Rapid and sensitive detection by PCR of mpcI gene in the rhizosphere

Abstract

A simple and sensitive detection system, using polymerase chain reaction (PCR) and a soil microcosm, was developed to detect a bacterial catabolic gene in the rhizosphere. The inoculated population of Alcaligenes eutrophus JMP134, a phenol and 2,4-dichlorophenoxy acetic acid utilizer, was readily detected by this technique, which permitted taking of samples from specific locations of root (including rhizosphere) and soil. The number of JMP134 viable cells (102–103 cells), typically picked up by the nitrocellulose filter strip method, yielded sufficient amount of the target DNA to be detected by PCR. Primers encoding metapyrocatechase I (MPC I; catechol 2,3-dioxygenase) enabled the discrimination of at least five viable cells of JMP134 among the indigenous microorganisms inhabiting bush bean roots. This simplified PCR detection procedure facilitated monitoring of the specific degradative gene in the rhizosphere in only 5 h.