Development of Quantitative Real-time PCR and Loop-mediated Isothermal Amplification (LAMP) Assays for Detection of <i>Microsporidium seriolae</i>

Abstract

Beko disease, caused by the Microsporidium seriolae infection, has been a problem in yellowtail aquaculture in western Japan. In recent years, severe cases of this disease have been confirmed, resulting in a significant decrease in product value due to mass mortality and residual cysts. Only polymerase chain reaction (PCR) assays have been reported so far as a detection method for the disease. In this study, quantitative real-time PCR (qPCR) and loop-mediated isothermal amplification (LAMP) methods have been optimized for M. seriolae detection in order to establish a more sensitive and rapid diagnosis. Target regions for each detection method were selected based on the nucleotide sequences obtained by the gene analysis of cysts in diseased fish. Primer sets for the qPCR and LAMP methods were designed, and the gene amplification efficiency of each method was evaluated. The results showed that the newly developed qPCR method could detect 1.4 copies of the target gene, and the LAMP method detected 100 copies within 15 minutes. In this study, the newly developed qPCR and LAMP assays were shown to be rapid and highly sensitive methods for quantitative detection of M. seriolae.

Evaluations of Lethal and Sub-lethal Toxicity of Febantel in the Juvenile Japanese Amberjack <i>Seriola quinqueradiata</i>

“…Thirty days from the beginning of the experiment (10 days after the last FBT administration in the 20-d group), all surviving fish were dissected, and visible M. seriolae cysts were counted in the fillet of left side of the body. In addition, qPCR was performed in 10 to 11 randomly selected fish from each group following the protocols described by Mekata et al (2021) and .…”

Specific and rapid detection of tilapia parvovirus using loop‐mediated isothermal amplification (LAMP) method

“…Although LAMP assays are reliable and sensitive in detecting TiPV‐infected fish, the qPCR assay had 100‐times higher sensitivity than the LAMP assay. Similarly, the sensitivity of the LAMP assay has been reported to be lower than the qPCR method for the detection of other fish pathogens, including TiLV (Phusantisampan et al, 2020), iridovirus (Yu et al, 2022), and Microsporidium seriolae (Mekata et al, 2021). Various factors, including differences in polymerase enzymes, the target positions of the primers, and the size of the amplicons, could affect amplification efficiency.…”

“…Similarly, the sensitivity of the LAMP assay has been reported to be lower than the qPCR method for the detection of other fish pathogens, including TiLV (Phusantisampan et al, 2020), iridovirus (Yu et al, 2022), and Microsporidium seriolae (Mekata et al, 2021).…”

Molecular and morphological description of a novel microsporidian Inodosporus fujiokai n. sp. infecting both salmonid fish and freshwater prawns

“…However, it has often been shown that M. seriolae and Kabatana species does not make a monophyletic clade (McGourty et al ., 2007 ; Casal et al ., 2010 ; Liu et al ., 2019 ). The available SSU rDNA sequences of M. seriolae in the INSDC database lack the first 300–400 nucleotides in the 5′ end (Bell et al ., 2001 ; Mekata et al ., 2021 ) and this may decrease phylogenetic resolution by shortening the resultant alignment dataset. In this study, we succeeded to determine the lacking region and obtained almost full length of the SSU rDNA (1328 bp) for M. seriolae .…”