Page 119 - 《渔业研究》2025年第5期

660 渔 业 研 究 第 47 卷

Establishment and preliminary application of fluorescence quantitative PCR
detection method for cone cell parasites in Larimichthys crocea

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GE Hui ，GUO Xiang ，WU Qisheng ，NING Yue ，YE Jun ，WU Liyun ，
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XIE Xinyi ，ZHANG Dongling ，HUANG Qiang ，WANG Weigang 4
(1. Key Laboratory of Cultivation and High-Value Utilization of Marine Organisms in Fujian Province,
Fisheries Research Institute of Fujian, Xiamen 361013, China;
2. Fisheries College, Jimei University, Xiamen 361021, China;
3. Xiapu Center for Marine and Fisheries Development, Ningde 355100, China;
4. Fisheries Technology Extension Center of Lianjiang County, Fuzhou 350599, China)
Abstract: [Introduction] From 2023 to 2025, Trypanosoma spp. have emerged as a significant threat to the
sustainable aquaculture of Larimichthys crocea, causing substantial economic losses. Current strategies for con-
trolling andpreventing trypanosomiasis in thisspecies remain limited. Early diagnosis is crucial for disease pre-
vention and control, but existing detection methods have limitations such as low sensitivity and cumbersome op-
eration. [Objective] This study aims to establish a rapid and accurate detection technology for Trypanosoma
spp. in L. crocea and explore its tissue distribution characteristics, providing support for early disease diagnosis
and epidemiological investigation. [Methods] Specific primers and TaqMan probes were designed based on the
conserved sequence of Trypanosoma spp. 18S rDNA in GenBank, and a fluorescence quantitative PCR (qPCR)
method was constructed; a standard curve was established through gradient dilution of recombinant plasmids to
verify the specificity, sensitivity, and repeatability of the method; samples of L. crocea from Sandu Island,
Xiapu, and Lianjiang breeding areas were collected to detect infection rates and analyze the differences in Tryp-
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anosoma spp. content in 9 tissues. [Results] The standard curve showed a good linear relationship (R =0.997),
with a detection limit of 25 copies/µL; the inter-group and intra-group cycle threshold variation coefficients
were respectively <2.0% and <1.5%, and the repeatability was stable; there was no cross-reaction with common
aquatic pathogens such as ciliates, Vibrio parahaemolyticus, and neurotropic virus, and the specificity was signi-
ficant. The overall infection rate of 60 samples was 93.33%, with the highest infection rate in Sandu Island
(100%); the tissue distribution showed that the content of Trypanosoma spp. in blood, heart, and spleen was sig-
nificantly higher than that in gills, liver, and kidneys, etc. (P<0.01). In addition, only a small number of Mugil
cephalus and Pagrus major were infected, with extremely low levels of pathogens. Among non-fish samples,
Undaria pinnatifida (Wakame) had the highest detection rate (93.33%), while a small amount of pathogens were
detected in netting and oysters, and no pathogens were detected in the remaining samples. [Conclusion] The
fluorescence quantitative PCR method established in the study has high specificity, sensitivity, and repeatability,
and can be used for early diagnosis of Trypanosoma spp. disease; the tendency of Trypanosoma spp. enrich-
ment in tissues such as blood, heart, and spleen provides a scientific basis for analyzing its pathogenic mechan-
ism and formulating targeted control strategies, and is of great significance for the healthy development of the L.
crocea aquaculture industry.
Key words: Larimichthys crocea; Trypanosoma spp.; fluorescence quantitative PCR; detection method; tissue
distribution