| Bacteria and phytoplasma are important plant pathogens, some of them have not been found in china and are or should be regulated as quarantine organisms.Conventional methods for the categorization, detection and identification of bacteria are mainly based on studying on their culture characterization and host ranges, which are not so precise, and more, phytoplasma and some bacteria can't be detected and identified due to their growth difficulties on media. Recent developments on molecular biology by using conservation sequences of 16SrDNA have greatly promoted the detection and identification of the two categories of pathogens. Real-time fluorescent PCR(RTF-PCR) is one of the most advanced technology developed in recent years throughout the world, the PCR-based technique with a fluorescent-labeled probe within the PCR reaction solution, the probe hybridize specifically to the PCR amplified product and release fluorescent signal which is monitored during the amplification. RTF-PCR greatly increases its detection specificity and sensitivity and can be completed within 2 hrs. It has been widely used for detection of human pathogen and plant fungal pathogen in recent years, but few were reported of its application on plant phytoplasma and bacteria pathogen.Phytoplasma, Liberobacter aslaticitm, Xanthomanas oryzae pv.orgzae and xothomonas oryzae pv. oryzioola were chosen as targets for studying on this paper, because they are all quarantine important pest, reprehensive of different taxis of the prokaryote and difficult in conventional detection: hard up for culture, or not - cultivable. RTF-PCR framework for phytoplasma and bacteria used specific hybridization probe TaqMan, which are designed based on the single nucleotide mutation or single nucleotide polymorphism(SNP). The method has been applied for detection for samples and proved to be simple, rapid,sensitive,accurate. It provide a new tool for detection and identification of plant microbe pathogen, it is especially useful for quarantine department and agriculture production. Main results or conclusions of the research are as fellows:1. A improved screening system for molecular identification of prokaryotes is suggested. The universal phytoplasma and bacterium primers and probes were designed based the conserved region of 16SrDNA, and the species or group specific primers and probes for were designed on the region where no mutation was found within the species or group, but mutationdid find among species or group.2. According to 16SrDNA, 6 TaqMan probes were designed and synthesized for RTF-PCR detection, including 1 universal phytoplasma probe, 4 specific phytoplasma probes, 1 universal bacteria probe and 1 liberobacter prove. Patents for all these probes are pending.3. 16S rDNA, 23S rDNA and 16S-23S rDNA spacer region of xanthemonas oryzae pv. oryzae and xanthomonas oryzae pv. oryzicola are sequenced are no mutation could be identified. So putative siderophore receptor gene was sequenced and used to separate the two bacteria, and specific TaqMan probe Bai (for X.o. pv.oryzae) and Tiao probe (for X.o pv. oryzicola) were designed. Patents for all two probes are pending.4. 16S rDNA gene of Chinese Liberobacter asiaticum was cloned and sequenced, the resut showed that it is a new strain of Asian L.a. species. The gene has registered in Gene bank (Code: AY192576)5. 16S rDNA gene of Chinese Paulownia witches'-broom phytoplasma was also cloned and sequenced, and the results shows that it is a new strain of phytoplasma 16Sr I-D. The gene has registered also in Gene bank (Code: AY192577)6. Pear decline disease is a quarantine object in china. The diseased sample was intercepted from the imported material , its 16S rDNA gene was cloned and sequenced, is 99.72% homogenous with that registered in Genebank.7. Different RTF-PCR methods were established for phytoplasma, Liberobaeter asiaticum, and x.o.pv. oryzae and x.o.pv. oryzicola respectively . The results showed that: all probes designed are very specific and effective; the...
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