1. Optimization of DAC-ELISA and IC-RT-PCR using the developed polyclonal antibody and one-step RT-PCR assays for detection of Indian citrus ringspot virus in kinnow orange of Punjab, India.
- Author
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Angira A, Baranwal VK, Ranjan A, and Choudhary N
- Subjects
- India, Rabbits, Animals, Reverse Transcriptase Polymerase Chain Reaction methods, Plant Leaves virology, Capsid Proteins genetics, Capsid Proteins immunology, Citrus virology, Flexiviridae genetics, Flexiviridae isolation & purification, Flexiviridae immunology, Citrus sinensis virology, Enzyme-Linked Immunosorbent Assay methods, Plant Diseases virology, Antibodies, Viral, Sensitivity and Specificity
- Abstract
Indian citrus ringspot virus (ICRSV), a member of the Mandarivirus genus, causes citrus ringspot disease, impacting kinnow orange quality and yield. Early and accurate detection methods are crucial before visible symptoms manifest in plants. In this study, a 507 bp partial coat protein gene (pCPG) segment was amplified from infected kinnow leaf tissues, cloned into a pET28a vector, and transformed into E. coli BL21(DE3) cells. Induced with IPTG, the cells overexpressed a recombinant partial coat protein (rpCP) of approximately 23 kDa, purified using Ni-NTA resin via affinity chromatography. Validated in western blot with an anti-His antibody, rpCP was used to generate an ICRSV-specific polyclonal antibody (PAb) in rabbits. PAb, optimized at 1:1000 dilution, successfully detected ICRSV in infected kinnow orange leaf extracts via DAC-ELISA and IC-RT-PCR assays. ICRSV was detectable in sample dilutions up to 1:640 and 1:10240 (w/v, g mL
-1 ) by DAC-ELISA and IC-RT-PCR, respectively. One-step RT-PCR assays were also optimized, confirming the presence of ICRSV by amplifying a 507 bp pCPG fragment from total RNA extracted from kinnow orange leaves, with dilution up to 1:5120 (w/v, g mL-1 ). The result demonstrated that IC-RT-PCR has a 16-fold and 2-fold higher sensitivity than DAC-ELISA and one-step RT-PCR assays., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier B.V. All rights reserved.)- Published
- 2024
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