[1]朱成科*,刘桂嘉*,张争世,等.黄颡鱼源维氏气单胞菌Aha和gyrB基因双重PCR检测方法的建立[J].中国预防兽医学报,2017,(10):810-814.[doi:10.3969/j.issn.1008-0425.201704002]
 ZHU Cheng-ke*,LIU Gui-jia*,ZHANG Zheng-shi,et al.Establishment of a duplex PCR assay based on Aha and gyrB genes for detection of Aeromonasveronii[J].Chinese journal of preventive veterinary medicine,2017,(10):810-814.[doi:10.3969/j.issn.1008-0425.201704002]
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黄颡鱼源维氏气单胞菌Aha和gyrB基因双重PCR检测方法的建立()
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2017年10
页码:
810-814
栏目:
诊断技术
出版日期:
2017-10-15

文章信息/Info

Title:
Establishment of a duplex PCR assay based on Aha and gyrB genes for detection of Aeromonasveronii
文章编号:
1008-0589(2017)10-0810-05
作者:
 

朱成科1*刘桂嘉1*张争世1李明朔2雷 骆1王 建1邓星星1周朝伟1郑宗林1*

 (1. 西南大学荣昌校区 水产系淡水鱼类资源与生殖发育教育部重点实验室/水产科学重庆市市级重点实验室,重庆 402460;
2. 西南大学荣昌校区 动物医学系,重庆 402460)
Author(s):
 

ZHU Cheng-ke1* LIU Gui-jia1* ZHANG Zheng-shi1 LI Ming-shuo2 LEI Luo1 WANG Jian1 DENG Xing-xing1 ZHOU Chao-wei1 ZHENG Zong-lin1*

 (1. Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Key Laboratory of Aquatics Science of Chongqing, Department of Fisheries in Rongchang Campus, Southwest University, Chongqing 402460, China;
2. Department of Veterinary Medicine in Rongchang Campus, Southwest University, Chongqing 402460, China)
关键词:
维氏气单胞菌Aha基因gyrB基因双重PCR检测
Keywords:
Aeromonas veronii  Aha  gyrB  duplex PCR  detection
分类号:
S852.61
DOI:
10.3969/j.issn.1008-0425.201704002
文献标志码:
A
摘要:
为建立一种快速、准确检测黄颡鱼源维氏气单胞菌(A.veronii)的方法,本研究根据A.veronii菌株RC110724黏附素基因(Aha)和促旋酶B亚单位基因(gyrB)序列设计引物,经条件优化建立了A.veronii的双重PCR检测方法。结果显示该方法可以同时扩增出A.veronii 419 bp和745 bp两条特异性的片段,而对嗜水气单胞菌、迟缓爱德华菌、副溶血性弧菌、麦氏弧菌、荧光假单胞菌、金黄色葡萄球菌、柱状黄杆菌、肺炎克雷伯氏菌、无乳链球菌、舒伯特气单胞菌扩增结果为阴性;该方法对A.veronii标准株ATCC35624和RC110724基因组DNA和菌体检测下限分别为6.6×10-3 ng/μL和3.2×102 cfu/mL。利用建立的双重PCR方法对30份临床样品进行检测,结果与细菌传统分离鉴定符合率为100 %。同时,双重PCR可以检出菌株RC110724人工感染的黄颡鱼肝、脾和肾组织中的细菌DNA,对肝和脾脏组织的样品检测效果最好。本研究建立的双重PCR检测方法特异性好,具有较高的检测灵敏性,可用于黄颡鱼等A.veronii感染病例的快速诊断和流行病学监测。
Abstract:
To develop a rapid method to detect pathogenic Aeromonas veronii in Pelteobagrus fulvidraco, a duplex PCR assay was established with two pairs of primers designed according to the Aha and gyrB of A.veronii strain RC110724. The results showed that two specific fragments about 419 bp and 745 bp were amplified from the genomic DNA extracted from A.veronii by the duplex PCR, which no cross-reactions with other bacteria such as Klebsiella pneumonia, Edwadsiella tarda, Vibrioparahe molyticus, Vibrio metschnikovii, Pseudomonas fluorescens, Staphylococcus aureus, Flavobacterium cloumnare, A.hydrophila, A.schuberti, Streptococcus agalactiae. The  detection limits of the method were 6.6×10-3 ng/μL for A.veronii ATCC35624 and3.2×102 cfu/mL for RC110724, respectively. Using the assay to detect 30 clinical samples, the results showed that the coincidence between bacterial isolation method and the duplex PCR was 100%. Meanwhile, the strains Rcll0724 DNA were detected in the tissues from liver, spleen, kindey and brain of P.fulvidraco affected by the A.veronii strain RC110724, especially in liver and spleen. In conclusion, the established duplex PCR in this study was specific, sensitive for the epidemiological surveillance of A.veronii infection in P.fulvidraco and other fish.

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(本文编辑:李 爽)

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更新日期/Last Update: 2017-11-06