Detection Of Three Latent Viruses In Pear Plants By RT-PCR And RT-LAMP Methods

In commercially cultivated pear trees, Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) are the most commonly occurred viral pathogens. These viruses are also important pathogens in apple. The mixed infections of two or more viruses are very common in cultivated pear plants, and can cause significant reduction in fruit quality and yields. Until now, no insect vector for these viruses has been found. The use of healthy propagation materials and plants is a very effective way for controlling the viral diseases. Therefore, establishment of a sensitive, reliable, fast and inexpensive detection methods for these viruses is of critical importance for the certification and quarantine programs of pear plants and germplasm. However, The high divergence of viruses, together with the inference of high contents of polysaccharide and polyphenol components in pear tissues, has impeded strongly the detection efficiency of pear viruses by RT-PCR methods.This study developed a multiplex RT-PCR assay for the simultaneous and efficient detection of three economically important viruses ACLSV, ASPV and ASGV infecting pear plants. Three primer pairs were used to amplify their fragments of247bp,358bp and500bp, respectively.The optimized mRT-PCR was evaluated by detecting those viruses in different tissues of pear and apple plants, and proved to be stable and sensitive.The sensitivity and specificity of the mRT-PCR assay for the three viruses were comparable to that of each uniplex RT-PCR. In conclusion, this mRT-PCR provides a useful tool for the routine and rapid detection and the differentiation of three pear viruses.Multiplex RT-PCR based on nested PCR technique was established, the nested-multiplex RT-PCR amplification method could detect the three latent virues in pear plants. Primers were designed in this study, M4as nested outer primer was designed in the Poly (A), nested multiplex RT-PCR inner primers were the same with multiplex-PCR has been established. Two sets of primers for the template has twice amplification which could greatly improve the efficiency of detection. The sensitivity is 105times of multiplex RT-PCR. This method has been applied in the virus-free seedlings rootstocks and achieved good results.ASGV detection method of loop-mediated isothermal amplification (LAMP) with high sensitivity has been established. Four LAMP primers were designed using PrimerExplorer software, and LAMP reaction system of dNTP concentration, Mg2+concentration and reaction temperature were optimized. The established LAMP has high specificity and the sensitivity of this method is1000times of RT-PCR. The reaction product of white precipitate of magnesium pyrophosphate could successfully observed by naked eye.