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ID:20417231
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页数:11页
时间:2018-10-13
《鼻气管鸟疫杆菌sybrgreenⅰ荧光定量pcr检测方法的建立及应用》由会员上传分享,免费在线阅读,更多相关内容在学术论文-天天文库。
1、鼻气管鸟疫杆菌SYBRGreenI荧光定量PCR检测方法的建立及应用摘要【目的】建立一种鼻气管鸟疫杆菌SYBRGreen1荧光定量PCR检测方法,并应用于临床样品检测。【方法】参照鼻气管鸟疫杆菌(GenBank登录号:JF810495.1)基因序列设计1对特异性引物,经PCR扩增,回收214bp的目的片段,与pMD18-TVector连接后转化到基因工程菌DH5ot屮,提取重组质粒,经PCR、酶切及测序鉴定后,筛选阳性质粒,倍比稀释作为模板,用于SYBRGreenI荧光定量PCR标准曲线的构建,并做敏感性试验、重复性试验和特异性试验。【结果】结果表明,标准曲线循环阈值与模板浓度呈良好的线性关
2、系,且标准曲线线性关系R2在0.999以上,产物Tm值在81.3-83.2°C之间,灵敏度为1.44xl02拷W/pL,特异性结果表明只能检测到鼻气管鸟疫杆菌的扩增曲线,重性试验变异系数小于0.7%,临床样品检出率为100%。【结论】木试验初步建立了检测鼻气管鸟疫杆菌的SYBRGreenI荧光定量PCR方法,为该病的早期诊断以及感染程度的定量分析奐定了基础。关键词:鼻气管鸟疫杆菌;SYBRGreenI;实时荧光定量PCRDevelopmentandapplicationofSYBRGreenIreal-timequantitativePCRtechniqueforOmithobacteriu
3、mrhinotrachealeWANGLi-rong,DIAOYou-xiangTANGYi,JUXiao-jun(CollegeofAnimalScienceandTechnology,ShandongAgriculturalUniversity,Tai’an,Shandong,271018,China)Abstract:【Objective】TheobjectiveofthestudyistoestablishamethodfordetectingOmithobacteriumrhinotrachealebySYBRGreenIfluorescencequantitativePCRde
4、tectionmethodandtoapplicationinclinicalsamplesdetection.[Method]ApairofspecificprimerswasdesignedaccordingtotheOmithobacteriumrhinotracheale(GenBankloginnumber:JF810495.1)genesequence.Thetarget214bpfragmentwasamplifiedinPCRgeneamplification,andinsertedintopMD18-Tvectorafterbeingpurified.Thentherest
5、ructuredvectorsweretransformedtoE.coliDH5a.PositiveplasmidwasextractedastemplatetoestablishSYBR-GreenIfluorescencequantitativePCRstandardcurveafterPCRidentifying,enzymecuttingandsequencing.Sensitivity,Reproducibilityandspecificityofthemethodweredetermined.【Result】Theresultsindicatedthatstandardcurv
6、eestablishedbyrecombinantplasmidshowedagoodlinearrelationshipbetweenthresholdcycleandtemplateconcentration.Thestandardcurvelinearrelationshipwasmorethan0.99.TheTmwas813-83.2andthesensitivedegreeis1.44xl02copiesper
7、iL,SpecificresultsshowedthatonlyamplificationcurveofOmithobacteriumrhinotrachealecanb
8、edetcctioncd,repeatabilitytestindicatedthevariationcoefficientislessthan0.7%;clinicalsampledetectionratewas100%.【Conclusion】ASYBRGreenIfluorescentquantitativePCRassayfordetectingOrnithobacteriumrhinotrachea
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