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熊向英,蔡小辉,彭银辉,黄国强,梁志辉,王志成.卵形鲳鲹主要致病链球菌多重PCR诊断技术的建立[J].广西科学,2016,23(1):35-40. [点击复制]
- XIONG Xiangying,CAI Xiaohui,PENG Yinhui,HUANG Guoqiang,LIANG Zhihui,WANG Zhicheng.Development of Multiplex PCR Assay for Detection of Main Streptococcosis Pathogens in Trachinoms ovatus[J].Guangxi Sciences,2016,23(1):35-40. [点击复制]
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| 卵形鲳鲹主要致病链球菌多重PCR诊断技术的建立 |
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熊向英, 蔡小辉, 彭银辉, 黄国强, 梁志辉, 王志成
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| (广西海洋研究所, 广西海洋生物技术重点实验室, 广西北海 536000) |
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| 摘要: |
| [目的]快速、准确地检测卵形鲳鲹(Trachinoms ovatus)感染无乳链球菌(Streptococcus agalactiae)、停乳链球菌(Streptococcus dysgalactiae)、海豚链球菌(Streptococcus iniae)和格式乳球菌(Lactococcus garvieae)4种主要的致病菌,为卵形鲳鲹的健康养殖提供依据。[方法]针对4种致病菌的特异基因设计特异性引物cfb-F/cfb-R,tuf-F/tuf-R,Si-F/Si-R,PLG-F/PLG-R,建立多重PCR反应体系,通过调试各引物对之间的最佳比例以及最适退火温度,对反应体系进行优化及灵敏度测试。2014年68月,应用该体系对北海及湛江各养殖场的发病卵形鲳鲹进行检测,然后用通用引物扩增16SrRNA基因,经测序、比对检测体系的准确性。[结果]反应体系中cfb-F/cfb-R,tuf-F/tuf-R,Si-F/Si-R,PLG-F/PLG-R的最适浓度分别为0.2μmol/L,0.08μmol/L,1.6μmol/L和0.4μmol/L;最优退火温度为54.3℃;4种目标菌的检测灵敏度为5×10-2 ng/μL;11份病原样品检测中,2份出现海豚链球菌的目的条带,4份出现无乳链球菌的目的条带,5份未见目的条带,和测序分析结果一致。[结论]多重PCR方法能代替传统的微生物检测方法特异、快速、灵敏地检测4种致病菌。 |
| 关键词: 多重PCR 链球菌病 无乳链球菌 停乳链球菌 格式乳球菌 海豚链球菌 |
| DOI：10.13656/j.cnki.gxkx.20160315.014 |
| 投稿时间：2015-06-15修订日期：2015-08-05 |
| 基金项目:广西面上基金项目(2012GXNSFAA053065),广西科技攻关项目(桂科攻1222013-2),广西区直属公益性科研院所基本科研业务费专项资金项目(GXIF-2012-02)和国家海洋局项目(201205028-4)资助。 |
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| Development of Multiplex PCR Assay for Detection of Main Streptococcosis Pathogens in Trachinoms ovatus |
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XIONG Xiangying, CAI Xiaohui, PENG Yinhui, HUANG Guoqiang, LIANG Zhihui, WANG Zhicheng
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| (Guangxi Key Laboratory of Marine Biotechnology, Guangxi Institute of Oceanology, Beihai, Guangxi, 536000, China) |
| Abstract: |
| [Objective] A method with multiplex PCR was developed for rapid and accurate detection of the four main streptococcosis pathogens of Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus iniae, and Lactococcus garvieae isolated from golden pompano Trachinotus ovatus, in order to provide theoretical foundation for healthy aquaculture of Trachinotus ovatus.[Methods] Each of the four pairs of oligonucleotide primers including cfbF/cfb-R, tuf-F/tuf-R, Si-F/Si-R, PLG-F/PLG-R exclusively amplified the target gene of the specific microorganism.The multiplex PCR was optimized by testing the proportion between the four sets of primers and the annealing temperature.And the sensitivity of the multiplex PCR assay was evaluated using pure DNA.A total of 11 pathogen samples were collected from cultured Trachinotus ovatus from June to August in 2014, and analyzed using the established multiplex PCR to test its applicability.To verify the results of the multiplex PCR, molecular identification was performed by amplification of 16S rRNA gene with universal primers.[Results] The optimized relative concentrations of the primers were 0.2μmol/L each of cfb-F/cfb-R, 0.08μmol/L each of tuf-F/tuf-R, 1.6μmol/L each of Si-F/Si-R and 0.4μmol/L each of PLG-F/PLG-R.The sensitivity of the multiplex PCR using purified DNA was 5×10-2ng/μL.The application results revealed that 2of the pathogen samples were S.iniae, 4samples were S.agalactiae and no products were amplified from the other 5pathogen samples.Sequencing of 16S rRNA and BLAST analysis were consistent with the results of the multiplex PCR.[Conclusion] The above results indicated that the multiplex PCR is a sensitive, specific and reproducible assay method for the simultaneous detection of four pathogenic bacteria that cause disease in golden pompano.Therefore, it could be a useful alternative to the conventional method for microbial detection. |
| Key words: multiplex PCR Streptococcosis Streptococcus agalactiae Streptococcus dysgalactiae Streptococcus iniae Lactococcus garvieae |
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