Establishment of multiplex immunocapture PCR for detection of banana streak virus

LU Yongsi; LIU Runpei; RAO Xueqin

doi:10.13300/j.cnki.hnlkxb.2025.01.018

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Establishment of multiplex immunocapture PCR for detection of banana streak virus

Plant protection | 更新时间：2026-11-05

- Establishment of multiplex immunocapture PCR for detection of banana streak virus
- Journal of Huazhong Agricultural University Vol. 44, Issue 1, Pages: 168-173(2025)
- 作者机构：
  
  华南农业大学植物保护学院/广东省微生物信号与病害防治重点实验室，广州510642
- 作者简介：
- 基金信息：
- DOI：10.13300/j.cnki.hnlkxb.2025.01.018
  CLC： S432.4
- Received：04 January 2024，
  
  Published：15 January 2025
- 稿件说明：
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卢咏思，刘润沛，饶雪琴.香蕉线条病毒多重免疫捕获PCR方法的建立及应用［J］.华中农业大学学报，2025，44（1）：168‐173.

LU Yongsi,LIU Runpei,RAO Xueqin.Establishment of multiplex immunocapture PCR for detection of banana streak virus[J].Journal of Huazhong Agricultural University,2025,44(01):168-173.
卢咏思，刘润沛，饶雪琴.香蕉线条病毒多重免疫捕获PCR方法的建立及应用［J］.华中农业大学学报，2025，44（1）：168‐173. DOI： 10.13300/j.cnki.hnlkxb.2025.01.018.

LU Yongsi,LIU Runpei,RAO Xueqin.Establishment of multiplex immunocapture PCR for detection of banana streak virus[J].Journal of Huazhong Agricultural University,2025,44(01):168-173. DOI： 10.13300/j.cnki.hnlkxb.2025.01.018.

摘要

为提高复合感染香蕉的香蕉线条病毒（banana streak virus，BSV）的诊断效率，保障香蕉产业的健康发展，设计基于香蕉线条病毒OL（banana streak OL virus，BSOLV）、香蕉线条病毒GF（banana streak GF virus，BSGFV）的外壳蛋白（coat protein，CP）基因和香蕉线条病毒IM（banana streak IM virus，BSIMV）的RNase H基因保守序列的特异引物，利用BSV多克隆抗血清捕获病毒为模板，通过对引物浓度、退火温度等条件的优化，建立能够同步检测BSOLV、BSGFV、BSIMV的多重免疫捕获PCR方法。研究结果显示，建成的最优体系为BSOLV、BSGFV、BSIMV上下游引物终浓度（μmol/L）比例0.5∶0.5∶0.5，退火温度为63 ℃。利用该多重免疫捕获PCR方法可以从1 mg/mL的香蕉粗提液中分别检测出BSOLV、BSGFV和BSIMV。所建立的多重免疫捕获PCR方法与引起相似症状的黄瓜花叶病毒（cucumber mosaic virus， CMV）无交叉反应。对15份田间香蕉样品进行检测，能够明显区分出复合侵染的样品，经测序验证，BSOLV、BSGFV和BSIMV的扩增产物与GenBank相应病毒同源率分别在94%以上。表明该多重免疫捕获PCR方法具有较高的敏感性和特异性，可应用于香蕉种苗以及田间香蕉样品中3种BSV的检测。

Abstract

To improve the diagnostic efficiency of banana streak virus （BSV） and promote the healthy development of the banana industry，a multiplex immunocapture PCR method was established for the simultaneous detection of BSOLV，BSGFV，and BSIMV by optimizing primer concentration，annealing temperature，and other parameters. The specific primers were designed based on the conserved sequences of the coat protein （CP） gene from the banana streak OL virus （BSOLV） and banana streak GF virus （BSGFV），and the RNase H gene of banana streak IM virus （BSIMV）. The viruses captured by the BSV polyclonal antisera served as templates. The results indicated that the optimal multiplex immunocapture PCR used to detect BSOLV，BSGFV，and BSIMV was achieved with final concentration ratios of 0.5 μmol/L∶0.5 μmol/L∶0.5 μmol/L for the upstream and downstream primers of BSOLV，BSGFV，and BSIMV，respectively，with an annealing temperature of 63 °C. BSOLV，BSGFV，and BSIMV could be detected from a crude banana extract of 1 mg/mL by the multiplex immunocapture PCR method. There was no cross-reaction with the cucumber mosaic virus （CMV），which causes symptoms similar to those caused by BSV. Fifteen field samples were analyzed using the multiplex immunocapture PCR method. The results showed that the banana samples co-infected with BSV were distinguishable，and the amplification products of BSOLV，BSGFV，and BSIMV were confirmed through sequencing. The nucleotide sequence identities of BSOLV，BSGFV，and BSIMV exceeded 94% when compared with the corresponding nucleotide sequences of these BSVs available in GenBank. The study demonstrates that the multiplex immunocapture PCR method exhibits high sensitivity and specificity，enabling the detection of the three BSVs in banana seedlings and field samples.

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references

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LU Yongsi

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