Utilization Of Real-time PCR For Establishing Detection System Of Cereal Cyst Nematode

Cereal cyst nematode is one of the most important worldwide soil-borne disease in wheat production, which is prevalent greater in wheat belts around China currently, causing serious problems about the securities of grain production in China. Heterodera avenae group contains 12 effective strains, since this disease reported in Hubei in 1989, two strains identified as Heterodera avenae and H.filipjevi in China. Since pathogenecity of different type of Cereal cyst nematode has significant difference, so cyst nematode detection and species identification play an important role in crop production. This study focused on Cereal cyst nematode using the molecule detection technology, in order to establish a kind of rapid, sensitive, and convenient method to detect Cereal cyst nematode. The discoveries of this study are the followings.According to analyzing ribosome DNA of H.avenae、H.filipjevi and H.glycine, three specific TaqMan-MGB probes and a pair of universal primer were designed based on partially ITS1-5.8S-1TS2 sequences of ribosome DNA about these three cyst nematode. With three specific probes and the universal primers, we established single real-time PCR detection system about H.avenae、H.filipjevi and H.glycine individually, and dual detection system of H.avenae and H.filipjevi. In order to make both the single and dual detection systems achieving best condition, we optimized the concentration of probes, primers and Taq DNA polymerase, screening the annealing temperature. Additionally, a better specificity, high sensitivity and effective target gene amplification were also required in our detection systems, the minimum detection limit was 1000 copies and the amplification efficiency stay between 0.95-1.05.Selected one best method about soil total DNA extraction from five ones, with pre-processing all the soil samples before DNA extraction, it could become easier to extract or purified the DNA of nematode in the soil stably, meanwhile this method was available in detecting the nematode in the soil rapidly.In this thesis, two standards were prepared described as plasmid DNA and nematode genome DNA, and we also tried to set up the standard of nematode genome DNA in soil. The plasmid sample was diluted to 1011～103 copies, and the real-time PCR three single detection results indicated that the linearity was good (R2=0.996、0.997、0.998), gene amplification was effective (E=1.02、1.02、1.00). Moreover, nematode genome DNA was diluted as gradient with a certain concentration of nematode DNA sample verified, and the detection results illustrated that amplification was associated with the sample concentration, the correlation coefficient was reach to 0.9181. In addition, standard culves of nematode genome DNA in soil was explored with the two methods, but failed to create effective standard curve.34 Cereal cyst nematode samples collected from Henan province were detected by qRT-PCR. The results indicated 23 cyst nematode samples from Xuchang, Yuzhou, Sanguo identified as H.avenae, which proportion was 67.7% of all samples; 8 cyst nematode samples from Xuchang, Lingjing identified as H.fllipjevi, which proportion was 23.5% of all samples; whereas 3 cyst nematode samples from Taichen Town of Linying County in City Luohe^ Nandun Town of Xiangcheng County and Pingdian Villiage of Shangshui County in Zhoukou identified tow types of Cereal cyst nematode, which proportion was 8.8% of all samples. By testing the mixed sample, we found that in the nematode samples from Taichen Town of Linying County in City Luohe, tow types of nematode almost at the same proportion, with H.avenae was a bit more than the H.filipjevi; however, in the nematode samples from Nandun Town of Xiangcheng County and Pingdian Villiage of Shangshui County in Zhoukou, H.avenae was much more than the H.filipjevi.