Development Of A Loop-mediated Isothermal Amplification Assay For Detection Of Aphelenchoides Besseyi And Aphelenchoides Ritzemabosi

Rice white tip nematode, Aphelenchoides besseyi(Christie, 1942) and chrysanthemum foliar nematode, Aphelenchoides ritzemabosi(Schwartz, 1911) Steiner & Buhrer, 1932 are important plant pathogenic nematodes which can harm many crops and they are plant quarantine pests in some countries or regions.Nematode of Aphelenchoides are similar from morphological which are identification difficult, complicated operation, time-consuming, high professional level of detection and identification requirements for personnel level with traditional morphological identification methods. So it is necessary to establish molecular methods to identify these two species of nematodes.Loop-mediated isothermal amplification(LAMP) is a DNA amplification method with advantages of high efficience, test results visible and no need of special equipment. The present study based A. besseyi and A. ritzemabosi 18 S ribosomal RNA gene are designed for these two kinds of nematodes LAMP specific primers, and using these two LAMP primers establishment with a high specificity, stability, sensitivity, energy and a practical A. besseyi and A. ritzemabosi LAMP detection system, the results are as follows. Designed A. besseyi LAMP primer B77 t and A. ritzemabosi LAMP primer R81 t, B77 t including two external primer,two inner primer and two loop primer, R81 t include two external primer, two inner primer and one loop primer; Establishment LAMP detection systems for A. besseyi and A. ritzemabosi and reaction results using calcein dye based on primer B77 t and primer R81 t. LAMP system of these two nematodes can detect specific nematodes from different orders, different families, different genera and belong to different kinds of A. besseyi and A. ritzemabosi, respectively, stable detection in identification of different populations of the same species of nematodes as well as individuals at different developmental instars of A. besseyi and A. ritzemabosi, detection sensitivity respectively 1/1000 and 1/100 single nematode DNA; In addition, these two LAMP system, the success of the direct object of the step can be omitted nematode isolated from the host plant is detected in the mixed sample types and plant tissue samples to achieve simple, accurate and rapid identification purposes.Identification and early diagnosis of this study A. besseyi and A. ritzemabosi provides a new method for rapid detection and accurate, sensitive, stable, the reaction results visible and practical advantages. Of Inspection and Quarantine, the rapid detection and identification of transporting quarantine and origin monitoring A. besseyi and A. ritzemabosi provides a good way to prevent these two kinds of plant pathogenic nematode dissemination of proliferation.