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Quantitative Real-Time Polymerase Chain Reaction Assay for Detection of Pectobacterium wasabiae Using YD Repeat Protein Gene-Based Primers

February 2012 , Volume 96 , Number  2
Pages  253 - 257

Myeong Ho Kim, Min Seok Cho, Byoung Kyu Kim, Hyeon Jin Choi, Jang Ho Hahn, ChangKug Kim, and Man Jung Kang, National Academy of Agricultural Science, Rural Development Administration, 441-707 Suwon, Republic of Korea; Seong Hwan Kim, Department of Microbiology, Dankook University, 330-714 Cheonan, Republic of Korea; and Dong Suk Park, National Academy of Agricultural Science, Rural Development Administration, Republic of Korea

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Accepted for publication 10 September 2011.

The aim of this study was to develop a quantitative polymerase chain reaction (qPCR) assay for specific detection of Pectobacterium wasabiae using a primer pair based on the YD repeat protein gene for amplification of a 140-bp DNA fragment from infected wasabi (Wasabia japonica), a member of the crucifer family. The soft rot caused by P. wasabiae is an emerging disease that is present in many wasabi-producing areas. However, specific and reliable methods for identifying the pathogen are not available. Therefore, a qPCR primer set for accurate diagnosis of P. wasabiae was developed from publically available genome sequences. A SYBR Green qPCR primer set was designed based on a YD repeat protein gene of P. wasabiae WPP163 because it is known that this gene is structurally diverse among species, pathovars, or subspecies. The specificity of the primer set was evaluated using genomic DNA from 5 isolates of P. wasabiae, 5 different species of Pectobacterium, and 16 other pathogenic reference bacteria. The primer set used in the PCR assay successfully amplified a 140-bp amplicon for all five P. wasabiae strains. No amplification was obtained from 29 other pathogenic bacteria. The assay was also able to detect at least two genomic DNA, or 3 CFU per reaction, when using calibrated cell suspension.

© 2012 The American Phytopathological Society