三重PCR检测鱼类致病性嗜水气单胞菌

Detecting pathogenic Aeromonas hydrophila in fish by triplex PCR

摘要:

【目的】建立一种能够快速准确地检测致病性嗜水气单胞菌的PCR方法。【方法】根据嗜水气单胞菌的16S rRNA、气溶素基因（aer）和丝氨酸蛋白酶基因（ahp）的保守序列设计了3对引物，然后进行了PCR反应条件的优化、特异性和敏感性的检测并与普通的细菌分离鉴定进行了临床样本和人工攻毒样本检出率的比较。【结果】该方法特异性好，只对致病性嗜水气单胞菌呈阳性扩增；敏感性高，最低可检测100fg的细菌DNA模版。对临床疑似黄鳝（Monopterus albus）样本的检出率为81.8%，高于细菌分离的40.9%；对人工攻毒鲫鱼(Carassius auratus)样本的检出率为87.5%，高于细菌分离的67.5%。【结论】本方法的成功建立，实现在同一反应管中同时对16SrRNA、aer和 ahp的检测，避免了只针对aer或 ahp单个毒力基因的PCR检测方法可能存在的漏检和误检，为致病性嗜水气单胞菌的诊断、大规模检疫、流行病学调查等提供了一种快速、准确而有效的检测方法。

Abstract:

[Objective] To develop a rapid PCR method to detect pathogenic Aeromonas hydrophila in fish. [Methods] For multiple PCR, three pairs of primers were designed based on the conservative sequences of 16SrRNA genes, aerolysin (aer) gens and serine-protease (ahp) genes of Aeromonas hydrophila. By optimization of PCR conditions and estimation of specificity, sensitivity, detection rate, a triplex PCR assay was established. [Results] The assay had a high specificity detecting only pathogenic strains of Aeromonas hydrophila but not other irrelative bacteria. The assay had a high sensitivity with the detection limit as low as 100fg, the detection rate of suspicious clinical samples by this assay was 81.8%, which was noticeably higher than that by bacterial isolation method (40.9%). The detection rate of mimic challenge samples by this assay was 87.5%, which was also noticeably higher than that by bacterial isolation method (67.5%). [Conclusion] The simultaneous detection of 16SrRNA gene and two virulent genes in one PCR assay could avoid missed detection possibly caused by PCR with single virulent gene, and provided a useful tool for rapid diagnosis, large-scale quarantine, and epidemiological investigation of the pathogenic Aeromonas hydrophila.