达卡气单胞菌PCR快速检测方法的建立
Establishment of a Rapid PCR Assay for Aeromonas dhakensis
钟可儿 1王静宇 1胡宝庆 2黄江峰 1周智勇 1徐先栋3
作者信息
- 1. 江西省水产科学研究所,江西南昌 330039
- 2. 南昌大学生命科学学院,江西南昌 330031
- 3. 江西省水产科学研究所,江西南昌 330039;南昌大学生命科学学院,江西南昌 330031
- 折叠
摘要
达卡气单胞菌(Aeromonas dhakensis)是一种危害严重的人兽共患病病原菌,可引起感染动物败血症和各脏器炎症.该病原的准确鉴定是防控相关疾病的基础.为建立一种针对达卡气单胞菌的特异性强、灵敏度高的快速PCR检测方法,以遗传标记DK1作为达卡气单胞菌的靶标,设计对应引物,优化PCR反应的退火温度,并评估方法的特异性及灵敏度.结果显示:该方法设计的引物对Ad-f/Ad-r在 65℃退火温度下可扩增出达卡气单胞菌的阳性条带(874 bp);将达卡气单胞菌菌液作 10 倍梯度稀释,检测最低限能够达到 1.38 CFU/μL,核酸检测最低限为 9.37×10-4 ng/μL;以嗜水气单胞菌(A.hydrophila)、维氏气单胞菌(A.veronii)等 20 种其他水产病原菌为模板进行PCR检测,均无特异性条带.综上所述,本研究建立的达卡气单胞菌PCR检测方法特异、灵敏,能够用于快速准确检测患病鱼体和环境样品中的达卡气单胞菌,为临床鉴定达卡气单胞菌提供了技术支撑.
Abstract
Aeromonas dhakensis(A.dhakensis)could cause septicaemia and organ inflammation in infected animals as a serious zoonotic pathogen.It is a precondition to prevent relevant diseases through accurate identification of the pathogen.In order to establish a rapid PCR method with high sensitivity and strong specificity for A.dhakensis,targeting at the genetic marker DK1 gene,corresponding primers were designed,and annealing temperature was optimized,followed by evaluation on the specificity and sensitivity of the method.The results showed that positive band(874 bp)of A.dhakensis could be amplified at the annealing temperature of 65℃by the primers Ad-f/Ad-r;the detection limit for A.dhakensis solution was 1.38 CFU/μL,and that for nucleic acids was 9.37×10-4 ng/μL;no specific bands were found by PCR assay taking 20 kinds of other aquatic pathogens(such as A.hydrophila and A.veronii)as templates.In conclusion,the established method,with strong specificity and high sensitivity,could be used to rapidly and accurately detect A.dhakensis from diseased fish and environmental samples,supporting clinical identification of the pathogen.
关键词
达卡气单胞菌/PCR检测/核心基因Key words
A.dhakensis/PCR assay/core gene引用本文复制引用
基金项目
江西省自然科学基金项目(20232BAB205070)
江西省现代农业产业技术体系建设专项(JXARS-03)
出版年
2024
国家科技期刊平台
NSTL
万方数据