土壤样本中菲利普孢囊线虫TaqMan实时荧光PCR快速检测及定量分析

doi:10.1016/j.jia.2022.09.016

Journal of Integrative Agriculture ›› 2023, Vol. 22 ›› Issue (8): 2591-2601.DOI: 10.1016/j.jia.2022.09.016

• • 上一篇

土壤样本中菲利普孢囊线虫TaqMan实时荧光PCR快速检测及定量分析

收稿日期:2022-03-23 接受日期:2022-08-06 出版日期:2023-08-20 发布日期:2022-08-06

Molecular diagnosis and direct quantification of cereal cyst nematode (Heterodera filipjevi) from field soil using TaqMan real-time PCR

JIAN Jin-zhuo^{1, 2}, HUANG Wen-kun¹, KONG Ling-an¹, JIAN Heng², Sulaiman ABDULSALAM^1,³, PENG De-liang¹, PENG Huan^1#

¹State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China
²Key Laboratory of Plant Pathology of Ministry of Agriculture and Rural Affairs, College of Plant Protection, China Agricultural University, Beijing 100193, P.R.China
³Division of Agricultural Colleges/Department of Crop Protection, Ahmadu Bello University, Zaria 810107, Nigeria

Received:2022-03-23 Accepted:2022-08-06 Online:2023-08-20 Published:2022-08-06
About author:JIAN Jin-zhuo, E-mail: jianjinzhuo@163.com; #Correspondence PENG Huan, Tel: +86-10-62815576, E-mail: hpeng@ippcaas.cn
Supported by:
This research was financially supported by the National Natural Science Foundation of China (31972247), the Science and Technology Innovation Project of the Chinese Academy of Agricultural Sciences (ASTIP-2016-IPP-04), and the Special Fund for Agro-scientific Research in the Public Interest, China (201503114).

摘要/Abstract

摘要：

【目的】菲利普孢囊线虫（Heterodera filipjevi）是全球小麦生产上的一种重要病原物，早期快速准确的检测对于小麦孢囊线虫病的防控至关重要。本研究基于TaqMan探针法的实时荧光定量PCR（real-time fluorescent quantitative PCR with TaqMan probes, TaqMan-qPCR）技术建立了一种直接从田间土壤中快速检测和定量分析H. filipjevi的方法。【方法】根据 H. filipjevi的RAPD-SCAR 序列（KC529338）设计TaqMan-qPCR特异性的引物和探针，利用已鉴定的27个线虫群体的DNA进行引物特异性检测；利用单条 J2 DNA稀释液（4^-0，4^-1，4^-2，4^-3，4^-4，4^-5，4^-6 和 4^-7）和单个雌虫DNA稀释液（10^-0，10^-1，10^-2，10^-3，10^-4，10^-5，10^-6和10^-7），以及基因组 DNA 的稀释液（100 μg.μL^-1，10 μg.μL^-1，1 μg.μL^-1，0.1 μg.μL^-1，0.01 μg.μL^-1，0.001 μg.μL^-1，100 pg.μL^-1 和 10 pg.μL^-1）对引物的灵敏度进行检测。通过人工将不同数量（0.1, 1, 10, 100和1000条）的 H. filipjevi添加到灭菌土中，建立了线虫数量的对数与CT值的标准曲线和回归方程，对32份田间土壤样品中H. filipjevi数量进行了评估，并对实时荧光定量PCR检测方法与传统定量方法进行了相关性分析。【结果】本研究建立的菲利普孢囊线虫TaqMan-qPCR检测技术具有极高的特异性和灵敏度，能够从13 种27 个线虫种群中特异的检测出菲利普孢囊线虫，检测阈值低至4^-3个单条J2 DNA、10^-3个单条雌虫DNA和0.01μg. μL^-1基因组DNA。田间土壤样品检测发现，实时荧光定量PCR定量检测方法与传统定量检测法呈现出较好的线性关系（R²= 0.8259）。【结论】本研究建立的TaqMan-qPCR检测H. filipjevi技术特异性强、灵敏度高，能广泛适用于田间土壤开展H. filipjevi快速检测和定量分析。

Abstract: Heterodera filipjevi continues to be a major threat to wheat production worldwide. Rapid detection and quantification of cyst nematodes are essential for more effective control against this nematode disease. In the present study, a TaqManminor groove binder (TaqMan-MGB) probe-based fluorescence quantitative real-time PCR (qPCR) was successfully developed and used for quantifying H. filipjevi from DNA extracts of soil. The primers and probe designed from the obtained RAPD-SCAR marker fragments of H. filipjevi showed high specificity to H. filipjevi using DNA from isolatesconfirmed species of 23 Heterodera spp., 1 Globodera spp. and 3 Pratylenchus spp. The qPCR assay is highly sensitive and provides improved H. filipjevi detection sensitivity of as low as 4^–3 single second-stage juvenile (J2) DNAs, 10^–3 female DNAs, and 0.01 μg μL^–1 genomic DNAs. A standard curve relating to the threshold cycle and log values of nematode numbers was generated and validated from artificially infested soils and was used to quantify H. filipjevi in naturally infested field soils. There was a high correlation between the H. filipjevi numbers estimated from 32 naturally infested field soils by both conventional methods and the numbers quantified using the qPCR assay. qPCR potentially provides a useful platform for the efficient detection and quantification of H. filipjevi directly from field soils and to quantify this species directly from DNA extracts of field soils

Key words: cereal cyst nematode , Heterodera filipjevi , molecular diagnosis , quantification , TaqMan real-time PCR

JIAN Jin-zhuo, HUANG Wen-kun, KONG Ling-an, JIAN Heng, Sulaiman ABDULSALAM, PENG De-liang, PENG Huan. 土壤样本中菲利普孢囊线虫TaqMan实时荧光PCR快速检测及定量分析[J]. Journal of Integrative Agriculture, 2023, 22(8): 2591-2601.

JIAN Jin-zhuo, HUANG Wen-kun, KONG Ling-an, JIAN Heng, Sulaiman ABDULSALAM, PENG De-liang, PENG Huan. Molecular diagnosis and direct quantification of cereal cyst nematode (Heterodera filipjevi) from field soil using TaqMan real-time PCR[J]. Journal of Integrative Agriculture, 2023, 22(8): 2591-2601.

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土壤样本中菲利普孢囊线虫TaqMan实时荧光PCR快速检测及定量分析

Molecular diagnosis and direct quantification of cereal cyst nematode (Heterodera filipjevi) from field soil using TaqMan real-time PCR

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