Location: Plant Genetic Resources Conservation Unit
Project Number: 6046-21000-012-016-A
Project Type: Cooperative Agreement
Start Date: May 15, 2020
End Date: Apr 30, 2022
Objective:
Objective 1. Development of nucleic acid-based detection tool for Peanut Clump Virus (PCV) and Indian Peanut Clump Virus (IPCV).
Sub-objective 1a: Obtain PCV/IPCV isolated from infected peanut leaf tissues from cooperators in Senegal and India.
Sub-objective 1b: Develop and standardize Reverse Transcriptome Polymerase Chain Reaction (RT-PCR) and Quantitative Reverse Transcriptome Polymerase Chain Reaction (qRT-PCR) protocol for detection of PCV/IPCV.
Sub-objective 1c: Development of conventional RT-PCR based detection of PCV/IPCV.
Approach:
Sub-objective 1a: Develop and standardize RT-PCR and qRT-PCR protocol for detection of PCV/IPCV.
To pursue the RT-PCR assays, coat protein (CP) and movement protein (MP) genes of PCV/IPCV will be artificially synthesized and an in vitro-transcript will be prepared. The gene sequences will be downloaded from the public database and most conserved gene sequence will be identified for artificial gene synthesis. Primers specific to the CP and MP gene will be designed based on the selected conserved PCV/IPCV sequences. Internal primers will be designed for qRT-PCR. The synthesized products will be used for the development of one-step RT-PCR and qRT-PCR protocol and sensitivity will be determined.
Sub-objective 1b: Obtain PCV/IPCV isolates from infected peanut leaf tissues from cooperators in Senegal and India.
In order to generate reliable and precise detection techniques, different isolates of PCV/IPCV will be collected from Senegal and India with collaborators. PCV/IPCV symptomatic samples will be collected from fields and lyophilized for future applications. Lyophilized samples and total RNA from infected samples will ship to the USA under APHIS permit. The diagnostic assay will be developed by the Co-PI in his Virology Laboratory, the University of Georgia, Tifton.
Sub-objective 1c: Development of conventional RT-PCR based detection of PCV/IPCV.
RT-PCR protocol will be optimized using total RNA from Indian PCV/IPCV isolates. Total RNA will be extracted from lyophilized samples using Trizol or plant RNA extraction kit and quality & quantity will be determined using Nanodrop. The amplified product of CP and MP will be cloned and sequenced for further confirmation.
The outcome of this research provides a new nucleic acid based molecular diagnostic tool for routine quarantine testing to detect PCV/IPCV and help protect the US peanut industry from these two seed-transmitted viruses. A possible pitfall of the proposed research may be that the viability of viral RNA in purified RNA and lyophilized leaf tissue could get degraded during the transportation from the source to the virology laboratory in the US. Numerous samples and replications will be processed to minimize the chances of degradation of virus RNA.
All of the protocols including primer sequences for detection of PCV/IPCV will be published for accessibility to interested researchers world-wide.
