Animals; D017931:DNA Primers; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D033561:Penaeidae / Q000821:virology*; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D046828:Roniviridae / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D017931:DNA Primers; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase