Real-time microchip PCR for detecting single-base differences in viral and human DNA
Real-time microchip PCR for detecting single-base differences in viral and human DNA
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DOI：
10.1021/ac971091u
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发表时间：
1998-05-01
影响因子：
7.4
通讯作者：
Belgrader, P
中科院分区：
文献类型：
--
作者：
Ibrahim, MS;Lofts, RS;Belgrader, P
本报告描述了实时5'核酸酶PCR检测，以快速区分单碱基多态性使用电池供电的微型分析热循环仪器（MATCI）。正痘病毒和人补体成分C6基因作为靶标，以证明使用MATCI诊断感染性疾病和遗传性疾病的可行性。在正痘病毒检测试剂中，设计了正痘病毒通用PCR引物，用于扩增骆驼痘、牛痘、猴痘和牛痘病毒中血凝素（HA）基因的266-281个碱基对（bp）片段。设计了一种牛痘病毒特异性荧光（TaqMan）探针，用于检测HA基因内的单碱基（A/G）取代。在C6基因测定中，从人基因组DNA中PCR扩增C6基因的73-bp片段，并使用TaqMan探针检测密码子98的第二位置的单碱基（A/C）多态性。MATCI能正确识别病毒DNA和人基因组DNA的核苷酸差异，并采用快速DNA制备方法，在1 h内实现了样品、人基因组DNA制备、DNA扩增和实时检测。
This report describes real-time 5' nuclease PCR assays to rapidly distinguish single-base polymorphism using a battery-powered miniature analytical thermal cycling instrument (MATCI). Orthopoxviruses and the human complement component C6 gene sewed as targets to demonstrate the feasibility of using the MATCI for diagnosis of infectious diseases and genetic disorders. In the Orthopoxvirus assay, consensus Orthopoxvirus PCR primers were designed to amplify 266-281 base-pair (bp) segments of the hemagglutinin (HA) gene in camelpox, cowpox, monkeypox, and vaccinia viruses. A vaccinia virus-specific fluorogenic (TaqMan) probe was designed to detect a single-base (A/G) substitution within the HA gene. In the C6 gene assay, a 73-bp segment of the C6 gene was PCR-amplified from human genomic DNA, and TaqMan probes were used to detect a single-base (A/C) polymorphism in the second position of codon 98. The MATCI correctly identified the nucleotide differences in both viral DNA and human genomic DNA, In addition, using a rapid DNA preparation method, it was possible to achieve sample, preparation of human genomic DNA, DNA amplification, and real-time detection in less than 1 h.