Development and validation of a multiplex reverse transcription PCR assay for simultaneous detection of three papaya viruses.

Decai Tuo,Peng Zhou,Xiaoying Li,Yong Yang,Pu Yan,Wentao Shen

doi:10.3390/v6103893

Abstract

Papaya ringspot virus (PRSV), Papaya leaf distortion mosaic virus (PLDMV), and Papaya mosaic virus (PapMV) produce similar symptoms in papaya. Each threatens commercial production of papaya on Hainan Island, China. In this study, a multiplex reverse transcription PCR assay was developed to detect simultaneously these three viruses by screening combinations of mixed primer pairs and optimizing the multiplex RT-PCR reaction conditions. A mixture of three specific primer pairs was used to amplify three distinct fragments of 613 bp from the P3 gene of PRSV, 355 bp from the CP gene of PLDMV, and 205 bp from the CP gene of PapMV, demonstrating the assay’s specificity. The sensitivity of the multiplex RT-PCR was evaluated by showing plasmids containing each of the viral target genes with 1.44 × 103, 1.79 × 103, and 1.91 × 102 copies for the three viruses could be detected successfully. The multiplex RT-PCR was applied successfully for detection of three viruses from 341 field samples collected from 18 counties of Hainan Island, China. Rates of single infections were 186/341 (54.5%), 93/341 (27.3%), and 3/341 (0.9%), for PRSV, PLDMV, and PapMV, respectively; 59/341 (17.3%) of the samples were co-infected with PRSV and PLDMV, which is the first time being reported in Hainan Island. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting multiple viruses in papaya and can be used for routine molecular diagnosis and epidemiological studies in papaya.

Highlights

Papaya (Carica papaya L.) is an important fruit crop grown widely in tropical and subtropical regions [1]
PRSV613-F/R + PLDMV355-F/R + PapMV205-F/R gave more clear and specific bands of target products compared to the rest combinations, and the primer combination was selected for further optimization in multiplex reverse transcription-polymerase chain reaction (RT-PCR) (Figure 1, Lane 10)
The uniplex and multiplex PCR and revere transcription (RT)-PCR protocols were standardized for the simultaneous detection of Papaya ringspot virus (PRSV), Papaya leaf distortion mosaic virus (PLDMV), and Papaya mosaic virus (PapMV), both as single and as mixed infections in papaya

Summary

Introduction

Papaya (Carica papaya L.) is an important fruit crop grown widely in tropical and subtropical regions [1]. Several viruses pose a serious threat to papaya production These include potyviruses, such as Papaya ringspot virus (PRSV) [2], Papaya leaf distortion mosaic virus (PLDMV). Among the known papaya viruses, PRSV, PLDMV, and PapMV have been detected on Hainan Island, China [5,14,15]. PRSV is considered the most widespread and destructive disease damaging papaya production in China [15]. PapMV has been considered of minor importance because it is rarely found in the field [17,18] These three viruses cause similar symptoms in papaya, such as mosaic, yellow-green leaf discoloration and distortion on leaves, water-soaking streaks on petioles, and ring-spots on fruits, making it difficult to distinguish among PLDMV, PRSV, and PapMV without further testing. To make rapid diagnoses and limit disease spreading, it is necessary to develop an effective and rapid detection method for these viral infections in papaya

Methods

Results

Conclusion