Establishment of loop-mediated isothermal amplification method (LAMP) for the detection of Vibrio nigripulchritudo in shrimp.
LAMP is a novel method that amplifies DNA with high specificity and rapidity under isothermal conditions. In this study, using the LAMP method, a diagnostic protocol was developed for the detection of Vibrio nigripulchritudo in shrimps. Vibrio nigripulchritudo is associated with distinct shrimp diseases (vibriosis) and is considered one of the threatening pathogens in shrimp industry. After initial cloning and sequencing of the intergenic spacer region (ITS) between 16S and 23S rRNA genes of V. nigripulchritudo, a set of four primers - two inner and two outer - were designed for use in the LAMP reaction. Reaction time and temperature were optimized for 60 min at 63 degrees C, respectively. The detection limit of V. nigripulchritudo by LAMP was 10(2) CFU mL(-1) but PCR could detect up to 10(3) CFU mL(-1). The LAMP method could detect the presence of V. nigripulchritudo from heart, lymphoid organ, and muscle of experimentally infected shrimps with V. nigripulchritudo. This study established a highly sensitive and a rapid diagnostic procedure for detection of V. nigripulchritudo in shrimps. The method developed in this study could be very useful for routine shrimp disease diagnostics.
Fall
Jean
J
Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan.
Chakraborty
Gunimala
G
Kono
Tomoya
T
Maeda
Minoru
M
Itami
Toshiaki
T
Sakai
Masahiro
M
D000818:Animals; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D021903:DNA, Ribosomal Spacer / Q000032:analysis; D020459:Genes, rRNA; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D033561:Penaeidae / Q000382:microbiology*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014733:Vibrio / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S