Development Of Real-time Reverse Transcription Polymerase Chain Reaction (RT-qPCR) For Detection Of3Pineapple Mealybug Wilt-associated Viruses

The Pineapple Mealybug Wilt Associated Virus(PMWaV) are a complex of positive single-stranded RNA viruses classified in the genus Ampelovirus, family Closteroviridae. Currently, there are five knowed virus isolated from pineapple plant all over the word. Mealybug wilt of pineapple(MWP) causes by PMWaV-2infecting and mealybug feeding is one of the most seriously disease and result in great yield impact. It had been reported that the incidence of MWP would up to60%and result in25-30%economic loss in Chian. Leading by college of Hawaii, the research of PMWaVs about morphological characteristics and classification, genome organization and detection methods had made great progress. Although scholars of domestic and overseas have established methods of Electron microscopy, ELISA, TBIA and general RT-PCR for PMWaVs detection, it’s rare to see the reasearch of detection PMWaVs by Real-Time Fluorescent Quantitative RT-PCR. In addition, the multiple Real-Time Fluorescent Quantitative RT-PCR for simultaneous detection kinds of PMWaVs has not been report worldwide. In the present study, in view the present of PMWaV-1,2,3in China, we collected pianapple sample with MWP form Institute of Tropical Fruit Trees, Hainan Academy of Agricultural Science and Fushan, Chengmai, Hainan. And then TaqMan probes and primers were designed and synthesized according to conserved sequence for the coat protein(CP) gene, respectively. Standard curves were established by recombinant plasmid preparation and the methods of single and triple Real-Time Fluorescent Quantitative RT-PCR for PMWaV-1,2,3have been development and optimization.The detailed procedures are described as follow:1) TaqMan probe and primers were designed and synthesized according to conserved sequence for the coat protein(CP) gene of PMWaV-1, and a method for detection and quantification of PMWaV-1was established and optimized in current study. The standard curve of the method was7=-3.307*log(x)+38.18, R2=0.998with the amplification efficiency of100.6%. It revealed that present method would detect the PMWaV-1specifically and the sensitivity was showing10times compare to the general RT-PCR, with LOD of4copies. This system provided an easy, specific, sensitive and good repeatability method for PMWaV-1detection and quantification. The result of samples detection showed that the PMWaV-1titres in older leaves of pineapple was higher than the PMWaV-1titres in the younger leaves, and the PMWaV-1titres in sucker was lower than leaves in pineapple plant.2)This study established a Real-Time Fluorescent Quantitative RT-PCR method with TaqMan probe based on specific primers of conserved nucleotide sequence of PMWaV-2coat protein gene. The standard curve of the method was F=-3.267*log(x)+43.33, R2=0.997with the amplification efficiency of102.3%. The results showed that the method could detect PMWaV-2specifically, and the sensitivity of Real-Time Fluorescent Quantitative RT-PCR is about10times higher than regular PCR, indicating a simple, specificity, high sensitivity, and reliable reproducibility detection method to PMWaV-2.3)TaqMan probe and primers were designd and synthesized according to conserved sequence for the CP gene of PMWaV-3, and a Real-Time Fluorescent Quantitative RT-PCR method for detecting PMWaV-3was established and optimized. The standard curve of the method was7=-3.160*log(x)+38.04, R2=0.992with the amplification efficiency of107.2%. The reasearch showed that the method could detection PMWaV-3specifically with LOD of303copies, indicating a simple, specificity, high sensitivity, and reliable reproducibility detection method to PMWaV-3.4)Three-times repeats experiment revealed that bBoth the variation coefficient of intra-assay and inter-assay for the single Real-Time Fluorescent Quantitative RT-PCR method were less than1.98%， indicating a simple, specificity, high sensitivity, and reliable reproducibility detection method for three PMWaVs.5)Specific primers and TaqMans with different fluorescent labels was designed and synthesized according to conserved sequence for the CP gene of PMWaV-1,2,3, respectively. And a method for simultaneous detection PMWaV-1,2,3had been develped and optimized. The standard curves of triple Real-Time Fluorescent Quantitative RT-PCR were7=-3.283*log(x)+39.02, R2=0.999with the amplification efficiency of101.7%(PMWaV-l),7=-3.393*log(x)+37.53, R2=0.998with the amplification efficiency of97.1%(PMWaV-2),7=-3.171*log(x)+38.48, R2=0.996with the amplification efficiency of106.7%(PMWaV-3), respectively. This implies that plasmid standard of three viruses could be amplificatied stably and shows a good linear relationship. Repeats experiment datas showed that standard deviation was below0.26and variable coefficient was below1.44%. Moreover, the teiple Real-Time Fluorescent Quantitative RT-PCR method could detection PMWaV-1,2,3as low as990copies,980copies and868copies, respectively. This system provided a fast, sensitive and sably method for simultaneous detection and quantification PMWaV-1,2,3.