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.
摘要：
印度柑橘环斑病毒（ICRSV），曼陀罗病毒属的成员，导致柑橘环斑病，影响金now橙的质量和产量。在植物出现可见症状之前，早期和准确的检测方法至关重要。在这项研究中,从感染的kinnow组织中扩增出507bp的部分外壳蛋白基因（pCPG）片段，克隆到pET28a载体中，并转化入大肠杆菌BL21(DE3)细胞。用IPTG诱导，细胞过表达约23kDa的重组部分外壳蛋白（rpCP），使用Ni-NTA树脂通过亲和层析纯化。用抗His抗体在westernblot中验证，rpCP用于在兔中产生ICRSV特异性多克隆抗体(PAb)。PAb,在1:1000稀释度优化，通过DAC-ELISA和ICRT-PCR方法成功检测了感染的金now橙叶提取物中的ICRSV。在高达1:640和1:10240(w/v，gmL-1）通过DAC-ELISA和IC-RT-PCR，分别。还优化了一步法RT-PCR检测方法,通过从金now橙叶提取的总RNA中扩增507bp的pCPG片段来确认ICRSV的存在，稀释度高达1:5120(w/v，gmL-1)。结果表明，IC-RT-PCR的灵敏度比DAC-ELISA和一步RT-PCR测定法高16倍和2倍。
