Development of TaqMan quantitative PCR assay for detection of Nocardia seriolae in fish and the environment

Yanxin Sun; Caiyi Wen; Zhuang Ma; Weimin Huang; Chun Liu; Wei Li; Shu Tang; Huajian Lin; Biao Jiang; Youlu Su

doi:10.1016/j.mimet.2022.106650

Development of TaqMan quantitative PCR assay for detection of Nocardia seriolae in fish and the environment

J Microbiol Methods. 2023 Feb:205:106650. doi: 10.1016/j.mimet.2022.106650. Epub 2022 Dec 5.

Authors

Yanxin Sun¹, Caiyi Wen¹, Zhuang Ma¹, Weimin Huang¹, Chun Liu¹, Wei Li¹, Shu Tang², Huajian Lin², Biao Jiang³, Youlu Su⁴

Affiliations

¹ Innovative Institute of Animal Healthy Breeding, College of Animal Sciences and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510222, Guangdong, China.
² Guangdong Animal Disease Control Center, Guangzhou 510222, Guangdong, China.
³ Innovative Institute of Animal Healthy Breeding, College of Animal Sciences and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510222, Guangdong, China. Electronic address: jiangbiao@zhku.edu.cn.
⁴ Innovative Institute of Animal Healthy Breeding, College of Animal Sciences and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510222, Guangdong, China. Electronic address: youlusu@zhku.edu.cn.

PMID: 36481430
DOI: 10.1016/j.mimet.2022.106650

Abstract

Nocardia seriolae is a gram-positive bacterium that causes nocardiosis, threatening fish farming. Advanced nocardiosis is challenging to control; thus, accurate detection methods of the causal agent in the early disease stage are required. In this study, we developed a TaqMan fluorescence quantitative PCR (qPCR) assay for quantitative detection of N. seriolae in fish tissues and water samples. A pair of highly specific primers and a TaqMan probe were designed based on the N. seriolae 16S23S rRNA internal transcribed spacer (ITS) region. A high correlation coefficient (R² = 0.998) of the standard curve with a 99.5% efficiency was obtained. The qPCR detection limit of the method was as low as 19.8 copies/μL, 1000 times more sensitive than conventional PCR, and has a good performance in the detection of cultured bacteria (y = -3.750× + 48.075, R² = 0.974). Even 1.42 CFU/mL N. seriolae collected from 500 mL of natural pond water can be detected. Furthermore, a linear model for the relationship between the log of bacteria load and Cq values in water was established (y = -3.239× + 40.978), and the R² value was 0.979. This assay was used for accurate N. seriolae detection in fish tissues, water samples, feeds and soils. This study provides a valuable tool for the early detection and control of nocardiosis in aquaculture.

Keywords: Bacterial load; Early monitoring; Environment pathogen; Nocardia seriolae; TaqMan real-time PCR.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Fish Diseases* / diagnosis
Fish Diseases* / microbiology
Fishes / microbiology
Nocardia Infections* / diagnosis
Nocardia Infections* / microbiology
Nocardia Infections* / veterinary
Nocardia* / genetics
Polymerase Chain Reaction

Supplementary concepts

Nocardia seriolae