| 王林,朱淑珠,王雅丽,何梅,李燏,宋常美.玛瑙红樱桃植原体的鉴定及快速检测体系的构建[J].中国南方果树,2025,54(3): |
| 玛瑙红樱桃植原体的鉴定及快速检测体系的构建 |
| Identification and Rapid Detection System of Phytoplasma in Prunus Pseudocerasus ‘Manaohong’ |
| 投稿时间：2024-05-16 修订日期：2024-07-30 |
| DOI： |
| 中文关键词: 植原体 ，序列比对， LAMP可视化， 纤维素滤纸条法 |
| 英文关键词:phytoplasma, sequence alignment, LAMP visualization, Cellulose filter paper strip method |
| 基金项目:国家自然科学基金地区基金（项目号：32160693）；贵州省林业科学技术研究项目（项目号：黔林科合[2020]）；贵阳学院学科团队建设项目（合同号2022-xk12） |
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| 中文摘要: |
| 本研究旨在确定导致贵州玛瑙红樱花变绿的病原体的种类并构建快速的检测方法。通过使用tuf基因的通用引物进行PCR扩增,并对扩增片段进行测序、Blast比对和同源性分析,进而构建系统进化树,确定了引起花变绿的植原体的分类地位。基于此tuf基因序列,设计了一对特异性引物,并对dNTPs、MgSO4、Bst2.0 DNA聚合酶浓度、引物比例以及反应程序进行了优化,构建了LAMP反应体系,同时结合滤纸条DNA快速提取方法,建立了一种不依赖PCR仪的、高灵敏度且简便快捷的检测技术。结果显示：玛瑙红樱桃花变绿植原体被鉴定为属于16SrV-B亚组的植物病原体；优化后的LAMP体系如下：DNTPs和MgSO4的最终浓度分别为1.4 mmol/L和10.0 mmol/L,内外引物的浓度比为1:10(均为10 μmol/L),外侧引物浓度为0.2 μmol/L,Bst2.0 DNA聚合酶的最终浓度为0.32 U/μL,DNA模板量为31.7 ng/μL,10× Isothermal Amplification Buffer用量为1×,在60℃下恒温反应50 min,此方法能有效检测出玛瑙红樱花变绿植原体,其灵敏度达到3.96×10-4 ng/μL,是传统PCR的100倍。本研究不仅明确了变绿植原体的分类地位,还建立了一种无需依赖高端设备的快速检测技术,为樱桃植原体疾病的诊断提供了新方法,有助于田间病害的早期监测与控制。 |
| 英文摘要: |
| The objective of this study was to identify the pathogen species responsible for flower greening in Prunus pseudocerasus ‘Manaohong’ and to develop a rapid detection method. Universal primers were used to amplify the tuf gene via PCR, followed by sequencing, Blast comparison, and phylogenetic analysis to determine homology and classify the Virescence phytoplasma. Based on the tuf gene sequence, specific primers were designed, and the concentrations of dNTPs, MgSO4, Bst2.0 DNA polymerase, and primer ratios were optimized. Additionally, an optimized LAMP reaction system was established. This detection method, which is independent of a PCR instrument, is highly sensitive, quick, and convenient. The results revealed that the Virescence phytoplasma in Prunus pseudocerasus ‘Manaohong’ belongs to sub-group 16SrV-B. The optimized LAMP system consists of the following final concentrations: 1.4 mmol/L dNTPs, 10.0 mmol/ MgSO4, an inner/outer primer concentration ratio of 1:10 (both at 10 μmol/L), an outer primer concentration of 0.2 μmol/L, a final concentration of Bst2.0 DNA polymerase of 0.32 U/μL, a DNA template concentration of 31.7 ng/μL, a volume of 10× Isothermal Amplification Buffer at 1×, and amplification carried out at 60°C for 50 min. The developed method effectively detected the Virescence phytoplasma in Prunus pseudocerasus ‘Manaohong’, exhibiting a sensitivity of up to 3.96 ×10-4 ng/μL, which is 100 times higher than traditional PCR methods. This study not only clarified the taxonomic status of the Virescence phytoplasma but also established a rapid detection method that does not rely on high-end equipment, enabling a new approach for diagnosing phytoplasma diseases in cherry trees and facilitating early monitoring and control of field diseases. |
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