Chu F K
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12208, USA.
Mol Cell Probes. 1998 Apr;12(2):93-9. doi: 10.1006/mcpr.1998.0150.
The potential of fatal outcome for patients afflicted with human ehrlichioses (HME and HGE) necessitates fast and accurate detection of the aetiologic agents and timely antibiotic treatment. A polymerase chain reaction (PCR)-based protocol is described that can detect as little as 10 copies of ehrlichial 16S rDNA and as few as 0.3 HGE-infected neutrophils. The method employs DNAzol for rapid DNA extraction from unfractionated whole blood in less than 1 h. For DNA amplification, highly specific oligonucleotide primers are designed that efficiently detect and distinguish between Ehrlichia chaffeensis and the HGE agent. These primers do not prime DNA extracted from closely related ehrlichial and rickettsial species. Although total DNA extracted from human blood contains nucleic acids that can be non-specifically amplified at moderate to high MgCI2 concentrations, such non-specific priming of non-ehrlichial DNA can be completely eliminated by lowering the MgCI2 concentration to 1 mM. Thus, this PCR-based procedure can detect and differentiate HGE and HME with speed, simplicity, specificity and sensitivity.
人类埃立克体病（HME和HGE）患者面临致命后果的可能性，使得快速准确地检测病原体并及时进行抗生素治疗成为必要。本文描述了一种基于聚合酶链反应（PCR）的方法，该方法能够检测低至10拷贝的埃立克体16S rDNA以及少至0.3个被HGE感染的中性粒细胞。该方法采用DNAzol在不到1小时的时间内从未经分离的全血中快速提取DNA。对于DNA扩增，设计了高度特异性的寡核苷酸引物，可有效检测并区分查菲埃立克体和HGE病原体。这些引物不会引发从密切相关的埃立克体和立克次体物种中提取的DNA的扩增。虽然从人血中提取的总DNA含有在中等至高MgCI2浓度下可被非特异性扩增的核酸，但通过将MgCI2浓度降低至1 mM，可完全消除非埃立克体DNA的这种非特异性引发。因此，这种基于PCR的方法能够快速、简便、特异且灵敏地检测和区分HGE和HME。
