Research And Application Of High-throughput Detection Techniques Based On Padlock Probe For Imported Tomato Pathogenic Bacteria

Tomato is one of the important economic vegetable in Chinese vegetable.Thediseases in tomato are more than40kinds,including three important bacterial species,namely, tomato bacterial spot disease (Pseudomonas syringae pv.tomato), tomatobacterial canker (Clavibacter michiganensis subsp. michiganensis) and tomatobacterial wilt (Ralstonia solanacearum race1). Cmm and Pst, which have occurredlocally in our country and caused serious economic losses, are quarantine pests ofEuropean and Mediterranean Plant Protection Orgnization (EPPO), and are alsochinese quarantine bacteria. In recent years, with the introduction of foreign excellenttomato seeds, the risk of tomato diseases introducing into our country has beenincreased. It is necessary for the entry of seeds and other propagating material toapply high throughput multi-pathogen detection technology.In this assay, Cmm，Pst and Rs1were selected as the research bacteria, accordingto the specific gene fragments of Cmm, Pst and Rs1obtained by Dreier, Fanelli, andLee, respectively, the T1and T2arms of their padlock probes were designed. And, theuniversal primers, hybridization probes and TaqMAN fluorescent probes were alsodesigned, according to the design principles of padlock probes. Cmm，Pst and Rs1were tested with four methods, which were the method of hyperbranched rolling circleamplification (HRCA), reverse dot-blot hybridization based on padlock probe,real-time PCR based on padlock probe,and DNA chip based on padlock probe. On thebasis of these studies, tomato samples intercepted in Fujian ports were tested forhigh-throughput detection.1, The detection technology of HCRA for Cmm, Pst and Rs1were established,and its specificity and sensitivity were also tested. The results showed that:(1) inHCRA optimization system, when using Taq DNA ligase, and using thermal cyclingconnection method, a good amplification effect could be got；the connection effectwas the best when final concentration of padlock probe was about8pmol/L；theamplification effect was the best at62℃；when using a10U exonuclease Ⅰ and 5Uexonuclease Ⅲ to digest the uncyclized padlock, the impact of linear padlockprobe could be removed effectively.(2) the specificity of HRCA was very high, onlythe target bacteria that could be detected specifically, while, the other strains weretested negative, the specificity of this method was compared with the conventionalPCR method, the result showed that the specificities of two methods were consistent.(3) the sensitivity of HRCA was very high, the minimum concentration of DNAdetected was500fg/μL, which was10-fold higher than that of conventional PCR.2, The high-throughpu detection technology for Cmm, Pst and Rs1wasestablished, using method of reverse dot-blot hybridization based on padlock probe.The results showed that:(1) multiple pathogens could be detected by this method inthe same reaction system.(2) the method of reverse dot-blot hybridization based onpadlock probe had high specificity, only the specific target bacteria that could bedetected from the test strains, the other strains tested negative.(3) the detectionsensitivity of reverse dot-blot hybridization based on padlock probe was about5pg/μL, which was coincident with that of conventional PCR.3, The detection technology of real-time PCR based on padlock probe for Cmm,Pst and Rs1was established, respectively.The results showed that:(1) Cmm, Pst andRs1could be detected from the tested strains by the method of real-time PCR basedon padlock probe, respectively.(2) the method of real-time PCR based on padlockprobe had high specificity, only the specific target bacteria that could be detected fromthe test strains, the other strains tested negative.(3) the detection sensitivity of thismethod was very high, the minimum concentration of detected DNA was50fg/μL,which was100-fold higher than that of conventional PCR.4, The detection method of DNA chip based on padlock probe was established.The results showed that:(1) Cmm, Pst and Rs1could be detected from the10testedstrains by this method, respectively.(2) the method of DNA chip based on padlockprobe had high specificity, only the specific target bacteria that could be detected fromthe test strains, the other strains tested negative.(3) the detection sensitivity of thismethod was high, the minimum concentration of DNA was about500fg/μL, whichwas10-fold higher than that of conventional PCR.(4) the target bacteria could bedetected from the tested bacteria by this method when padlock probes were linked between mixed lock probes and mixed DNA, the assay of DNA chip based on padlockprobe was specific and sensitive, and was suitable for high-throughput detection ofpathogens, it would be an effective tool for detecting Cmm, Pst and Rs1in entry-exitphytosanitary.5, In application detection for tomato samples, the simulated tomato seedscontaminated by Cmm were tested by the method of HRCA, and45collected sampleswere tested by the method of reverse dot-blot hybridization based on padlock probe,real-time PCR based on padlock probe, and DNA chip based on padlock probe,respectively. The results showed that:(1) Cmm could be detected by the method ofHRCA from the simulated tomato seeds contaminated by Cmm, which could beinferred that the contaminated tomato seeds could be detected and the simulationdetection method had the practical significance.(2) in the samples detection for CMMwith the method of real-time PCR based on padlock probe,5samples tested positivefrom45samples, which were2samples from Japan(Jap1214, Jap1102),2samplesfrom Yongtai(Yongt1001, Yongt1002) and1sample from Minqing (Minq1001), theother strains were tested negative.(3) in the detection for Cmm,Pst and Rs1with themethod of reverse dot-blot hybridization based on padlock probe,5Cmm samples,5Pst samples and8Rs1samples tested positive from the45samples, the other samplestested negative.(4) in the detection for Cmm,Pst and Rs1with the method of DNAchip based on padlock probe,5Cmm samples,5Pst samples and8Rs1samplestested positive from the45samples, the other samples tested negative.(5) tomatosamples could be detected with three methods, which were the real-time PCR, reversedot-blot hybridization and DNA chip based on padlock probe, and the test results bythe three methods were identical, testing and verifying mutually the detectionreliability.