鲁氏耶尔森菌qPCR快速检测方法的建立和应用

鲁氏耶尔森菌(Yersinia ruckeri)是导致虹鳟(Oncorhynchus mykiss)肠炎红嘴病的病原。本研究以鲁氏耶尔森菌毒力因子rup A基因为靶标设计1对特异性引物,以含有rup A基因部分序列的重组质粒为标准品构建标准曲线,优化建立检测鲁氏耶尔森菌的SYBR Green I实时荧光定量PCR检测方法,并对腹腔注射鲁氏耶尔森菌菌悬液的虹鳟肝、肾、脾、血液样品进行检测。结果显示,设计的引物具有良好的种间特异性,标准曲线线性方程为y=–3.3766x+40.012(R~2=0.9958);最低检测限为57 copy/μL,较常规PCR的灵敏度高出约100倍。应用检测结果表明,本方法可准确检测被鲁氏耶尔森菌感染的虹鳟样品。研究表明,所建立的qPCR方法具有特异、灵敏、快速、定量的优点,可用于快速诊断虹鳟肠炎红嘴病早期病症及定量检测鲁氏耶尔森菌。

Development and application of a rapid qPCR assay for detecting Yersinia ruckeri

Enteric redmouth disease (ERM) is an emerging problem in aquaculture all over the world. ERM is caused by the gram-negative bacterial pathogen Yersinia ruckeri, which can infect salmonids and several other fish taxa and cause clinical signs of hemorrhaging on the body surface and in the intestine. Fish suffering from ERM exhibit exophthalmos, darkening skin, subcutaneous hemorrhage of the mouth and throat, perianal swelling with yellow fluid, and other deleterious outcomes up to and including death. When ERM appears in an aquaculture fa-cility, a large number of fish may be affected over a short period of time. Various antibiotics are available for the treatment of ERM and vaccines can also be used in the treatment and prevention of this disease. Several methods such as ultrastructural examination and LAMP have been developed for ERM detection. However, these assays are generally laborious and time-consuming, and are not sufficiently sensitive. To establish a rapid and quantitative method for the detection of Y. ruckeri, a pair of specific primers was designed and synthesized based on the viru-lent gene rupA. A recombinant plasmid containing part of the rupA gene was used as a standard to construct a standard curve. SYBR Green I real-time quantitative PCR assay was established for the detection of Y. ruckeri by optimizing experimental conditions. The established qPCR method was also applied to the detection of Y. ruckeri in tissues of artificially-infected rainbow trout. Our results showed that the designed primers had good interspeci-fic specificity. The quantitative linear equation was y =-3.3766x+40.012 (R2=0.9958). The detection limit of qPCR method was 57 copies/μL, which is approximately 100-fold greater than conventional PCR. This qPCR method can accurately detect Y. ruckeri in rainbow trout. These results suggest that our qPCR method has the advantages of specificity, sensitivity, rapidity, and quantification, and can be used for rapid diagnosis of early-stage disease and quantitative detection of Y. ruckeri.