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首页> 外文期刊>Clinical and diagnostic laboratory immunology >A rapid and gentle method for isolation of genomic DNA from pathogenic Nocardia spp.
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A rapid and gentle method for isolation of genomic DNA from pathogenic Nocardia spp.

机译：一种快速温和的从致病诺卡氏菌属物种中分离基因组DNA的方法。

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摘要

The lack of simple and efficient methods for extraction of DNA from Nocardia spp. has hampered molecular manipulation of the DNA for diagnostic purposes. In the present study, a method for the rapid extraction of undegraded genomic nocardial DNA was established. Briefly, 14 pathogenic Nocardia strains were grown at 37 degrees C for 3 to 5 days in Sauton broth containing 0.05% Tween 80. Subsequently, the cultures were treated for 48 h with 1.2 mg of cycloserine per ml (final concentration). Cells were then harvested by centrifugation and treated with a lysis solution containing 3 mg of lysozyme per ml. This was followed by the addition of proteinase K and sodium dodecyl sulfate to final concentrations of 0.2 mg/ml and 0.5%, respectively, and incubation for 1 h at 50 degrees C. DNA was precipitated with isopropanol after phenol-chloroform-isoamyl alcohol extractions and RNase treated before being quantitated and analyzed by agarose gel electrophoresis. The average undegraded DNA yields obtained were 101 micrograms for Nocardia brasiliensis and 121 micrograms for N. asteroides. This DNA was suitable for restriction endonuclease digestion and PCR amplification, which are methods being applied to the characterization and diagnosis of slowly growing organisms such as Nocardia spp.
机译：缺乏从诺卡氏菌属中提取DNA的简单有效方法。为了诊断目的，已经阻碍了DNA的分子操作。在本研究中，建立了一种快速提取未降解基因组心肌DNA的方法。简而言之，将14种致病诺卡氏菌菌株在含有0.05％Tween 80的Sauton肉汤中于37°C生长3至5天。随后，将培养物用1.2 mg环丝氨酸/ ml（终浓度）处理48小时。然后通过离心收集细胞，并用每毫升含3 mg溶菌酶的裂解液处理。随后添加蛋白酶K和十二烷基硫酸钠至终浓度分别为0.2 mg / ml和0.5％，并在50摄氏度下孵育1小时。在苯酚-氯仿-异戊醇萃取后，DNA用异丙醇沉淀RNase处理后再进行琼脂糖凝胶电泳定量和分析。对于巴西诺卡氏菌，获得的平均未降解DNA产量为101微克，对小行星猪笼草为121微克。该DNA适用于限制性核酸内切酶消化和PCR扩增，这是用于表征和诊断缓慢生长的生物如诺卡氏菌的方法。

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