期刊
JOURNAL OF CLINICAL MICROBIOLOGY
卷 56, 期 12, 页码 -出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/JCM.01061-18
关键词
extraction yield; ccfDNA; extraction methods; viral diagnostics; viral DNA processing
类别
资金
- Lifespan/Tufts/Brown Center for AIDS Research [P30AI042853]
- ALSAC
An element essential for PCR detection of microbial agents in many sample types is the extraction step, designed to purify nucleic acids. Despite the importance of this step, yields have not been extensively compared across methods to determine whether the method used contributes to quantitative differences and the lack of commutability seen with existing clinical methods. This may in part explain why plasma and blood viral load assays have proven difficult to standardize. Also, studies have identified small DNA fragments of <200 bp in plasma (cell-free DNA [cfDNA)), which may include significant quantities of viral DNA. Our study evaluated extraction yields for 11 commercially available extraction methods, including 4 new methods designed to isolate cfDNA. Solutions of DNA fragments with sizes ranging from 50 to 1,500 bp were extracted, and then the eluates were tested by droplet digital PCR to determine the DNA fragment yield for each method. The results demonstrated a wide range of extraction yields across the variety of methods/instruments used, with the 50- and 100-bp fragment sizes showing especially inconsistent quantitative results and poor yields of less than 20%. Slightly higher, more consistent yields were seen with 2 of the 4 circulating cell-free extraction kits. These results demonstrate a significant need for further evaluation of nucleic acid yields across the variety of extraction platforms and highlight the poor extraction yields of small DNA fragments by existing methods. Further work is necessary to determine the impact of this inconsistency across instruments and the relevance of the low yields for smaller DNA fragments in clinical virology testing.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据