Degrange V, Bardin R
Laboratoire d'Ecologie Microbienne, URA Centre National de la Recherche Scientifique 1450, Université Claude Bernard Lyon, Villeurbanne, France.
Appl Environ Microbiol. 1995 Jun;61(6):2093-8. doi: 10.1128/aem.61.6.2093-2098.1995.
Although the biological conversion of nitrite to nitrate is a well-known process, studies of Nitrobacter populations are hindered by their physiological characteristics. This report describes a new method for detecting and counting Nitrobacter populations in situ with the PCR. Two primers from the 16S rRNA gene were used to generate a 397-bp fragment by amplification of Nitrobacter species DNA. No signal was detected from their phylogenetic neighbors or the common soil bacteria tested. Extraction and purification steps were optimized for minimal loss and maximal purity of soil DNA. The detection threshold and accuracy of the molecular method were determined from soil inoculated with 10, 10(2), or 10(3) Nitrobacter hamburgensis cells per g of soil. Counts were also done by the most-probable-number (MPN)-Griess and fluorescent antibody methods. PCR had a lower detection threshold (10(2) Nitrobacter cells per g of soil) than did the MPN-Griess or fluorescent antibody method. When PCR amplification was coupled with the MPN method, the counting rate reached 65 to 72% of inoculated Nitrobacter cells. Tested on nonsterile soil, this rapid procedure was proved efficient.
虽然亚硝酸盐向硝酸盐的生物转化是一个广为人知的过程，但硝化细菌种群的研究却因其生理特性而受到阻碍。本报告描述了一种利用聚合酶链反应（PCR）原位检测和计数硝化细菌种群的新方法。使用来自16S rRNA基因的两种引物，通过扩增硝化细菌物种的DNA来生成一个397 bp的片段。在所测试的其系统发育近缘种或常见土壤细菌中未检测到信号。针对土壤DNA的最小损失和最大纯度对提取和纯化步骤进行了优化。分子方法的检测阈值和准确性是通过向每克土壤接种10、10²或10³个汉堡硝化细菌细胞的土壤来确定的。计数也通过最可能数（MPN）-格里斯法和荧光抗体法进行。与MPN-格里斯法或荧光抗体法相比，PCR的检测阈值更低（每克土壤10²个硝化细菌细胞）。当PCR扩增与MPN方法相结合时，计数率达到接种硝化细菌细胞的65%至72%。在非无菌土壤上进行测试，证明了这种快速方法是有效的。
