Ibrahim M S, Lofts R S, Jahrling P B, Henchal E A, Weedn V W, Northrup M A, Belgrader P
United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702, USA.
Anal Chem. 1998 May 1;70(9):2013-7. doi: 10.1021/ac971091u.
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 served 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.
本报告描述了使用电池供电的微型分析热循环仪（MATCI）进行实时5'核酸酶PCR检测，以快速区分单碱基多态性。正痘病毒和人类补体成分C6基因作为靶标，以证明使用MATCI诊断传染病和遗传疾病的可行性。在正痘病毒检测中，设计了通用的正痘病毒PCR引物，以扩增骆驼痘病毒、牛痘病毒、猴痘病毒和痘苗病毒中血凝素（HA）基因的266 - 281个碱基对（bp）片段。设计了一种痘苗病毒特异性荧光（TaqMan）探针，以检测HA基因内的单碱基（A/G）替换。在C6基因检测中，从人类基因组DNA中PCR扩增出C6基因的一个73 bp片段，并使用TaqMan探针检测密码子98第二位的单碱基（A/C）多态性。MATCI正确识别了病毒DNA和人类基因组DNA中的核苷酸差异。此外，使用快速DNA制备方法，可以在不到1小时的时间内完成样品、人类基因组DNA的制备、DNA扩增和实时检测。
