A Simplified Multiplex PCR Assay for Simultaneous Detection of Six Viruses Infecting Diverse Chilli Species in India and Its Application in Field Diagnosis
Abstract:Chilli is infected by at least 65 viruses globally, with a mixed infection of multiple viruses leading to severe losses being a common occurrence. A simple diagnostic procedure that can identify multiple viruses at once is required to track their spread, initiate management measures and manage them using virus-free planting supplies. The present study, for the first time, reports a simplified and robust multiplex PCR (mPCR) assay for the simultaneous detection of five RNA viruses, capsicum chlorosis orthotospo… Show more
“…sustainable development of the pepper industry. In pepper, previous studies have developed multiplex RT-PCR protocols that could detect two or more viruses simultaneously in one reaction such as ChiVMV and CMV(Priyoda et al, 2018) or CMV, TMV, PMMoV, PVY, and tomato spotted wilt virus (TSWV)(Nemes & Salánki, 2020) or capsicum chlorosis orthotospovirus (CaCV), ChiVMV, large cardamom chirke virus (LCCV), CMV, PMMoV, and chilli leaf curl virus (ChiLCV)(Priyoda et al, 2022). Note that those multiplex RT-PCR methods did not include PVMV.…”
Viral diseases cause severe losses commonly and become one of main global limiting factors in pepper production. Chilli veinal mottle virus (ChiVMV), pepper veinal mottle virus (PVMV), cucumber mosaic virus (CMV), and pepper mild mottle virus (PMMoV) are the dominant viruses damaging pepper in South China. To detect these viruses efficiently, a developed multiplex reverse transcription polymerase chain reaction (RT-PCR) method was established. Four pairs of specific primers were used to amplify a 990 bp product for PMMoV, a 923 bp product for ChiVMV, a 823 bp product for PVMV, and a 682 bp product for CMV. The optimal primer concentration, the optimal reaction annealing temperature and cycle number were determined. The developed multiplex RT-PCR could detect PMMoV, ChiVMV, CMV and PVMV from cDNA diluted up to 10–4, 10–4, 10–3 and 10–3, respectively. Then it was sucessfully used to detect virus infection from eight mixed pepper leaf samples. The result were basically consistent with that of monoplex RT-PCR. Additionally, seventy-five diseased samples collected from nine major pepper growing regions in Guangxi were analyzed by the developed multiplex RT-PCR. The results showed that most of samples were coinfected by two or more viruses, and ChiVMV and PVMV had relatively higher total detection rates of 65.33% and 60.00% respectivlely. The results also indicated that the four viruses infecting pepper weren't evenly distributed in Guangxi. In a conclusion, the developed multiplex RT-PCR here will be a efficient tool for routine molecular diagnosis of PMMoV, ChiVMV, PVMV and CMV.
“…sustainable development of the pepper industry. In pepper, previous studies have developed multiplex RT-PCR protocols that could detect two or more viruses simultaneously in one reaction such as ChiVMV and CMV(Priyoda et al, 2018) or CMV, TMV, PMMoV, PVY, and tomato spotted wilt virus (TSWV)(Nemes & Salánki, 2020) or capsicum chlorosis orthotospovirus (CaCV), ChiVMV, large cardamom chirke virus (LCCV), CMV, PMMoV, and chilli leaf curl virus (ChiLCV)(Priyoda et al, 2022). Note that those multiplex RT-PCR methods did not include PVMV.…”
Viral diseases cause severe losses commonly and become one of main global limiting factors in pepper production. Chilli veinal mottle virus (ChiVMV), pepper veinal mottle virus (PVMV), cucumber mosaic virus (CMV), and pepper mild mottle virus (PMMoV) are the dominant viruses damaging pepper in South China. To detect these viruses efficiently, a developed multiplex reverse transcription polymerase chain reaction (RT-PCR) method was established. Four pairs of specific primers were used to amplify a 990 bp product for PMMoV, a 923 bp product for ChiVMV, a 823 bp product for PVMV, and a 682 bp product for CMV. The optimal primer concentration, the optimal reaction annealing temperature and cycle number were determined. The developed multiplex RT-PCR could detect PMMoV, ChiVMV, CMV and PVMV from cDNA diluted up to 10–4, 10–4, 10–3 and 10–3, respectively. Then it was sucessfully used to detect virus infection from eight mixed pepper leaf samples. The result were basically consistent with that of monoplex RT-PCR. Additionally, seventy-five diseased samples collected from nine major pepper growing regions in Guangxi were analyzed by the developed multiplex RT-PCR. The results showed that most of samples were coinfected by two or more viruses, and ChiVMV and PVMV had relatively higher total detection rates of 65.33% and 60.00% respectivlely. The results also indicated that the four viruses infecting pepper weren't evenly distributed in Guangxi. In a conclusion, the developed multiplex RT-PCR here will be a efficient tool for routine molecular diagnosis of PMMoV, ChiVMV, PVMV and CMV.
Accurate and timely diagnosis of plant viral infections plays a key role in effective disease control and maintaining agricultural productivity. Recent advances in the diagnosis of plant viruses have significantly expanded our ability to detect and monitor viral pathogens in agricultural crops. This review discusses the latest advances in diagnostic technologies, including both traditional methods and the latest innovations. Conventional methods such as enzyme-linked immunosorbent assay and DNA amplification-based assays remain widely used due to their reliability and accuracy. However, diagnostics such as next-generation sequencing and CRISPR-based detection offer faster, more sensitive and specific virus detection. The review highlights the main advantages and limitations of detection systems used in plant viral diagnostics including conventional methods, biosensor technologies and advanced sequence-based techniques. In addition, it also discusses the effectiveness of commercially available diagnostic tools and challenges facing modern diagnostic techniques as well as future directions for improving informed disease management strategies. Understanding the main features of available diagnostic methodologies would enable stakeholders to choose optimal management strategies against viral threats and ensure global food security.
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