The Study Of Molecular Methods In Tomato Ringspot Virus And Genically Modified Organisms Detection

Tomato ring spot virus (ToRSV) , which result in serious loss in many crops, is distributed extensively in America and Europe,such as the United States,France and Russia .ToRSV has not been found in China by now and is an important quarantine plant virus (on the Al list).A pair of oligonucleotide primers corresponding to the ToRSV RNA1 replicase region was synthesized and was successfully used to amplify the target region of the grape isolate. The amplified fragment was cloned into the vector of PMD18-T then sequenced. The sequence is 449nt in length and shows 85% homologous to the grape isolate deposited in the GeneBank. RT-PCR is a fast and sensitive method for ToRSV detection, but non-detectable and false positive result is often occurred. A novel hybridization capture RT-PCR-ELISA(HC-RT-PCR-ELISA) was developed based on the above mentioned RT-PCR, The main contributions to the development of the method are that a solid primer is successfully bound to PCR tube wall specially for aimed RNA capture in order to raise RNA purification and reduce time consumption. DIG labeled probe hybridization with solid PCR product was performed as well as electrophoresis of liquid product to reduce false positive result.Compare to routine RT-PCR, HC-RT-PCR-ELISA is a more sensitive (about 10 times to RT-PCR) besides unpurified nucleotide requirement, lOug infected plant organism can be detected by solid hybridization. Due to the step of hybridization was added the false positive results reduced .On the base of the HC-RT-PCR-ELISA. hybridization capture real time fluorescent PCR(HC-RT-F-PCR) was established to detect ToRSV. A Taqman probe labeled by a quenching and fluorescent was added to the PCR reaction buffer. More and more fluorescent signal can be collected with the PCR reaction carry on. The second method is more automatized and much less time consumption (only 3 hours from nucleotide hybridization capture to result found). Themost important is that contamination from PCR product can be easily avoided.The two methods, HC-RT-PCR-ELISA and HC-RT-F-PCR, were successfully used to detect the virus from the imported grape from France. In four grapes ToRSV was detected from six ones, the same result was achieved with the two methods. The former is also used to detect squash maize virus (SqMV) and genomically modified organisms (GMOs),In the detection of SqMV, an another pair of primers were synthesized for Nest PCR in order to validate the results of HC-RT-PCR-ELISA. But in the detection of GMOs, The capture nucleotide is much longer than that in ToRSV and SqMV.