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一种快速比色环介导等温扩增检测方法，用于检测引起水稻纹枯病的立枯丝核菌 AG-1IA。

A rapid colorimetric LAMP assay for detection of Rhizoctonia solani AG-1 IA causing sheath blight of rice.

机构信息

ICAR-National Bureau of Agriculturally Important Microorganisms (NBAIM), Kushmaur, Maunath Bhanjan, Uttar Pradesh, 275103, India.

ICAR-Indian Institute of Sugarcane Research, Lucknow, 226002, India.

出版信息

Sci Rep. 2020 Dec 16;10(1):22022. doi: 10.1038/s41598-020-79117-0.

DOI:10.1038/s41598-020-79117-0
PMID:33328516
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7744555/
Abstract

Rhizoctonia solani is one of the most devastating pathogens. R. solani AG-1 IA causes sheath blight in rice, maize, and other Gramineous plants. Accurate identification is essential for the effective management of this pathogen. In the present study, a set of four primers were designed viz. RSPG1, RSPG2, RSPG4, and RSPG5 for polygalacturonase (PG) gene, an important virulence factor in phytopathogenic fungi. All four primer sets showed specific amplification of 300 bp (RSPG1F/R), 375 bp (RSPG2F/R), 500 bp (RSPG4F/R) and 336 bp (RSPG5F/R) amplicons. q-PCR detection using each primer sets could detect up to 10 pg of DNA. We also designed six primers (RS_pg_F3_1/RS_pg_B3_1, RS_pg_FIP_1.1/RS-pg_BIP_1.1, and RS_pg_LF_1/RS_pg_LB_1) for PG gene. Further, a colorimetric LAMP assay developed yielded visual confirmation of the pathogen within 45 min of sample collection when coupled with rapid high throughput template preparation method (rHTTP) from infected samples. The sensitivity of the LAMP assay was as low as 1.65 fg/µl of template DNA and could effectively detect R. solani AG-1 IA from diseased plant tissues and soil samples. The LAMP assay was highly specific for R. solani as it did not show any amplification with other AG groups of R. solani and closely related fungal and bacterial outgroups. This study will help in designing an effective point of care diagnostic method for early monitoring of R. solani and thereby planning timely preventive measures against the pathogen.

摘要

腐霉菌是最具破坏性的病原体之一。腐霉菌 AG-1 IA 引起水稻、玉米和其他禾本科植物的叶鞘腐烂。准确的鉴定对于有效管理这种病原体至关重要。在本研究中，设计了一组四个引物，即用于多聚半乳糖醛酸酶（PG）基因的 RSPG1、RSPG2、RSPG4 和 RSPG5，PG 基因是植物病原真菌中一个重要的毒力因子。这四组引物都显示出 300 bp（RSPG1F/R）、375 bp（RSPG2F/R）、500 bp（RSPG4F/R）和 336 bp（RSPG5F/R）扩增子的特异性扩增。使用每个引物组的 q-PCR 检测可以检测到高达 10 pg 的 DNA。我们还为 PG 基因设计了六个引物（RS_pg_F3_1/RS_pg_B3_1、RS_pg_FIP_1.1/RS-pg_BIP_1.1 和 RS_pg_LF_1/RS_pg_LB_1）。此外，开发的比色环介导等温扩增（LAMP）检测法在结合从感染样本中快速高通量模板制备方法（rHTTP）时，可以在 45 分钟内对样本进行病原体的可视化确认。LAMP 检测的灵敏度低至模板 DNA 的 1.65 fg/µl，并且可以从患病植物组织和土壤样本中有效检测到腐霉菌 AG-1 IA。LAMP 检测对腐霉菌具有高度特异性，因为它与腐霉菌的其他 AG 组以及密切相关的真菌和细菌外群没有任何扩增。这项研究将有助于设计一种有效的即时护理诊断方法，用于早期监测腐霉菌，从而及时制定针对该病原体的预防措施。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fb3/7744555/1cabb34923f3/41598_2020_79117_Fig7_HTML.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fb3/7744555/bc2cbee85930/41598_2020_79117_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fb3/7744555/8ee6728bddd3/41598_2020_79117_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fb3/7744555/1cabb34923f3/41598_2020_79117_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fb3/7744555/49687140304f/41598_2020_79117_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fb3/7744555/b1fc2560b47f/41598_2020_79117_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fb3/7744555/909689efb893/41598_2020_79117_Fig3_HTML.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fb3/7744555/bc2cbee85930/41598_2020_79117_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fb3/7744555/8ee6728bddd3/41598_2020_79117_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fb3/7744555/1cabb34923f3/41598_2020_79117_Fig7_HTML.jpg

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