鼻气管鸟疫杆菌sybrgreenⅰ荧光定量pcr检测方法的建立及应用

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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