| The pathogen of Citrus HuangLongBing (HLB) is phloem-limited bacteria genus Candidatus Liberibacters, which was previousely reported difficult for in vitro cultivation. It's spreaded by Diaphorina citri and the scion.The only method to control the disease is strengthening the quarantine measures, nurtureing and promoting the use of non-viral seedlings, and early detecting or eradicating of diseased plants. The effective implementation of these control methods must coordinate with a fast, accurate and practical detection method of HLB bacterium.The rolling circle amplification system for HLB based on padlock probe was developed on the basis of PCR technique. It has more advantages in specificity and sensitivity. Padlock probe is unique single-long-chain oligonucleotides, constituting with the sequences that complementary with target sequence and the link sequence as two parts. The sequences that complementary with target sequence are at both ends of the probe, the link sequence is in the middle. The specificity of padlock probe is decided by the sequences at both ends, and only when the sequences at both ends is fully complement with the target sequences, the padlock probe can connect into a loop with the role of the ligase. When there is no target sequences or not fully complementary, the padlock probes only exist at the linear form. Because of the unique design, padlock probe can be applied to a variety of amplification methods and multi-target detection.In this study, padlock probe was designed according to the specific sequence of Candidatus Liberobacters. The rolling circle amplification primers were designed on the basis of link sequence. The study established the rolling circle amplification system of HLB by optimizing the link system and procedures, exonuclease digestion system and procedures, and rolling circle amplification system. As compared with conventional PCR, rolling circle amplification system only amplify the Candidatus Liberibacter, other pathogens, such as citrus tristeza virus (CTV), citrus exocortis viroid (CEVd) are not amplified. The result of specificity is the same with PCR. Comparing with conventional PCR, rolling circle amplification technology has a higher sensitivity. The range of concentration of positive by using rolling circle amplification detection system is from 100 pg/μl to 100 fg/μl, the least concentration can be detected out was 100 fg/μl. The conventional PCR detection system can detect the concentration of positive bacteria from 100 pg/μl to 1 pg/μl, the least concentration can be detected out was 1 pg/μl. The rolling circle amplification system for the detection of citrus Huanglongbing bacterium established in this paper has good specificity and sensitivity, which provided a new molecular detection technology for dynamic monitoring and control of HLB in our country.Expect for HLB, citrus tristeza virus and citrus exocortis viroid are also important pathogens of citrus, which were often mixed occurred on citus. In order to achieve rapid and accurate detection of these pathogens, on the basis of standard RT-PCR of HLB, CTV and CEVd, we established a multiple RT-PCR detection system of HLB and CTV, and then optimized the multiplex RT-PCR detection system of CTV, HLB, and CEVd. Because there are many factors affect the multiple RT-PCR, in this study we optimized many aspects of the multiple RT-PCR, such as the selection of primers, amplification system, and amplification protocol and so on. In order to achieve the best amplification results, when establishing the CTV, HLB multiple RT-PCR system, we mainly optimized the major factor-annealing temperature, and confirmed the sensitivity by practice use.10 samples out of totally 20 citrus samples confirmed to be mixed infected. In the established CTV, HLB, CEVd multiplex RT-PCR detection system, we mainly optimized the combination of the primer's concentration.
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