Comparison of Packed Beds and Qiagen Columns for Recovering Trace Amounts of B. anthracis DNA from Liquid Suspensions

Sorensen, K ; Arroyo, E ; Erler, A ; Christian, A T ; Camp, D ; Wheeler, E K

Lawrence Livermore National Laboratory

关键词: Amplification;    Lawrence Livermore National Laboratory;    Aerosols;    Polymerase Chain Reaction;    Processing;   

DOI  :  10.2172/973645
RP-ID  :  UCRL-TR-222387
RP-ID  :  W-7405-ENG-48
RP-ID  :  973645

美国|英语

来源: UNT Digital Library

【 摘 要 】

The goal of this work was to optimize and evaluate LLNL's in-bed amplification technology to improve the level of detection for suspensions containing trace amounts of anthracis DNA. The binding/cleaning performance of the packed bed is compared to the conventional commercial approach; Qiagen column cleanup and elution, followed by detection through an ex-situ amplification process. Five liquid suspensions were spiked with B.anthracis DNA in concentration series. These suspensions were: (1) water, (2) water with EDTA, (3) dirty water from carpet extraction, (4) dirty carpet extraction with phosphate buffered saline (PBS) plus 0.1% Tween 20 plus 0.1% gelatin, and (5) a subway aerosol collected in water. Each suspension matrix was spiked with DNA and injected (in replicate) into either Qiagen Microcolumns (using the kit processing instructions) or LLNL's packed bed (using the LLNL in-bed purification and amplification protocol). The process output was assayed by quantitative polymerase chain reaction (QPCR). Table ES-1 shows the level of DNA (pg per 100 uL of input suspension) that resulted in successful amplification for all reactions (X=Y), and the level for which at least one of the reactions was successful (X>0). For each suspension and DNA concentration, there were Y QPCR assays of which X showed successful amplification. LLNL's packed bed technology outperformed Qiagen Microcolumns for all five suspensions, typically by one order of magnitude in both the limit of assured detection (all reactions positive), and the lower limit of detection (some reactions positive).