New Detection Technology For Xanthomonos Axonopodis Pv. Citri Based On Padlock Probe

Citrus bacterial canker Disease (CBCD), caused by Xanthomonas axonopodis pv. citri(Xac), is a serious disease of most citrus species and cultivars in many citrus-producing areas in the world. For the lack of resistant cultivars and specific chemicals, many measures have to be adopted to control CBCD for a long time, for example, enhanced construction of non-quarantine area and phytosanitary system against the spread of Xac, eradication and mass destruction methods for infected plants. Fast and robust methods for diagnostic and detective purpose are necessary needed to support the monitoring of epidemic situation of non-quarantine area and customhouse detection system for citrus materials.A lot of methods have been used to detect plant pathogen, for instance visual method, pathogen cultivation and pathogenicity detection, phage biologically assay, enzyme-linked immune-sorbent assay (ELISA), dot immune-binding assay (DIA), DNA-bases hybridization, genomic fingerprints, polymerase chain reaction(PCR). The application of PCR and real-time PCR, specially, provides for the detection and quarantine of plant pathogen a more rapid, more precise, more sensitive and more convenient method. All these methods, except PCR, however, can only detect single target. Though duplex PCR and even mulriple PCR, which are difficult to be optimized, can be used to detect multiplex targets, the competition and restrain among the different primer pairs influence directly the specificity and stability of the detection.Padlock probes are single-strand oligonucleotides with complementary ends to adjacent target sequences. The link sequence of the padlock probe is not necessary for target detection and so can be used for the binding region of universal primers. The special structure of the padlock probe, which can be amplified and detected by many means, thus meets the need of specificity and multianalysis of detection.Objective: Develop for Xanthomonas axonopodis pv. citri (Xac) a bran new rolling circle amplification detection system by using padlock probe and rolling circle amplification technique. With Tilletia controversa Kühn (TCK) as reference strain, The detection system based on padlock probe and reverse dot blot hybridization is established, which has the expectation of providing new customhouse detection means for plant quarantine and allowing multiplex detection of different targets in the future due to the great specificity and sensitivity of the detection system. Methods: Padlock probe for Xanthomonas axonopodis pv. citri (Xac) and Tilletia controversa Kühn (TCK) was designed respectively based on the sequence of the unique hypothetic protein gene in complete genome of Xac and TCK, and amplification primers ware designed according to the universal linking sequence of padlock probe; the ligation system and procedure, the digest system and rolling circle amplification system were all optimized, and then RCA detection method for Xac, whose specificity and sensitivity were tested compared with conventional PCR, was developed; Optimized the process of sample preparation, and practicability and stability of RCA method was then tested by detecting the samples collected from field; Xac and TCK were detected simultaneously using reverse dot blot hybridization based on padlock probe and ligase-dependent PCR; Specificity and sensitivity of the system were tested.Experiment Results: Detection system of rolling circle amplification (RCA) for Xac was established and optimized based on the padlock probe designed. Results show that the system can detect Xac and its DNA specifically, while other plant pathogens and bacteria attached on the surface of citrus leaves can not be detected, which is accordant with conventional PCR. This indicates the detection system has great specificity. The detection sensitivity of RCA is 20 cfu/μL for Xac pathogens and 10.8 fg/μL for cloned DNA fragment, which is a little higher than the sensitivity of conventional PCR. Inhibitory factors are removed effectively by improving the process of sample preparation and then leaf samples collected from orange orchards were detected with both RCA and conventional PCR. The result shows that the Xac positive percentage is no striking dissimilarity between the two methods (P>0.01). The specificity of padlock probe for TCK was also tested with both RCA and conventional PCR and the result shows great coherence between two methods, indicating non-false detection. Cloned plasmid of the unique hypothetic protein gene of Xac and(or) TCK can be detected by reverse dot blot hybridization based on padlock probe and ligase-dependent PCR and the detection sensitivity for Xac target mixed with TCK target is 1.08 fg/μL, which provides the feasibility of establishing the system for multiplex detection.