恶臭假单胞菌PCR检测方法的建立及应用
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摘要
以恶臭假单胞菌(ACCC100085)为材料,采用假单胞菌显色培养基,对恶臭假单胞菌进行菌株分离,并依据《伯杰氏细菌鉴定手册》,对分离菌株进行生理生化学鉴定。根据GeneBank数据库恶臭假单胞菌CarA基因序列设计引物,建立恶臭假单胞菌PCR检测方法。该方法能扩增恶臭假单胞菌161 bp特异性片段,对于恶臭假单胞菌纯培养物基因组DNA的最低检出限为23.6×10~(-3)μg/mL,对2010年以来49批次入境环保菌剂样品进行检测,阳性率为100%,与传统细菌分离检测结果完全相符。该方法具有较高的灵敏度和特异性,能检测入境环保菌剂中恶臭假单胞菌。
With Pseudomonas putida(ACCC100085)as material, Pseudomonas putida were isolated from CHROMagar?Pseudomonas. Based on Bergey's Manual of Bacterial Identification, isolated strains were carried on the physiological-chemical identification. According to the CarA gene of Pseudomonas putida from GeneBank, a pair of primers were designed to establish a PCR assay. Specific PCR products of 161 bp for Pseudomonas putida were amplified by the assay. Simultaneously,the detection limit Genomic DNA of the single PCR was 23.6×10~(-3)μg/mL. Detecting 49 samples entry microbial blends Pseudomonas putida in the environmental protection since 2010 by single PCR,the positive rate was 100%,and the results were identical with the results by conventional microbiology method. The method of the single PCR could detect entry microbial blends Pseudomonas putida in the environmental protection with high sensitivity and specificity.
With Pseudomonas putida(ACCC100085)as material, Pseudomonas putida were isolated from CHROMagar?Pseudomonas. Based on Bergey's Manual of Bacterial Identification, isolated strains were carried on the physiological-chemical identification. According to the CarA gene of Pseudomonas putida from GeneBank, a pair of primers were designed to establish a PCR assay. Specific PCR products of 161 bp for Pseudomonas putida were amplified by the assay. Simultaneously,the detection limit Genomic DNA of the single PCR was 23.6×10~(-3)μg/mL. Detecting 49 samples entry microbial blends Pseudomonas putida in the environmental protection since 2010 by single PCR,the positive rate was 100%,and the results were identical with the results by conventional microbiology method. The method of the single PCR could detect entry microbial blends Pseudomonas putida in the environmental protection with high sensitivity and specificity.
引文
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[3] L Barth Reller, Melvin P, Weinstein. Detection and identification of microorganisms by gene amplification and sequencing[J]. Medical Microbiology, 2007,44(8):1108-1114.
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[8]文娅.一种新型环保微生物菌剂的研制及处理污水的初步研究[D].北京:北京林业大学,2012.Wen Ya. Studies on a New-combined Environment Microbial Agent and Its Effects on Treating Polluted Water[D].Beijing:Beijing Forestry University, 2012.
[9]进出口环保用微生物菌剂环境安全管理办法[J].司法业务文选,2010(26):13-19.Import and export of environmental security measures for the administration of environmental protection with microbial agents[J]. Selected Works of Judicial Business, 2010(26):13-19.
[10]维护环境安全从源头加强进出口环保用微生物菌剂管理[N].中国环境报,2010-05-11.Maintain Environmental Safety from the Source in Order to Strengthen the Management of Import and Export of Environmental Protection with Microbial Agents[N]. China Environment News, 2010-05-11.
[11] R E布坎南, N E吉本斯.伯杰细菌鉴定手册[M].第八版.北京:科学出版社, 1984:279-280.Re Buchanan, Ne Gibbons. Bergey’s Manual of Determinative Bacteriology[M]. 8th Edition. Beijing:Science Press,1984:279-280.
[12]吴兴海,杨伟克.利用PCR技术快速检测玉米细菌性枯萎病菌[J].甘肃农业大学学报, 2007,42(5):88-91.Wu Xinghai, Yang Weike. Application of PCR method in rapid identification of Pantoea stewartii[J]. Journal of Gansu Agricultural University, 2007,42(5):88-91.