Tsai Yu-Li, Le Joanne Y, Olson Betty H
Department of Environmental Health Science and Policy, University of California, Irvine 92697, USA.
Can J Microbiol. 2003 Jun;49(6):391-8. doi: 10.1139/w03-048.
A magnetic capture hybridization - polymerase chain reaction (MCH-PCR) method was used to increase the detection sensitivity of the enterotoxin gene LTIIa, used as a biomarker for waste in environmental samples. The samples were collected from cow lagoons of different farms and from environmental waters. Total DNA was extracted from colonies grown on mTEC medium or directly from environmental samples. The cow-specific Escherichia coli LTIIa gene was used as a DNA marker. A LTIIa-specific oligonucleotide probe was designed to capture the LTIIa marker during the MCH, followed by PCR. Varying levels of humic acid were added to the DNA extracts to evaluate the sensitivity and effectiveness of MCH-PCR. The minimal detection limit of MCH-PCR for the LTIIa gene was 2.5 ag/muL DNA. In the presence of humic acid, MCH-PCR was able to increase the detection sensitivity 10 000-fold over that of conventional PCR. The MCH-PCR could also detect one cell with the LTIIa DNA marker in a 1-L seeded environmental water sample. Results in this study indicate that MCH-PCR is more sensitive than nested PCR in testing environmental samples.
采用磁捕获杂交-聚合酶链反应（MCH-PCR）方法提高肠毒素基因LTIIa的检测灵敏度，该基因用作环境样品中废弃物的生物标志物。样品取自不同农场的奶牛泻湖和环境水体。总DNA从在mTEC培养基上生长的菌落中提取，或直接从环境样品中提取。牛特异性大肠杆菌LTIIa基因用作DNA标记。设计了一种LTIIa特异性寡核苷酸探针，用于在MCH过程中捕获LTIIa标记，随后进行PCR。向DNA提取物中添加不同水平的腐殖酸，以评估MCH-PCR的灵敏度和有效性。MCH-PCR对LTIIa基因的最低检测限为2.5 ag/μL DNA。在存在腐殖酸的情况下，MCH-PCR能够将检测灵敏度提高到比传统PCR高10000倍。MCH-PCR还能够在1 L接种的环境水样中检测到一个带有LTIIa DNA标记的细胞。本研究结果表明，在检测环境样品时，MCH-PCR比巢式PCR更灵敏。
