期刊文献+

香鱼假单胞菌SYBR GreenⅠ荧光定量PCR检测方法的建立 被引量:7

Development of a SYBR GreenⅠreal-time PCR assay for specific detection of Pseudomonas plecoglossicida
下载PDF
导出
摘要 为建立香鱼假单胞菌的快速定量检测方法,本研究根据Gen Bank中登录的rpo D基因序列设计了一对特异性引物,以该菌基因组DNA为模板通过PCR扩增其180 bp的rop D基因片段,并克隆于p MD18-T载体中,以纯化的重组质粒为标准品建立了香鱼假单胞菌SYBR GreenⅠ荧光定量PCR检测方法。结果显示在1.9×103拷贝/μL^1.9×108拷贝/μL范围内呈现良好的线性关系,相关系数为0.999,灵敏度可达1.9×103拷贝/μL;该方法对恶臭假单胞菌、荧光假单胞菌、创伤弧菌等病原菌DNA扩增结果均为阴性,特异性良好;组内和组间变异系数均小于2%,具有较高的重复性和稳定性。本研究建立的方法不仅能够快速检测香鱼假单胞菌,还能够对该病原菌感染的动态变化进行定量研究,为大黄鱼香鱼假单胞菌感染导致的内脏白点病的早期诊断及防控提供一个新的检测技术。 To develop a specific assay for detection of Pseudomonas plecoglossicida, the SYBR green I real-time PCR assay were established with a pair of primers targeting rpoD gene of P.plecoglossicida. Then, a standard curve was constructed with series dilution of the recombinant plasmids containing the rpoD gene. The correlation coefficient of standard curve constructed using the threshold cycle (CT) versus log10 copy numbers of standard plasmid samples showed a good linearity (R2=0.999), and the minimum level of detection was 1.9×10^3 copies/μL. The specificity of the assay was confirmed by the lack of cross-amplification with DNA templates from P.putica, Vibrio alginolyticus, V.parahaemolyticus, Streptococcus iniae, Aeromonas hyclrophilia, V. v-uln- ificus, Shcwanella alga, P.fluorescens and Nocardia seriolae. The coefficients of variance (CV) were 1.29% and 1.70% for the intra- and inter-assay, respectively, which indicated a good reliability and reproducibility. This detection assay provided a rapid means to quantitatively accessing the presence of P.plecoglossicida, which can be a useful tool for the quantification and monitoring of this bacterial pathogen in farmed fish, as well as the diagnosis of the P.plecoglossicida-related fish disease.
出处 《中国预防兽医学报》 CAS CSCD 北大核心 2015年第1期35-39,共5页 Chinese Journal of Preventive Veterinary Medicine
基金 长江学者和创新团队发展计划(IRT0734) 农业部公益性行业科研专项(200903029) 浙江省自然科学基金(LY13C190005) 宁波市科技创新团队项目(2013B82012)
关键词 rpoD基因 香鱼假单胞菌 荧光定量PCR SYBR Green rpoD gene Pseudomonas plecoglossicida real-time PCR SYBR Green I
  • 相关文献

参考文献7

二级参考文献93

共引文献128

同被引文献128

  • 1胡娇,张飞,徐晓津,苏永全,覃映雪,马英,张艺,韩坤煌,鄢庆枇.大黄鱼(Pseudosciaena crocea)内脏白点病病原分离鉴定及致病性研究[J].海洋与湖沼,2014,45(2):409-417. 被引量:28
  • 2谢书秋,刘振勇.闽东大黄鱼养殖现状分析与发展对策[J].福建水产,2006,28(3):95-97. 被引量:11
  • 3郑华,陈法霖,欧阳志云,方治国,王效科,苗鸿.不同森林土壤微生物群落对Biolog-GN板碳源的利用[J].环境科学,2007,28(5):1126-1130. 被引量:69
  • 4Landman W J. Is Mycoplasma synoviae outrunning Mycoplasma gallisepticum? A viewpoint from the Netherlands [J]. Avian Pathol, 2014, 43(1): 2-8.
  • 5Brown D R, Whitcomb R F, Bradbury J M. Revised minimal standards for description of new species of the class Mollicutes (division Tenericutes) [J]. Int J SystEvolMicrobiol, 2007, 57(Pt 11): 2703-2719.
  • 6Garcia M, Ikuta N, Levisohn S, et al. Evaluation and comparison of various PCR methods for detection of Mycoplasma gallisepticum infection in chickens [J]. Avian Dis, 2005, 49(1):125-132.
  • 7Kleven S H. Control of avian mycoplasma infections in commercial poultry [J]. Avian Dis, 2008, 52(3): 367-374.
  • 8Kahya S, Temelli S, Eyigor A, et al. Real-time PCR culture and serology for the diagnosis of Mycoplasma gallisepticum in chicken breeder flocks [J]. Vet Microbiol, 2010, 144(3-4): 319-324.
  • 9Salisch H, Hinz K H, Graack H D, et al. A comparison of a commercial PCR-based test to culture methods for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in concurrently infected chickens [J]. Avian Pathol, 1998, 27(2): 142-147.
  • 10Feberwee A, Mekkes D R, de Wit J J, et al. Comparison of culture, PCR, and different serologic tests for detection of Mycoplasma gallisepticum and Mycoplasma synoviae infections [J]. Avian Dis, 2005, 49(2): 260-268.

引证文献7

二级引证文献30

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部