DETECTION AND ENUMERATION OF BACTERIA IN SOIL BY DIRECT DNA EXTRACTION AND POLYMERASE CHAIN-REACTION
DETECTION AND ENUMERATION OF BACTERIA IN SOIL BY DIRECT DNA EXTRACTION AND POLYMERASE CHAIN-REACTION
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DOI：
10.1128/aem.58.9.2717-2722.1992
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发表时间：
1992-09-01
影响因子：
4.4
通讯作者：
SIMONET, P
中科院分区：
文献类型：
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作者：
PICARD, C;PONSONNET, C;SIMONET, P
为了建立一种快速、特异的土壤细菌检测方法，对聚合酶链反应（PCR）方法进行了改进。该方案的每个步骤，包括直接裂解细胞，DNA纯化和PCR扩增，进行了优化。为了提高裂解的效率，一个步骤，特别是对一些抵抗经典技术的微生物的关键，我们使用小的土壤样品（100毫克）和各种裂解处理，包括超声处理，微波加热，和热冲击。通过最多三个Elutip d柱实现核酸的纯化。最后，通过使用加强剂条件、较低变性温度和添加甲酰胺的双相方案优化PCR扩增。使用两种微生物作为模型：根癌土壤杆菌（Agrobacterium tumefaciens），其在所使用的土壤中天然不存在并且被接种以校准方案的有效性，和弗兰克氏菌属（Frankia spp.），一种土壤中的放线菌。用特异性引物对A. tumefaciens的16S核糖体基因的可变区或Frankia spp.对接种的A.当接种量为10（7）至10（3）个细胞时，常规获得根癌农杆菌菌株。此外，我们观察到的接种物的大小和PCR反应的结果之间的强相关性允许的协议的有效性进行评估，在土壤样品中存在的微生物细胞的数量计数。这使我们能够估计Frankia spp的土著人口。在0.2 × 10（5）个基因组（即，可扩增的靶序列）。
In order to develop a rapid and specific detection test for bacteria in soil, we improved a method based on the polymerase chain reaction (PCR). Each step of the protocol, including direct lysis of cells, DNA purification, and PCR amplification, was optimized. To increase the efficiency of lysis, a step particularly critical for some microorganisms which resist classical techniques, we used small soil samples (100 mg) and various lytic treatments, including sonication, microwave heating, and thermal shocks. Purification of nucleic acids was achieved by passage through up to three Elutip d columns. Finally, PCR amplifications were optimized via biphasic protocols using booster conditions, lower denaturation temperatures, and addition of formamide. Two microorganisms were used as models: Agrobacterium tumefaciens, which is naturally absent from the soil used and was inoculated to calibrate the validity of the protocol, and Frankia spp., an actinomycete indigenous to the soil used. Specific primers were characterized either in the plasmid-borne vir genes for A. tumefaciens or in the variable regions of the 16S ribosomal gene for Frankia spp. Specific detection of the inoculated A. tumefaciens strain was routinely obtained when inocula ranged from 10(7) to 10(3) cells. Moreover, the strong correlation we observed between the size of the inocula and the results of the PCR reactions permitted assessment of the validity of the protocol in enumerating the number of microbial cells present in a soil sample. This allowed us to estimate the indigenous population of Frankia spp. at 0.2 x 10(5) genomes (i.e., amplifiable target sequences) per g of soil.