玉米孢囊线虫SCAR-PCR快速分子检测技术

doi:10.3864/j.issn.0578-1752.2022.17.006

摘要/Abstract

摘要：

【目的】玉米孢囊线虫（Heterodera zeae）为孢囊线虫属定居型半内寄生线虫,可侵染多种禾本科作物的根部,玉米孢囊线虫的发生和传播扩散将会对我国玉米生产造成严重威胁。本研究旨在建立玉米孢囊线虫的快速分子检测体系,以期从近缘属种可以准确地检测出玉米孢囊线虫,为玉米孢囊线虫的监测预警和防控打下技术基础。【方法】从我国河南、河北、甘肃、山东、湖南、广西和北京共收集20个玉米孢囊线虫近缘属种群体作为供试线虫;选取13条含有10个碱基的随机引物,采用RPAD技术对供试孢囊线虫进行多态性分析,筛出玉米孢囊线虫特异性RPAD片段,并将其转化为玉米孢囊线虫SCAR-PCR引物;采用PCR方法测试玉米孢囊线虫特异性引物的准确性以及检测技术体系的稳定性、灵敏度和实效性。【结果】通过RAPD比对分析,发现随机引物OPA03能在玉米孢囊线虫基因组DNA中扩增出一条468 bp的特异性片段;将该片段回收测序,根据片段序列信息,利用Primer 5.0设计一对特异性引物HzF1/HzR1;特异性检测结果表明,HzF1/HzR1仅在玉米孢囊线虫群体中扩增出大小为393 bp的特异性片段,其他5种16个孢囊群体（禾谷孢囊线虫、菲利普孢囊线虫、大豆孢囊线虫、旱稻孢囊线虫和甜菜孢囊线虫）和4个其他种群（水稻干尖线虫、马铃薯腐烂茎线虫、落选短体线虫和咖啡短体线虫）均没有扩增出目的条带;以含有玉米孢囊线虫的混合种群孢囊DNA为模板时,特异性引物HzF1/HzR1也能扩增出来单一明亮的393 bp的特异片段,而不含玉米孢囊线虫的混合种群不能扩增出该片段;该体系实现了玉米孢囊线虫准确、稳定的检测目的;对建立的玉米孢囊线虫快速检测技术体系灵敏度和实用范围测试,表明该检测体系对玉米孢囊线虫单个孢囊和单条幼虫均有灵敏的检测能力,检出值不低于1/2 000个孢囊和1/80条2龄幼虫。【结论】本研究建立的玉米孢囊线虫SCAR-PCR快速分子检测技术,可用于玉米孢囊线虫单一样本和混合种群的快速检测,对玉米孢囊线虫的孢囊和2龄幼虫均有灵敏的检测能力,检测引物特异性强,检测方法便捷稳定,灵敏度高。

关键词: 玉米孢囊线虫, RAPD, 特异性检测, SCAR-PCR

Abstract:

【Objective】 Corn cyst nematode (Heterodera zeae) is one of the sedentary semi-endoparasitic nematode of Heterodera spp., infects the roots of various gramineae crops. The occurrence and spread of H. zeae will pose a serious threat to corn yield production. The objective of this study is to establish the rapid and accurate molecular detection system for H. zeae from the related cyst nematode species, which will lay a technical foundation for the monitoring, early warning and prevention and control of H. zeae.【Method】 A total of 20 cyst nematode populations of H. zeae related species were collected as the nematode samples, from Henan, Hebei, Gansu, Shandong, Hunan, Guangxi and Beijing. Thirteen random primers containing 10 bases were selected, and RPAD technique was used to analyze the polymorphism of the tested nematode samples. The specific RPAD fragment was screened and transformed to SCAR-PCR primers of H. zeae. PCR was used to test the accuracy of specific primers for H. zeae and the stability, sensitivity and effectiveness of the detection technology system.【Result】 By comparative analysis of the RAPD results, one specific fragment of 468 bp was produced by primer OPA03. The fragment was recovered for sequencing. According to the fragment sequencing information, a pair of specific primers HzF1/HzR1 was designed. The specific primers HzF1 and HzR1 specificity test results showed that, one 393 bp specific fragment was amplified from H. zeae, no target bands were amplified in the other 16 populations of 5 related Heterodera spp. species (H. avenae, H. filipjevi, H. glycines, H. elachista and H. schachtii) and 4 populations of other species (Aphelenchoides besseyi, Ditylenchus destructor, Pratylenchus neglectus, P. coffeae). Furthermore, the 393 bp specific fragment was also amplified from the 6 related Heterodera spp. species mixed DNA. Meanwhile, there were no target bands when the mixed population without H. zeae. The system realized the accurate and stable detection of H. zeae, and the sensitivity and practical range of the established rapid detection technology system were tested. The results showed that the detection system was sensitive to single cyst and single 2nd stage juvenile (J2) of H. zeae. The minimum detection thresholds were 1/2 000 of single cyst or 1/80 of single J2, respectively.【Conclusion】 The SCAR-PCR rapid molecular detection technique established in this study can be used for the rapid detection of single samples and mixed populations of H. zeae. It has sensitive detection ability for both the cysts and the J2 of H. zeae, with strong primer specificity, convenient and stable detection method and high sensitivity.

Key words: Heterodera zeae, RAPD, specific detection, SCAR-PCR

崔江宽,任豪豪,曹梦园,陈昆圆,周博,蒋士君,汤继华. 玉米孢囊线虫SCAR-PCR快速分子检测技术[J]. 中国农业科学, 2022, 55(17): 3334-3342.

CUI JiangKuan,REN HaoHao,CAO MengYuan,CHEN KunYuan,ZHOU Bo,JIANG ShiJun,TANG JiHua. SCAR-PCR Rapid Molecular Detection Technology of Heterodera zeae[J]. Scientia Agricultura Sinica, 2022, 55(17): 3334-3342.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2022.17.006

https://www.chinaagrisci.com/CN/Y2022/V55/I17/3334

图/表 7

表1

用于玉米孢囊线虫SCAR-PCR特异性检测的线虫种群"

种群编号 Population code	种类 Species	来源 Origin	寄主植物 Host plant
Hz01	玉米孢囊线虫H. zeae	广西来宾Laibin, Guangxi	玉米Zea mays
Hz02	玉米孢囊线虫H. zeae	河南省濮阳市清丰县Qingfeng County, Puyang, Henan	玉米Zea mays
Hz03	玉米孢囊线虫H. zeae	河南省许昌市长葛Changge, Xuchang, Henan	玉米Zea mays
Hz04	玉米孢囊线虫H. zeae	河南省许昌市禹州Yuzhou, Xuchang, Henan	玉米Zea mays
Ha01	禾谷孢囊线虫H. avenae	河南省周口市扶沟县Fugou County, Zhoukou, Henan	小麦Triticum aestivum
Ha02	禾谷孢囊线虫H. avenae	河南省洛阳市偃师Yanshi, Luoyang, Henan	小麦Triticum aestivum
Ha03	禾谷孢囊线虫H. avenae	河北省沙河市Shahe, Hebei	小麦Triticum aestivum
Ha04	禾谷孢囊线虫H. avenae	山东省烟台市莱州Laizhou, Yantai, Shandong	小麦Triticum aestivum
Hf01	菲利普孢囊线虫H. filipjevi	河南省商丘市虞城县Yucheng County, Shangqiu, Henan	小麦Triticum aestivum
Hf02	菲利普孢囊线虫H. filipjevi	河南省洛阳市栾川县Luanchuan County, Luoyang, Henan	小麦Triticum aestivum
Hf03	菲利普孢囊线虫H. filipjevi	河北省邯郸市Handan, Hebei	小麦Triticum aestivum
Hf04	菲利普孢囊线虫H. filipjevi	山东省滕州市Tengzhou, Shandong	小麦Triticum aestivum
He01	旱稻孢囊线虫H. elachista	河南省信阳市潢川县Huangchuan County, Xinyang, Henan	水稻Oryza sativa
He02	旱稻孢囊线虫H. elachista	河南省新乡市获嘉县Huojia County, Xinxiang, Henan	水稻Oryza sativa
He03	旱稻孢囊线虫H. elachista	湖南省长沙市Changsha, Hunan	水稻Oryza sativa
Hg01	大豆孢囊线虫H. glycines	河南省周口市淮阳县Huaiyang County, Zhoukou, Henan	大豆Glycine max
Hg02	大豆孢囊线虫H. glycines	河南省南阳市内乡县Neixiang County, Nanyang, Henan	大豆Glycine max
Hg03	大豆孢囊线虫H. glycines	甘肃省庆阳市Qingyang, Gansu	大豆Glycine max
Hg04	大豆孢囊线虫H. glycines	河北省廊坊市Langfang, Hebei	大豆Glycine max
Hs01	甜菜孢囊线虫H. schachtii	中国农业科学院Chinese Academy of Agricultural Sciences	甜菜Beta vulgaris
Ab01	水稻干尖线虫A. besseyi	河南农业大学Henan Agricultural University	水稻Oryza sativa
Dd01	马铃薯腐烂茎线虫D. destructor	河南农业大学Henan Agricultural University	马铃薯Solanum tuberosum
Pn01	落选短体线虫P. neglectus	河南农业大学Henan Agricultural University	小麦Triticum aestivum
Pc01	咖啡短体线虫P. coffeae	河南农业大学Henan Agricultural University	小麦Triticum aestivum

表1

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)
OPA02	TGCCGAGCTG
OPA03	AGTCAGCCAC
OPA06	GGACCCTGAC
OPA09	GGGTAACGCC
OPA13	CAGCACCCAC
OPA18	AGGTGACCGT
OPB15	GGAGGGTGTT
OPC06	AAGACCCCTC
OPD13	GGGGTGACGA
OPG06	GTGCCTAACC
OPG08	TCACGTCCAC
OPG10	AGGGCCGTCT
OPK16	GAGCGTCGAA