| Tobacco bacterial wilt disease,caused by Ralstonia solanacearum, is destructive to tobacco throughout the world. R. solanacearum is a kind of soil-borne pathogen, which is widespread in tropical, subtropical and warm temperate regions of the world and no effective methods are found to control the disease now. R. solanacearum, existing in soil along with the diseased plant debris, becomes the mainly infection source of plant bacterial wilt disease. R. solanacearum entries into plants mostly by injured roots, and moves rapidly in the vascular bundles, then results in plants to wither even die in a short time. Since now there are no effective bactericides and resistant varieties and it is very difficult to control tobacco bacterial wilt disease, quickly and exactly detecting the number of R. solanacearum in its latent form in soil is an urgent measure to prevent the development of the tobacco bacterial wilt disease.The traditional detection methods have many disadvantages such as long detecting cycle, complicated operation and low sensitivity, which do not meet the requirement of the rapid disease-detection. With the development of molecular biological technology, Polymerase Chain Reaction (PCR) has been used for detection of pathogenic microorganism extensively because of its speedability, specificity and sensitivity. In this research, we successfully established the conventional PCR and Real-time PCR (RTi-PCR) for quantitative analysis of R. solanacearum in the soil, which has been developed into fast detection kit primarily.In this study, the specific primer pair R.sol1/R.sol2 based on the unique nucleotide sequence of R. solanacearum was designed. The PCR condition was optimized and the detection system was developed with 331bp of amplification product from R. solanacearum. The system has many advantages including better specificity and higher sensitivity, and the detecting sensitivity was up to 100 fg/μL. The result showed that the system can be applied for detecting R. solanacearum in soil.The extraction method of soil genomic DNA was modified and improved, which can eliminate the PCR inhibitor substances, such as humane acid, phenolics, heavy metal etc. The protocol utilised to extract genomic DNA suitable for PCR amplification from soil enabled the extraction of total nucleic acids in a 2.5 h period.The Real-time PCR detection system for quantitative assay of tobacco bacterial wilt in soil was developed. Similarly, the primer pair R.sol1/R.sol2 was used to optimize and establish the SYBR Green I RTi-PCR system. TaqMan probe R.sol-probe and the primer pair R.sol1/R.sol2 were used to develop the TaqMan probe RTi-PCR system. In two systems above, RTi-PCR standard curve was constructed with a series of 10-fold dilutions of recombinant plasmid with the 331 bp fragment from R. solanacearum, and the detection limit was 1.2 fg/μL, which was higher in sensitivity than conventional PCR detection system. The two systems could be applied to detect the number of R. solanacearum in soil quantitatively and accurately and the disease can be diagnosed early.In this study, 35 soil samples coming from tobacco-cultivated fields in different areas of Chongqing including Pengshui, Qianjiang and Youyang were detected with the two systems. The results illustrated that the systems we have established were stable and reliable.The stable detection kit for R. solanacearum was developed primarily by the vacuum freeze-drying technology and stabilizing agents for nucleic acid and enzyme. The kit may be kept and transported in room temperature, and it is simple to operate. The detection kit is helpful to realize the speedability detect of R. solanacearum in soil.
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