Development and Application of the Quantitative Detection of Papaya ringspot potyvirus Based on Real-Time RT-PCR Technique
Date Issued
2006
Date
2006
Author(s)
Lee, Yi-Shia
DOI
zh-TW
Abstract
Papaya ringspot is one of the most destructive diseases of papaya and it is a limiting factor for papaya industry. This disease first occurred in Taiwan in 1975, and it has spreading widely throughout all of papaya-cultivated areas in Taiwan. It is caused by Papaya ringspot virus (PRSV), which belongs to Potyvirus, Potyviridae. According to the different symptoms in papaya hosts, PRSV was divided into three major strains including SM (severe mottling), DF (severe mottling with leaf-deformation) and SMN (severe mottling with necrosis and quick decline) strains. SM was a predominant strain of PRSV in the field of Taiwan several years ago. However, DF and SMN have recently become newly rising dominant strains. Almost full-length of genomic sequences of three PRSV strains were determined in the previous study. Continuous sequencing was conducted in this study. The complete nucleotide sequences (10326 bases) of three strains were determined by application of 5’ RACE and 3’ RACE systems. They encode a polyprotein of 3344 amino acids with a 5’ untranslated region of 85 nucleotides and a 3’ untranslated region of 209 nucleotides. The results of alignment of full genomic nucleotide sequences demonstrated that SM is 97.3% identical to DF, and SMN is 97.0% and 96.8% identical to SM and DF. Most genes in the PRSV genome among the three strains are 95~100% homologous, but the P1 gene and 5’ UTR region are about 95% and 92% homologous among three different strains, respectively. Phylogenic analysis of various international PRSV isolates revealed that SM, DF and SMN are close to the Yung-Kang isolate (another PRSV isolate from Taiwan), but far away from the Thailand and Hawaii isolates. The rapid assay based on reverse transcription-polymerase chain reaction (RT-PCR) with the primer pair PRSV-857 was developed for the detection of PRSV in previous study. Another primer pair PRSV-829 was devised in our study, and it has been proven to achieve better specificity and sensitivity in PRSV detections. In addition, the real-time RT-PCR technology was applied for quantitative monitoring of different PRSV strains in papaya hosts. Both “SYBR Green” and “TaqMan primer/probe” methods were adopted to develop the quantitative detection of PRSV with real-time RT-PCR, and “TaqMan primer/probe” method obtained more satisfactory results. A TaqMan primer/probe combination (named TP-PRSV), selected from the conserved regions of coat protein gene, was designed for the common detections of PRSV. Three primer/probe kits (named TP-DF, TP-SM, and TP-SMN), selected from the variable regions of P1 gene, were also developed for the strain-specific quantitative detection of the DF, SM and SMN strain, respectively. Our devised real-time RT-PCR assays were further applied to monitor the virus multiplicative dynamics in papaya hosts in the inoculation tests with different PRSV strains. When the papaya hosts (cultivar FR) were inoculated with the DF strain, PRSV could be detected 10 days after inoculation. The replication curve (DF) was increasing linearly from the 10th to 16th day, stationary from the 16th to 22th day, and reaching to the peak 24 days after inoculation. When the papayas were inoculated with the SMN strain, PRSV could be detected 10 days after inoculation. The replication curve (SMN) was increasing linearly from the 10th to 18th day, and reaching to the peak 18 days after inoculation. When the papayas were inoculated with both DF and SMN strains, DF could be detected 16 days after inoculation. The replication curve (DF) was increasing linearly from the 16th to 22th day, and reaching to the peak 22 days after inoculation. On the other hand, SMN could be detected 18 days after inoculation. The replication curve (SMN) was increasing linearly from the 18th to 22th day, and reaching to the peak 22 days after inoculation. The development of real-time RT-PCR assays of PRSV is helpful to the ecological studies of papaya ringspot disease.
Subjects
木瓜輪點病毒
定量偵測技術
Papaya ringspot potyvirus
real-time RT-PCR
Type
other
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