The Establishment of PMA-qPCR for Detecting Bacterial Angular Leaf Spot on Cucumber and Its Preliminary Application

doi:10.13560/j.cnki.biotech.bull.1985.2016.02.009

Abstract

Abstract: Bacterial angular leaf spot on cucumber is a common bacterial disease for cucumber, and its causative pathogen is Pseudomonas syringae pv.lachrymans.Currently this disease has caused a worldwide serious economic loss. Duo to the feature of PMA dyes that may distinguish whether the bacterial cells are alive or dead, PMA-qPCR was established by combining it with fluorescence quantitative PCR(qPCR)technology.(1)We compared the sequences of gene gap1 in different strains of the bacteria in GenBank, and found out the conservative area of gene gap1, and designed a pair of specific primers Dxf1 and Dxr1 for this bacterium according to the sequences of conservative area of gene gap1.Then we applied the qPCR method to do amplification using the genome of 5 non-Pseudomonas syringae pv.Lachrymans as template, and only P. syringae pv.lachrymans showed the positive result, proving that primers had specificity.(2)We used the gene gap1 as the target gene to construct a clone vector that was inserted into the competent cells of Escherichia coli.Then the E.coli cells were cultured to have a lot of copies.Using the extracted plasmids as the standard sample, and it was diluted to seven gradients of 5×10¹-5×10⁷ and then drew a standard curve.The amplification efficiency obtained from the standard curve was 96.6%, and the coefficients of variation both within groups and between groups were less than 2%, showing a fine repeatability of the standard curve.(3)We applied the PMA-qPCR method to detect the actual samples and gained a Ct value of 27.99.Based the linear correlation between initial template from the standard curve and Ct values, we measured 7.69×10² copies viable cells per 100 mg sick leaves.This study showed that PMA-qPCR method could quickly identify and quantify the number of living bacteria in actual samples, and provided a technical support for the prevention and control of bacterial angular leaf spot on cucumber.

Key words: bacterial angular leaf spot on cucumber, PMA-qPCR, Pseudomonas syringae, detection, viable cell

KONG Wei-wen, LI Yun-long, WANG Jing-qi, LIU Ting-ting, CHEN Nan, JIN Yi, HE Xiao-qing. The Establishment of PMA-qPCR for Detecting Bacterial Angular Leaf Spot on Cucumber and Its Preliminary Application[J]. Biotechnology Bulletin, 2016, 32(2): 70-75.

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