PCR amplification of catechol 2,3-dioxygenase gene sequences from naturally occurring hydrocarbon degrading bacteria isolated from petroleum hydrocarbon contaminated groundwater
PCR amplification of catechol 2,3-dioxygenase gene sequences from naturally occurring hydrocarbon degrading bacteria isolated from petroleum hydrocarbon contaminated groundwater
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PCR 扩增从石油碳氢化合物污染的地下水中分离出的天然碳氢化合物降解细菌的儿茶酚 2,3-双加氧酶基因序列
DOI：
10.1111/j.1574-6941.1996.tb00193.x
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发表时间：
1996
影响因子：
4.2
通讯作者：
S. Walia
中科院分区：
文献类型：
--
作者：
B. Joshi;S. Walia
摘要采用聚合酶链反应扩增技术检测了汽油污染地下水中天然细菌邻苯二酚2，3-双加氧酶（CD）基因序列。用于PCR测定的引物选自xylE、nahH和pAW 313的CD基因保守区的DNA序列。CD结构基因内部的30 bp DNA序列（ICP 313）用作探针以鉴定扩增的DNA片段。通过用Stu I和Bg I限制酶消化DNA片段，产生预期大小的DNA片段，确认扩增的DNA片段的身份。使用从多种细菌分离株分离的DNA测试PCR测定的特异性，所述细菌分离株包括大肠杆菌ATCC 25922、粪肠杆菌ATCC 29212、铜绿假单胞菌ATCC 27853、金黄色葡萄球菌ATCC 25923、金黄色葡萄球菌ATCC 29213、玫瑰色微球菌ATCC 516、粘质沙雷氏菌ATCC 60、肺炎克雷伯氏菌ATCC 13883、金黄杆菌ATCC 29896、葡萄酒色菌ATCC 17899、睾丸酮丛毛单胞菌ATCC 11996、发孢甲基弯菌OB 3b、枯草芽孢杆菌ATCC 21697、真养产碱杆菌ATCC 17698、球型节杆菌ATCC 35698、恶臭假单胞菌mt-2 ATCC 33015、醋酸钙不动杆菌ATCC 31012，和恶臭假单胞菌ATCC 17484。在缺乏CD活性的细菌分离株中未观察到DNA扩增。我们的引物扩增CD基因序列在天然存在的烃降解细菌分离的地下水污染的汽油。PCR扩增的检测极限为每ml 10 −2个大肠杆菌（pAW 313）细胞。将先前表征的烃降解菌恶臭假单胞菌OU 83用作阳性对照。以汽油污染的地下水样品为模板，直接提取DNA，验证了PCR扩增检测环境样品中CD特异基因型的有效性。从6个汽油污染地下水样品中的5个样品中扩增出CD基因特异性DNA片段。以汽油污染地下水直接提取的DNA为模板，经限制性内切酶酶切和30 bp内探针Southern杂交（ICP 313）鉴定了PCR扩增DNA的同一性和特异性。基于这项研究中提出的证据，我们报告的第一次，有用的PCR扩增检测的CD基因序列在汽油污染的地下水。
Abstract Polymerase chain reaction amplification was used to detect catechol 2,3-dioxygenase (CD) gene sequences in the native bacterial populations present in gasoline contaminated groundwater samples. The primers used for the PCR assay were selected from the DNA sequences of the conserved region of the CD gene of xylE, nahH, and pAW313. A 30 bp DNA sequence (ICP313) internal to the structural gene of CD was used as a probe to identify the amplified DNA fragment. The identity of the amplified DNA fragment was confirmed by digesting the DNA fragment with Stu I and Bg I restriction enzymes which produced DNA fragments of expected sizes. The specificity of the PCR assay was tested using DNA isolated from a variety of bacterial isolates which include Escherichia coli ATCC 25922, Enterobacter faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 29213, Micrococcus roseus ATCC 516, Serratia marcescens ATCC 60, Klebsiella pneumoniae ATCC 13883, Chryseobacterium gleum ATCC 29896, Chromatium vinosum ATCC 17899, Comamonas testosteroni ATCC 11996, Methylosinus trichosporium OB3b, Bacillus subtilis ATCC 21697, Alcaligenes eutrophus ATCC 17698, Arthrobacter globiniformis ATCC 35698, Pseudomonas putida mt-2 ATCC 33015, Acinetobacter calcoaceticus ATCC 31012, and Pseudomonas putida ATCC 17484. Amplification of DNA was not observed in bacterial isolates lacking CD activity. Our primers amplified CD gene sequences in naturally occurring hydrocarbon degrading bacteria isolated from groundwater contaminated with gasoline. The detection limit of the PCR amplification was 10 −2 cells of Escherichia coli (pAW313) per ml. A previously characterized hydrocarbon degrader Pseudomonas putida OU83 was used as a positive control. The usefulness of PCR amplification for the detection of CD specific genotypes in the environmental samples was verified using DNA directly extracted from gasoline contaminated groundwater samples as a template. A CD gene specific DNA fragment was amplified from 5 out of 6 gasoline contaminated groundwater samples. The identity and specificity of the PCR amplified DNA generated using the DNA directly extracted from the gasoline contaminated groundwater as template was confirmed by restriction enzyme digestion and Southern hybridization using the 30 bp internal probe (ICP313). Based on the evidence presented in this study, we report for the first time, usefulness of PCR amplification for the detection of CD gene sequences in gasoline contaminated groundwater.