| Tilletia indica Mitra (TIM) is one of the most serious fungal diseases which cause Karnal bunt of wheat. This fungal disease not only leads to the severe crop production losses, but also affects the quality of the flour. It is important quarantine pathogen both in China and abroad. Tilletia walkeri Castlebury (TW) parasitize in ryegrass, it is the most important similar specie of Tilletia indica Mitra. Both morphological and molecular characters are extremely similar to TIM. In addition, TW often appearance in wheat seed result in great interference of the identification of TIM. There are no reports that have been found about TIM and TW in China. With increasing of the wheat entrance, the entry and transmission risk of both smuts will be increased. It is significance to establish simply, quickly, exactly detection method to preclude the entry of those pathogens.There are many identification methods for TIM and TW, for example: morphology identification, molecular detection and so on. The molecular detection based on PCR is sensitivity, specially, simply and quickly detection method for inspection and quarantine work. PCR technology can be used to multiplex detection, but multiplex PCR have one mortal restriction that the interference in different primers and probes in one reaction tube. This restriction result in degrade of detection stability and accurate. More and more molecular detection methods have been used to institution of standard detection methods because of its superiority. But the short research of standard reference materials seriously restricted the use of those standard methods. Padlock probe is a single strand oligonucleotide with special structure. The 5′and 3′terminal of padlock probe are used to hybridize with target sequences, and the link sequence in the middle of padlock probe can be used to universal primers design. It is especially appropriate for multiplex detection and can effectually avoid the problem of multiplex PCR. Therefore, two specificity padlock probes had been designed for TIM and TW, and two rolling circle amplification methods had been built for TIM and TW. Based on the padlock probes and the rolling circle amplification methods a multiplex Gene chip method had been researched and built. In the same time, the padlock probes had been designed for Tilletia controversa Kune (TCK), Tilletia tritici Wolff (TCT) and Tilletia laevis Kühn (TLa). Combine those padlock probes with rolling circle amplification methods a multiplex Gene chip method had been researched for five target detection.The detection sensitivity of rolling circle amplification is 9pg/μL for TIM standard molecule, 0.245ng/μL for TIM DNA, and 14.5pg/μL for TW standard molecule, 0.255ng/μL for TW DNA. The multiplex Gene chip method can specifically detect TIM and TW. The detection sensitivity of multiplex Gene chip method is 24.5pg/μL for TIM DNA and 2.55pg/μL for TW DNA.Two standard molecules were constructed by molecular clone technology of TIM and TW. The standard molecule for TIM contained two DNA fragments, one about 2297bp DNA sequence from mitochondrial DNA, one ITS sequence about 710bp and the sequencing result show highly homology with sequence of TIM from GenBank (>99.5%). The standard molecule for TW contained two DNA fragments, one about 2.3kb DNA sequence from mitochondrial DNA, one ITS sequence about 710bp and the sequencing result show highly homology with sequence of TW from GenBank (>99.8%). The performance detection of two standard molecules shows good homogeneity and specificity, specially, the detection result show good stability after long time keep in room condition.To our knowledge, this is the first reported application of rolling circle amplification to test of TIM and TW. These detection methods researchs provide the specificity and exactly new molecular diagnosis tools for import of wheat, and have quarantine significance for protecting wheat growth in China.
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