發(fā)布時(shí)間：2019-05-21 18:19

【摘要】：為建立蘋果屬冬生疫霉Phytophthora hibernali、丁香疫霉P.syringae和栗黑水疫霉P.cambivora 3種檢疫性疫霉的同步分子檢測(cè)方法,根據(jù)疫霉屬18S rRNA,ITS,HSP90和Ypt1基因分別設(shè)計(jì)通用引物,冬生疫霉、丁香疫霉及栗黑水疫霉特異引物,建立了四重PCR檢測(cè)方法,并進(jìn)行了靈敏度和模擬帶菌測(cè)試.建立了可同時(shí)檢測(cè)蘋果屬上冬生疫霉、丁香疫霉和栗黑水疫霉的特異四重PCR檢測(cè)體系:在20μL反應(yīng)體系中,最佳引物濃度組合為10μmol/L的18SUF/18SUR,PHSF/PHSR,PCSF/PCSR和PSSF/PSSR分別為0.2,0.6,0.8,1.0μL;最佳退火溫度和退火時(shí)間分別為63℃和20s.該體系擴(kuò)增冬生疫霉出現(xiàn)884bp的18S rRNA條帶和232bp的ITS基因特異帶,丁香疫霉出現(xiàn)884bp的18S rRNA條帶和683bp的HSP90基因特異帶,擴(kuò)增栗黑水疫霉出現(xiàn)884bp的18S rRNA條帶和314bp的Ypt1基因特異帶,對(duì)照菌只出現(xiàn)18S rRNA條帶;四重PCR反應(yīng)體系檢測(cè)靈敏度低于單重PCR;模擬帶菌試驗(yàn)可同時(shí)擴(kuò)增出4個(gè)片段.表明該四重PCR檢測(cè)方法能實(shí)現(xiàn)冬生疫霉、丁香疫霉和栗黑水疫霉的同步特異檢測(cè),實(shí)現(xiàn)蘋果屬類水果及種苗檢疫性疫霉的快速檢測(cè).
[Abstract]:In order to establish a synchronous molecular method for the detection of three kinds of quarantine Phytophthora, Phytophthora hibernali, clove P.syringae and P.cambivora, according to the 18s rRNA,ITS,HSP90 and Ypt1 genes of Phytophthora, general primers were designed for the detection of Phytophthora. A four-fold PCR method was established for the detection of Phytophthora clove and Phytophthora mollissima with specific primers, and the sensitivity and simulated carrying test were carried out. A specific quadruple PCR system for the simultaneous detection of Phytophthora, Phytophthora clove and Phytophthora mollissima was established. In the 20 渭 L reaction system, the optimum primer concentration was 18SUF 鈮，

本文編號(hào)：2482291