PDF(Pubmed)
Abstract：
Kaposi\'s sarcoma (KS) is a cancer affecting skin and internal organs for which the Kaposi\'s sarcoma associated herpesvirus (KSHV) is a necessary cause. Previous work has pursued KS diagnosis by quantifying KSHV DNA in skin biopsies using a point-of-care (POC) device which performs quantitative loop-mediated isothermal amplification (LAMP). These previous studies revealed that extracting DNA from patient biopsies was the rate limiting step in an otherwise rapid process. In this study, a simplified, POC-compatible alkaline DNA extraction, ColdSHOT, was optimized for 0.75 mm human skin punch biopsies. The optimized ColdSHOT extraction consistently produced 40,000+ copies of DNA per 5 µl reaction from 3 mg samples-a yield comparable to standard spin column extractions-within 1 h without significant equipment. The DNA yield was estimated sufficient for KSHV detection from KS-positive patient biopsies, and the LAMP assay was not affected by non-target tissue in the unpurified samples. Furthermore, the yields achieved via ColdSHOT were robust to sample storage in phosphate-buffered saline (PBS) or Tris-EDTA (TE) buffer prior to DNA extraction, and the DNA sample was stable after extraction. The results presented in this study indicate that the ColdSHOT DNA extraction could be implemented to simplify and accelerate the LAMP-based diagnosis of Kaposi\'s sarcoma using submillimeter biopsy samples.
摘要：
卡波西肉瘤（KS）是一种影响皮肤和内脏器官的癌症，卡波西肉瘤相关疱疹病毒（KSHV）是其必要原因。先前的工作通过使用即时护理（POC）设备对皮肤活检中的KSHVDNA进行定量来进行KS诊断，该设备执行定量环介导的等温扩增（LAMP）。这些先前的研究表明，从患者活检中提取DNA是其他快速过程中的限速步骤。在这项研究中,一个简化的，与POC相容的碱性DNA提取，冷射,针对0.75mm人类皮肤穿孔活检进行了优化。优化的ColdSHOT提取在没有重要设备的情况下，在1小时内从3mg样品中每5μl反应始终产生40,000+个DNA拷贝-产量与标准旋转柱提取相当。估计DNA产量足以从KS阳性患者活检中检测到KSHV，在未纯化的样品中,LAMP测定不受非靶组织的影响。此外，通过ColdSHOT获得的产量对于在DNA提取之前在磷酸盐缓冲盐水（PBS）或Tris-EDTA（TE）缓冲液中的样品储存是稳健的，提取后DNA样品稳定。这项研究的结果表明，可以使用亚毫米活检样本进行ColdSHOTDNA提取，以简化和加速基于LAMP的卡波西肉瘤诊断。
