Rapid detection and identification of the bacterium Pantoea stewartii in maize by TaqMan^® real-time PCR assay targeting the cpsD gene

Authors: Tambong, J.T.¹; Mwange, K.N.¹; Bergeron, M.²; Ding, T.²; Mandy, F.²; Reid, L.M.³; Zhu, X.³

Source: Journal of Applied Microbiology, Volume 104, Number 5, May 2008 , pp. 1525-1537(13)

Buy & download fulltext article:

You have access to the full text article on a website external to ingentaconnect.

Please click here to view this article on Wiley Online Library.

You may be required to register and activate access on Wiley Online Library before you can obtain the full text. If you have any queries please visit Wiley Online Library

Abstract:

Aims:

The development and evaluation of a sensitive and specific TaqMan^® real-time polymerase chain reaction (PCR) for the detection and identification of Pantoea stewartii on maize. Methods and Results:

A TaqMan^®-based real-time PCR assay targeting the cpsD gene enabling specific detection of P. stewartii in maize leaves and seeds was developed. Under optimal conditions, the selected primers and probe were specific for the detection of all 14 reference P. stewartii strains by real-time PCR. The 32 non-Panteoa and eight other Pantoea strains tested negative. The TaqMan^® PCR assay detected 1 pg of purified DNA and 10⁴P. stewartii colony forming units per millilitre (10 cells per reaction) in pure cultures consisting of 92·0% intact (viable) cells. Direct processing of leaf lesions and seeds by the real-time PCR detected 10 and 50 P. stewartii cells per reaction respectively. TaqMan^® real-time PCR results were validated by dilution plating of macerates and PCR-based subcloning followed by DNA sequencing. Conclusions:

The real-time PCR assay described is a rapid, reliable and more sensitive tool for the detection of P. stewartii. Significance and Impact of the study:

This real-time PCR assay would avoid false-negative results and reduce the time required for certifying maize seed shipments.

Keywords: corn; Erwinia stewartii; flow cytometry; Stewart's wilt; Zea mays

Document Type: Research article

DOI: http://dx.doi.org/10.1111/j.1365-2672.2007.03674.x

Affiliations: 1: Environmental Health Program (Biodiversity), Agriculture and Agri-Food Canada, Ottawa, ON, Canada 2: National HIV Immunology Laboratory, Public Health Agency, Centre for Infectious Disease Prevention and Control, Tunney's Pasture, Ottawa, ON, Canada 3: BioProducts and BioProcesses, Agriculture and Agri-Food Canada, Ottawa, ON, Canada

Publication date: 2008-05-01