Development of rapid simple and sensitive real-time reverse transcriptase loop-mediated isothermal amplification method (RT-LAMP) to detect viral desease (PRDV YHV IHHNV and TSV) of penaeid shrimp

Okugawa Shogo, . and Kidchakan Supamattaya, . and Sakai Masahiro, . and Yoshida Teruty, . and Sudhakaran Raja, . and Itami Toshiaki, . and Kono Tomoya, . and Mekata Tohru, . and Inada Mari, . Development of rapid simple and sensitive real-time reverse transcriptase loop-mediated isothermal amplification method (RT-LAMP) to detect viral desease (PRDV YHV IHHNV and TSV) of penaeid shrimp. pp. 561-575. ISSN 0116-6514

A one-step single tube real-time accelerated loop-mediated isothermal amplification (real-time LAMP) assay was developed separately to detect major shrimp viral diseases such as penaeid rod shaped DNA virus (PRDV) hypodermal and haematopoietic necrosis virus (IHHNV) Taura syndrome virus (TSV) and yellow head virus (YHV). Real-time LAMP method is more sensitive than other conventional PCR RT-PCR and LAMP methods. The applicability of this assay was validated with plenty of viral samples collected from Japan and Thailand. Highly conserved regions of each viral genome developed separately were used to design the real-time LAMP primers. The real-time LAMP assay reported in this study is simple and rapid where specific amplification is obtained for PRDV IHHNV TSV and YHV in 60 min under isothermal conditions at 63C employing six distinct sequences of the target gene. The quantification of viral load in the infected samples was determined from the standard curve based on their threshold time required for turbidity to occur in the reaction by precipitation of magnesium pyrophosphate. Sensitivity analysis revealed that all of these viruses can be detected up to 100 copies of template DNA rendering it ten-fold more sensitive than conventional LAMP assay.

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Abstract

A one-step single tube real-time accelerated loop-mediated isothermal amplification (real-time LAMP) assay was developed separately to detect major shrimp viral diseases such as penaeid rod shaped DNA virus (PRDV) hypodermal and haematopoietic necrosis virus (IHHNV) Taura syndrome virus (TSV) and yellow head virus (YHV). Real-time LAMP method is more sensitive than other conventional PCR RT-PCR and LAMP methods. The applicability of this assay was validated with plenty of viral samples collected from Japan and Thailand. Highly conserved regions of each viral genome developed separately were used to design the real-time LAMP primers. The real-time LAMP assay reported in this study is simple and rapid where specific amplification is obtained for PRDV IHHNV TSV and YHV in 60 min under isothermal conditions at 63C employing six distinct sequences of the target gene. The quantification of viral load in the infected samples was determined from the standard curve based on their threshold time required for turbidity to occur in the reaction by precipitation of magnesium pyrophosphate. Sensitivity analysis revealed that all of these viruses can be detected up to 100 copies of template DNA rendering it ten-fold more sensitive than conventional LAMP assay.

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Item Type:	Article
AGROVOC Term:	Viral diseases
AGROVOC Term:	Prawns and shrimps
AGROVOC Term:	Haematopoietic necrosis virus
AGROVOC Term:	Genomes
AGROVOC Term:	Precipitation
AGROVOC Term:	Genes
AGROVOC Term:	Turbidity
AGROVOC Term:	Penaeus monodon
AGROVOC Term:	Infection
AGROVOC Term:	DNA
Depositing User:	Ms. Suzila Mohamad Kasim
Last Modified:	24 Apr 2025 06:28
URI:	http://webagris.upm.edu.my/id/eprint/23768

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