Establishment and Application of TaqMan Fluorescence Quantitative PCR Detection Method for Dickeya dadantii
SHEN Xiao-Ling1,2, YI Jian-Ping3, WANG Rong-Zhou4, QIAN Jun-Ting3, LI Yan-Min4, YAO Hai-Feng5, LOU Bing-Gan1,2,*
1 Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China; 2 Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Hangzhou 310058, China; 3 Shanghai Entry-Exit Inspection and Quarantine Bureau, Shanghai 200135, China; 4 Zhejiang Provincial Bureau of Plant Protection and Quarantine, Hangzhou 310020, China; 5 Hangzhou Lin'an Agricultural Technology Extension Center, Lin'an 311300, China
Abstract：Bacterial stem and root rot caused by Dickeya dadantii is proved to be a serious disease, which is harmful to sweet potato (Ipomoea batatas) production. It can damage the stems, leaves, petioles and seed tubers, infecting and forming brown water-stained lesions. Now, the disease occurs in Zhejiang, Guangdong, Heibei, Henan and other provinces. Long-distance transportation of the seed tuber and germchit are the main way to spread the disease to the disease-free zone. Therefore, strict quarantine measures are the key to ensure healthy production in disease-free areas.An accurate, sensitive and rapid detection method is a powerful tool to guarantee strictly quarantining and insurance for researching on the occurrence of disease epidemics. Compared with closely related species, the flagellin protein gene (fliC) sequence of D. dadantii encoding the flagellin with high specificity which makes it to be a suitable target for molecular detection. With the fliC gene as the target, specific primers and TaqMan probes were designed, thus the method for fluorescence quantitative PCR detection (TaqMan qRT-PCR) of D. dadantii was established in this study. The method showed good specificity against D. dadantii based on the test results, and it could be effectively distinguished from bacteria of different species in the same genus and different genera. The method had high sensitivity and the detection sensitivity of the bacterial DNA was up to 280 fg/μL, which was 1 000 times of that of routine PCR. The detection sensitivity of bacterial liquid was up to 2 CFU/μL (CFU: colony forming units), the sensitivity was 100 times higher than the routine PCR detection method. Using NGM (nematode growth medium) to isolate suspected sweet potato stem and root rot samples, it was found that D. dadantii could form dark brown to blue colonies on the medium, which could significantly improve the efficiency of separation and purification of pathogenic bacteria. The actual samples were tested on 70 sweet potato and soil samples using the constructed TaqMan qRT-PCR method, routine PCR method and separation culture method based on NGM medium. Results showed that the positive detection rates of TaqMan qRT-PCR, routine PCR, and isolation culture method were 91.4%, 77.1%, and 71.4%, respectively. TaqMan qRT-PCR detection was more accurate, and the detection time was shortened from 96 h to 1.5 h. It could be seen that the quantitative PCR detection system based on TaqMan probe established in this study coul d be effectively used for rapid detection of suspected samples of sweet potato stem and root rot, diseased bodies, field soil, seed tuber and seedlings, and provided a fast and accurate molecular detection method of sweet potato stem and root rot for inspection and quarantine department, which was of great significance for effectively controlling the spread of the disease. This method can also be used for the quantitative detection of the pathogenic bacteria of sweet potato stem and root rot in suspect samples, providing technical support for studying the relationship between the population of pathogenic bacteria and disease severity in soil.
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