Rapid Detection Of Four Important Phytopathogenic Bacteria In Leguminosae Using Padlock Probes-based Liquichip

With the rapid growth of China’s imports of Leguminosae seeds, the risk of quarantine bacteria carried on seeds also was increased, which seriously impact on the production of beans industry. The objective of this study was to develop a rapid, high-throughput and multiplex detection of Pseudomonas savastanoi pv. phaseolicola (PSPH), Pseudomonas syringae pv. pisi (PSPI), Curtobacterium flaccumfaciens pv. flaccumfaciens (CFF), and Xanthomonas campestris pv. phaseoli(XCP) using padlock probes and liquichip, which should be an alternative to the traditional detection methods of individual bacteria, and resolve of invasive alien pests risk management and prevention and control and other problems, the major conclusions are as follows:1) This was the first time to design the padlock probes of PSPH, PSPI, CFF, and XCP, then optimize the conditions of ligation, digestion reaction, rolling circle expansion increase reaction system, and develop 4 singlet circle amplification detection assays for the PSPH, PSPI, CFF, and XCP, respectively. We also assess their specificity and sensitivity. The results show that the 4 singlet circle amplification detection assays could high-specifically detect the corresponding specific target bacteria, other negative control strains were unable to check the detection. For sensitivity of the PSPI, PSPH, CFF singlet rolling circle amplification system are the same as that of conventional PCR,600 fg/μL, 600fg/μL and 4.34pg/μL, respectively; and the sensitivity of XCP rolling circle amplification is ten fold higher than that of conventional PCR, which is 38.3fg/μL,2) We firstly established high-throughput and multiplex detection of Pseudomonas savastanoi pv. phaseolicola, Pseudomonas syringae pv. pisi, Curtobacterium flaccumfaciens pv. flaccumfaciens, and Xanthomonas campestris pv. phaseoli using padlock probes and liquichip, which achieve the same reaction system of rapid simultaneous detection of four kinds of important high-throughput detection of bacteria, and to assess the specificity, sensitivity and stability of the detection system. The results showed this quadruple assay could specifically detect 100% of target pathogens, detecting pea bacterial blight bacteria DNA and sensitivity of the bacterial suspension were 4.85 pg/μL and 1.6×104 cfu/ml, detection bean Phytophthora halo bacteria DNA and sensitivity of the bacterial suspension was 874 fg/μL and 1.5×10 cfu/ml, detection bean common bacterial blight bacteria DNA and sensitivity of the bacterial suspension were 486fg/μL and 2.3×103cfu/ml, detection of bacterial bean wilt pathogen DNA and the sensitivity of the bacterial suspension were 1.06 pg/μL and 1.1 × 104 cfu/ml; retest intraclass coefficient of variation of less than 7.2%, with good repeatability.3) Assay sensitivity using extracts of healthy seeds spiked with cells of PSPI、PSPH、 CFF、XCP, conventional PCR can only detect 100 -fold dilution of Xanthomonas campestris pv. phaseoli, result of other dilution treatments and target bacterias were negative; bead-based multiplex suspension array can be simultaneously detected 100,101 times the processing of PSPI、PSPH、CFF、XCP in one reaction,,102 times in the process was detected cells of CFF、XCP, the results indicate that bead-based multiplex suspension array suitable for the actual contaminated seed testing than conventional PCR, and detection of higher sensitivity and efficiency.