刺激隐核虫PCR检测方法的建立
Establishment of PCR amplification detection for Cryptocaryon irritans
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摘要: 根据已知刺激隐核虫ITS序列设计引物,扩增刺激隐核虫ITS部分序列,并进行克隆、测序及序列分析建立刺激隐核虫PCR快速检测方法,检测DNA片段长度为387 bp,与福建长乐CL1209(KC550300)、福建福鼎FD1210(KC550302)、广东番禺PYH4.12株(DQ270010)、日本Wakayama分离株(AB608054)、台湾嘉义Chiayi株(AF490381)等序列同源性为100%,检测灵敏度为2个虫体。应用建立方法对2011-2013年收集到的50份患刺激隐核虫病的大黄鱼样品进行检测,结果与根据临床检测结果的符合率为100%。该检测方法的建立,为刺激隐核虫病的监测和诊断奠定了快速、简便、高通量的检测方法基础。Abstract: Specific primers were designed based on the DNA sequence of internal transcribed spacer 1 (ITS-1). The genomic DNA of Cryptocaryon irritans，isolated from culturing Pseudosciaena crocea in Luoyuan of Fujian，used as a template DNA to amplify part of the gene fragment of ITS-1，which had a 387 bp band when electrophoresed on an agarose gel. Then the PCR amplification products were cloned，sequenced and analyzed. Homology comparison indicated that they shared 100 % identity with the gene sequence of CL1209 strain ( KC550300)，FD 1210 ( KC550302) ， PYH 4.12 strain ( DQ270010) ，Wakayama strain (AB608054)， and Chiayi strain (AF490381) . PCR amplification for the detection of Cryptocaryon irritans was established， which was limited to 2 polypides， then being applied to detect the samples of the C. irritans monitoring during the last three years (2011-2013 ) . The results were 100% conformed to clinical test results. It revealed that this PCR amplification method was quick，sensitive reproducible and applicable for the detection of Cryptocaryon irritans.
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