本文選題：實(shí)時(shí)熒光定量PCR　切入點(diǎn)：TaqMan探針　出處：《生物技術(shù)通報(bào)》2016年11期 　論文類型：期刊論文

【摘要】：在TaqMan探針法實(shí)時(shí)熒光定量PCR中,排除由引物探針聚合延伸引起的假陽性問題以及由引物二聚體(Primer Dimer,PD)引起的假陰性問題。首先對(duì)不同探針進(jìn)行引物探針聚合實(shí)驗(yàn);然后通過雙重PCR的干擾實(shí)驗(yàn)選擇合適的內(nèi)標(biāo)基因,并使用內(nèi)標(biāo)質(zhì)粒檢測(cè)驗(yàn)證中部同序引物排除PD干擾的實(shí)用性;最后比較探針法不同體系檢測(cè)HBV基因的靈敏度。結(jié)果表明,引物與探針聚合實(shí)驗(yàn)中,含有反義堿基的HBVP4在重復(fù)實(shí)驗(yàn)中未出現(xiàn)假陽性;競(jìng)爭(zhēng)性雙重PCR和非競(jìng)爭(zhēng)性雙重PCR兩模板開始出現(xiàn)干擾作用的濃度差分別為20倍和100倍;對(duì)于內(nèi)標(biāo)基因,使用中部同序引物對(duì)以及普通引物對(duì)分別可以檢測(cè)到10-9和10-8稀釋度;3種HBV基因檢測(cè)體系,使用普通引物對(duì)可以檢測(cè)到Ct33左右,加入內(nèi)標(biāo)系統(tǒng)和使用中部同序引物對(duì)均可檢測(cè)到Ct35左右。在TaqMan-分子信標(biāo)結(jié)構(gòu)調(diào)整的基礎(chǔ)上引入反義堿基可以在排除由引物和探針聚合延伸引起的假陽性問題;在探針法中,使用中部同序引物對(duì)和加入內(nèi)標(biāo)系統(tǒng)均可降低PD的干擾程度,提升檢測(cè)的靈敏度。
[Abstract]:In real-time fluorescent quantitative PCR with TaqMan probe method, the false positive problem caused by the extension of primer probe polymerization and the false negative problem caused by primer dimer dimer dimer were eliminated. First, primer probe polymerization experiments were carried out for different probes. Then the suitable internal standard gene was selected by double PCR interference experiment, and the practicability of eliminating PD interference was verified by using internal standard plasmid detection. Finally, the sensitivity of probe method for detecting HBV gene in different systems was compared. In primer and probe polymerization, HBVP4 containing antisense bases did not appear false positive in repeated experiments, the concentration difference of interference between competitive double PCR and non-competitive double PCR began to be 20 times and 100 times respectively. Using middle sequence primer pairs and common primer pairs, 10-9 and 10-8 dilution of HBV genes could be detected, and Ct33 could be detected by common primer pairs. Ct35 can be detected by adding internal standard system and using middle sequence primer pairs. Introducing antisense bases on the basis of structural adjustment of TaqMan- molecular beacons can eliminate false positive problems caused by the extension of primer and probe polymerization. Using the central sequence primer pair and adding the internal standard system can reduce the interference of PD and improve the sensitivity of detection.
【作者單位】： 中國人民解放軍總醫(yī)院臨檢科;溫州醫(yī)科大學(xué)檢驗(yàn)醫(yī)學(xué)院生命科學(xué)學(xué)院;北京泰格瑞分子檢驗(yàn)有限公司;
【基金】：國家重大科學(xué)儀器設(shè)備開發(fā)專項(xiàng)(2012YQ03026107)
【分類號(hào)】：R440

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