Mahbubani M H, Schaefer F W, Jones D D, Bej A K
Department of Biology, Miles College, Birmingham, AL 35216, USA.
Curr Microbiol. 1998 Feb;36(2):107-13. doi: 10.1007/s002849900288.
Genomic DNA was extracted either directly from Giardia muris cysts seeded into environmental surface waters or from cysts isolated by immunomagnetic beads (IMB). A 0.171-kbp segment of the giardin gene was PCR-amplified following "direct extraction" of Giardia DNA from seeded Cahaba river water concentrate with moderate turbidity (780 JTU's), but DNA purified from seeded Colorado river water concentrates with high turbidity (2 x 10(5) JTUs) failed to amplify. However, if the cysts were first separated by the IMB approach from seeded Cahaba or Colorado river waters, and the DNA released by a freeze-boil Chelex(R)100 treatment, detection of G. muris by PCR amplification could be achieved at a sensitivity of 3 x 10(0) or 3 x 10(1) cysts/ml, respectively. If, however, the G. muris cysts used to seed even moderately turbid river waters (780 JTUs) were formalin treated (which is conventionally used for microscopic examination), neither direct extraction nor IMB purification methods yielded amplifiable DNA. Use of immunomagnetic beads to separate Giardia cysts from complex matrices of environmental surface waters followed by DNA release and PCR amplification of the target giardin gene improved the reliability of detection of this pathogen with the required sensitivity.
基因组DNA要么直接从接种到环境地表水的鼠贾第虫包囊中提取，要么从通过免疫磁珠（IMB）分离的包囊中提取。在用中度浊度（780 JTU）的接种卡哈巴河水浓缩物对贾第虫DNA进行“直接提取”后，对贾第虫素基因的0.171 kbp片段进行了PCR扩增，但从高浊度（2×10⁵ JTU）的接种科罗拉多河水浓缩物中纯化的DNA未能扩增。然而，如果首先通过IMB方法从接种的卡哈巴河或科罗拉多河水中分离包囊，并用冻煮Chelex®100处理释放DNA，则通过PCR扩增检测鼠贾第虫的灵敏度分别可达3×10⁰或3×10¹个包囊/毫升。然而，如果用于接种甚至中度浊度河水（780 JTU）的鼠贾第虫包囊经过福尔马林处理（这是传统上用于显微镜检查的方法），则直接提取法和IMB纯化法均无法产生可扩增的DNA。使用免疫磁珠从环境地表水的复杂基质中分离贾第虫包囊，然后释放DNA并对目标贾第虫素基因进行PCR扩增，提高了以所需灵敏度检测这种病原体的可靠性。
