JIA-2021-1306 葡萄根结线虫PCR快速分子检测技术研究

doi:10.1016/j.jia.2022.08.100

Journal of Integrative Agriculture ›› 2022, Vol. 21 ›› Issue (11): 3408-3416.DOI: 10.1016/j.jia.2022.08.100

所属专题：线虫合辑Nematology

• • 上一篇

JIA-2021-1306 葡萄根结线虫PCR快速分子检测技术研究

收稿日期:2021-08-13 接受日期:2022-02-13 出版日期:2022-11-01 发布日期:2022-02-13

Study on PCR rapid molecular detection technique of Meloidogyne vitis

YANG Yan-mei^1,², LIU Pei³, LI Hong-mei⁴, PENG Huan⁵, DU Xia^1,², DONG Ye^1,², HU Xian-qi^1,²

¹College of Plant Protection, Yunnan Agricultural University, Kunming 650201. P.R.China
²State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, P.R.China
³Institute of Agricultural Environment & Resources, Yunnan Academy of Agricultural Sciences, Kunming 650205, P.R.China
⁴International College, Yunnan Agricultural University, Kunming 650201, P.R.China
⁵State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China

Received:2021-08-13 Accepted:2022-02-13 Online:2022-11-01 Published:2022-02-13
About author:YANG Yan-mei, E-mail: yym_yunnong@163.com; Correspondence HU Xian-qi, Tel: +86-871-65220998, Fax: +86-871-65227695, E-mail: xqhoo@126.com
Supported by:
This research was supported by the grants from the National Key Research and Development Program of China (2018YFD0201202 and 2017YFD0200601).

摘要/Abstract

摘要：

葡萄根结线虫（Meloidogyne vitis）是在云南省葡萄根部发现的一种新的根结线虫种类，该线虫在侵染地高密度存在，已对葡萄造成严重损害。葡萄根结线虫病的发生可能对我国葡萄产业发展构成威胁，为了建立一种快速、可靠的葡萄根结线虫特异性分子检测方法，本研究以葡萄根结线虫核糖体DNA内转录间隔区(ribosomal DNA-internal transcribed spacer，rDNA-ITS)基因片段为靶标，设计筛选葡萄根结线虫种特异性检测引物，通过优化反应体系，并对所设计引物的可靠性、特异性及灵敏性进行检测验证，建立了葡萄根结线虫PCR快速分子检测技术体系。结果表明，优化后的引物最佳退火温度为53℃，该引物能够对不同龄期葡萄根结线虫进行检测；特异性检测结果表明，本研究建立的PCR分子检测技术能够从葡萄根结线虫中扩增获得长度为174 bp的特异性片段，选用的5种非靶标根结线虫则无任何扩增条带，从而将葡萄根结线虫和其他5种非靶标根结线虫有效区分开；灵敏度检测结果表明，该PCR分子检测技术能够有效的检测单头2龄幼虫和10^-⁴头雌虫的DNA；此外，该PCR分子检测技术能够从混合线虫种群中特异性地检测出葡萄根结线虫，并能够有效检测出土壤中的葡萄根结线虫，检测灵敏度为0.5 g土壤中可检测出2头2龄幼虫或一头雄虫。本研究建立的快速、灵敏、特异的PCR分子检测技术可用于葡萄根结线虫单头2龄幼虫的直接鉴定、混合线虫群体中葡萄根结线虫的检测及0.5 g土壤中2头2龄幼虫或一头雄虫的直接检测。本研究建立的PCR分子检测技术能够准确、快速地检测出葡萄根结线虫，将为葡萄根结线虫的发生危害调查和高效绿色防控策略的制定提供技术支撑。

Abstract: Meloidogyne vitis is a new root-knot nematode parasitic on grape root in Yunnan Province, China. In order to establish a rapid, reliable and specific molecular detection method for M. vitis, the species-specific primers were designed with rDNA-ITS (ribosomal DNA internal transcribed spacer) gene fragment as the target. The reaction system was optimized and the reliability, specificity and sensitivity of primer were testified, therefore, a rapid PCR detection method for M. vitis was established. The result showed that the optimal annealing temperature of the primers was 53°C, which was suitable for the detection of different life stages of M. vitis. Specificity test showed that the specific fragment size of 174 bp was obtained from M. vitis, but other five non-target nematodes did not have any amplification bands, thus effectively distinguish M. vitis and the other five species, and could specifically detect the M. vitis from mixed populations. Sensitivity test showed that this PCR technique could detect the DNA of a single second-stage juvenile (J₂) and 10^–4female. Futhermore, this PCR technique could be used to detect directly M. vitis from soil samples. The rapid, sensitive and specific PCR molecular detection technique could be used for the direct identification of a single J₂ of M. vitis and the detection of M. vitis in mixed nematode populations and the detection of two J₂s or one male in 0.5 g soil samples, which will provide technical support for the investigation of the occurrence and damage of M. vitis and the formulation of efficient green control strategies.

Key words: Meloidogyne vitis , PCR rapid detection , reliability , specificity , sensitivity

. JIA-2021-1306 葡萄根结线虫PCR快速分子检测技术研究[J]. Journal of Integrative Agriculture, 2022, 21(11): 3408-3416.

YANG Yan-mei, LIU Pei, LI Hong-mei, PENG Huan, DU Xia, DONG Ye, HU Xian-qi. Study on PCR rapid molecular detection technique of Meloidogyne vitis[J]. Journal of Integrative Agriculture, 2022, 21(11): 3408-3416.

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JIA-2021-1306 葡萄根结线虫PCR快速分子检测技术研究

Study on PCR rapid molecular detection technique of Meloidogyne vitis

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