CN103397085A - 一种穿刺短体线虫检测试剂盒及其检测方法 - Google Patents

一种穿刺短体线虫检测试剂盒及其检测方法 Download PDF

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CN103397085A
CN103397085A CN201310250165XA CN201310250165A CN103397085A CN 103397085 A CN103397085 A CN 103397085A CN 201310250165X A CN201310250165X A CN 201310250165XA CN 201310250165 A CN201310250165 A CN 201310250165A CN 103397085 A CN103397085 A CN 103397085A
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张裕君
王金成
张瑞峰
魏亚东
廖芳
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Tianjin Entry Exit Inspection and Quarantine Bureau of Animals Plants and Food Inspection Center
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Abstract

本发明“一种穿刺短体线虫检测试剂盒及其检测方法”涉及植物病虫害检疫领域。通过设计针对穿刺短体线虫18S特异序列的特异引物，应用PCR扩增技术，实现了穿刺短体线虫的快速检测。该方法特异性强，灵敏度高和稳定性好，对于提高穿刺短体线虫检测的准确性、缩短检测时间具有重要的作用。

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一种穿刺短体线虫检测试剂盒及其检测方法
技术领域
本发明涉及植物病虫害检疫领域，提供了一种利用PCR扩增技术快速检测穿刺短体线虫的方法及其试剂盒，适用于口岸检验检疫实验室应用。 
背景技术
穿刺短体线虫(Pratylenchus penetrans)属于短体科、短体亚科、短体线虫属，寄主范围广，报道有350余种植物寄主，主要危害小麦、玉米、大麦、马铃薯等重要经济作物及百合、郁金香等等多种花卉，目前是我国进境植物检疫性有害生物，是我国进口种苗类商品重点检测对象之一。穿刺短体线虫为可迁移的内寄生线虫，世界各地都有分布，全球所有温带地区的国家以及我国的辽宁省、山东省都有报道，该种线虫可直接危害植物的根系、根茎、块根和块茎等地下部分，严重时甚至引起寄主死亡。因为穿刺短体线虫在根与土壤之间可迁移，又为细菌、真菌的进一步侵染提供可能，该线虫可伴随贸易性的种苗花卉、鳞茎块茎种球及介质传播扩散。 
目前国内针对穿刺短体线虫的检测方法还是依靠经典形态学的方法，受穿刺短体线虫生长发育期、虫口数量的限制，造成该种线虫的鉴定依赖经验，并且存在鉴定周期长等方面的不足，不能满足进口种苗商品快速通关的需要。 
本研究通过巧妙地设计针对穿刺短体线虫的18S特异序列的特异引物，应用PCR扩增技术，实现了穿刺短体线虫的高效快速检测。该方法特异性强，灵敏度高和稳定性好，对于提高线虫检测的准确性、缩短检测时间具有重要的作用。适合口岸检验检疫部门运用和推广。 
发明内容
本发明需要解决的技术问题是提供用于检测穿刺短体线虫的特异性引物 
本发明需要解决的另一问题是提供包含上述引物的PCR检测试剂盒。 
本发明用于检测穿刺短体线虫的PCR引物序列为： 
上游引物P.pen-F：5’-TAATTTACTTGATCTTGACAAACT-3’ 
下游引物P.pen-R：5’-GTATCGGACATCAACACCTG-3’ 
本发明的穿刺短体线虫检测试剂盒，包括以下成分： 
(1)穿刺短体线虫DNA模板和阴性对照咖啡短体线虫等DNA模板 
(2)穿刺短体线虫上下游引物： 
上游引物P.pen-F：5’-TAATTTACTTGATCTTGACAAACT-3’ 
下游引物P.pen-R：5’-GTATCGGACATCAACACCTG-3’ 
(3)10×Taq PCR Buffer，DDH2O，2.5mM dNTP，5U/μl Taq酶 
(4)判定结果：扩增产物经电泳检测出现634bp特异条带，则说明该样品含有穿刺短体线虫，若无扩增产物或出现大小不一致的条带，则说明该样品中不含有穿刺短体线虫。 
与现有技术相比，本发明的进步在于：检测速度快，节省时间，特异性强，灵敏度高，稳定性好，适合口岸植物检疫实验室快速鉴定穿刺短体线虫。 
附图说明
图1为穿刺短体线虫试剂盒特异性测试结果，1、2、3号样品为咖啡短体线虫、4、5号样品为斯氏短体线虫、6、7号样品为落选短体线虫、8品为伤残短体线虫，9、10、11、12号样品为穿刺短体线虫，从图1可以看出，只有穿刺短体线虫样品能扩增出634bp大小特异性单一条带，其他对照线虫均无扩增产物，用本试剂盒能准确地检测出穿刺短体线虫。 
具体实施方式
PCR引物设计： 
1    GAACGGCTCA TTACAACAGC TATAATTTAC TTGATCTTGA CAAACTTACA TGGATAACTG 
                                    P.pen-F 
61   AGGTAATTCT TGAGCTAATA CATGCACCAA AGCTTTGACC GTAAGGGAAG AGCGCATTTA 
121  TTGGAACAAA ACCAAACGGC TTCGGCTGTT CCCAGTTGAA TCAGAATAAC TCAGCTGATC 
181  GTACGGTCTT GCACCGACGA CGTGTCTTTC AAGTATCTGC TTTATCAACT TTCGATGGTA 
241  GGGTATCTGC CTACCATGGT GGTGACGGAT AACGGAGGAT CAGGGTTCGA CTCCGGAGAA 
301  GGGGCCTGAG AAATGGCCAC TACGTCTAAG GATGGCAGCA GGCGCGCAAA TTACCCACTC 
361  TCAGAATTTG GAGGAGGTAG TGACGAGAAA TAACGAGGTT GATCTCTTCT GAGGCCAACC 
421  ATCGGAATGG GTACAATTTA AACCCTTTAA CGAGTATCTA TGAGAGGGCA AGTCTGGTGC 
481  CAGCAGCCGC GGTAATTCCA GCTCTCAAAA TGCATAGAAT TATTGCTGCG GTTAAAAAGC 
541  TCGTAGTTGG GTCTGCAGCC GGAGCTCCGG TCCATCTTCG GATGTGTACT GGATGTCTTC 
601  GGCTTTCTGA CAGTGTTCGG CTCTGTGCCT CTCAACAGGT GTGGATGTCC GATACTGTAA 
                                                   P·pen-R 
661  GTTTACTTTG AATAAATCAG AGTGCTCTAA ACAGGCGTTT TGCTTGAATG ATTGTGCATG 
根据截获的穿刺短体线虫18S测序序列，并经GeneBank比对，利用引物设计软件，与其它线虫差异位点设计一组引物，片段大小为634bp，由Invitrogen(上海英俊生物技术有限公司)公司合成： 
上游引物P.pen-F：5’-TAATTTACTTGATCTTGACAAACT-3’ 
下游引物P.pen-R：5’-GTATCGGACATCAACACCTG-3’ 
标本来源 
本实验测试了12个线虫种群，其中包括4个穿刺短体线虫种群，3个咖啡短体线虫，2个斯氏短体线虫，2个落选短体线虫，1个伤残短体线虫种群，见下表： 
Figure BSA00000914582200031
实施例1、穿刺短体线虫的检测 
DNA提取：按照TIANamp Genomic DNA Kit组织基因组DNA提取试剂盒说明书进行操作，提取线虫基因组DNA溶于50uL TE中，-20℃保存备用。 
PCR扩增：以无菌水作为空白对照，分别以P.cof1、P.cof2、P.cof3、P.scr1、P.scr2、P.neg1、P.neg2、P.vul1、P.pen1、P.pen2、P.pen3和P.pen4线虫基因组DNA作为模板，进行PCR扩增。 
PCR反应体系： 
PCR反应条件：94℃预变性1min，94℃变性30s，58℃退火30s，72℃延伸40s，35个循环，72℃延伸5min。 
结果： 
从图1结果可以看出，9、10、11、12号样品为穿刺短体线虫，均出现634bp大小特异性单一条带，1、2、3号样品为咖啡短体线虫、4、5号样品为斯氏短体线虫、6、7号样品为落选短体线虫、8品为伤残短体线虫，均无扩增产物。结果表明，本发明中的穿刺短体线虫特异引物只能从穿刺短体线虫样品中扩增出634bp的特异单一条带，对其它同属线虫如咖啡短体线虫(P.coffeae)、斯氏短体线虫(P.scribneri)、落选短体线虫(P.neglectus)、伤残短体线虫(P.vulnus)等等阴性对照都没有出现扩增产物，显示了该引物良好的特异性。 
Figure ISA00000914582400011

Claims (3)

1.一种用于穿刺短体线虫检测的特异性引物，其特征在于，序列为： 
上游引物P.pen-F：5’-TAATTTACTTGATCTTGACAAACT-3’ 
下游引物P.pen-R：5’-GTATCGGACATCAACACCTG-3’ 
2.一种用于检测穿刺短体线虫的试剂盒其特征在于，包括如下成分： 
(1)阳性对照穿刺短体线虫DNA模板和阴性对照线虫DNA模板 
(2)穿刺短体线虫上下游引物 
上游引物P.pen-F：5’-TAATTTACTTGATCTTGACAAACT-3’ 
下游引物P.pen-R：5’-GATAGACCAGAGCTCCGGCT-3’ 
(3)10×Taq PCR Buffer，ddH2O，2.5mM dNTP，5U/μl Taq酶 
3.一种穿刺短体线虫的快速检测方法，包括如下步骤： 
Figure DEST_PATH_FSB0000113627980000011
(2)PCR反应条件：95℃预变性5min，95℃变性30s，58℃退火30s，72℃延伸40s，35个循环，72℃延伸5min。 
(3)判定结果：扩增产物经电泳检测出现634 bp特异条带，则说明该样品含有穿刺短体线虫，若无扩增产物或出现大小不一致的条带，则说明该样品中不含有穿刺短体线虫。 
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103789427A (zh) * 2014-01-23 2014-05-14 宁波检验检疫科学技术研究院 日本短体线虫的pcr检测试剂及试剂盒
CN105331736A (zh) * 2015-12-15 2016-02-17 林康艺 一种快速检测落选短体线虫的环等温扩增引物及其应用
CN105331735A (zh) * 2015-12-15 2016-02-17 林康艺 一种快速检测伤残短体线虫的环等温扩增引物及其应用
CN105349688A (zh) * 2015-12-15 2016-02-24 林康艺 一种快速检测穿刺短体线虫的环等温扩增引物及其应用
CN105349687A (zh) * 2015-12-15 2016-02-24 林康艺 一种快速检测桑尼短体线虫的环等温扩增引物及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
王扬等: "柑桔半穿刺线虫与短体线虫rDNA的ITS-RFLP研究", 《莱阳农学院学报》, vol. 21, no. 02, 30 June 2004 (2004-06-30) *
陈晓玲等: "咖啡短体线虫分子鉴定", 《植物检疫》, vol. 23, no. 02, 15 March 2009 (2009-03-15) *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103789427A (zh) * 2014-01-23 2014-05-14 宁波检验检疫科学技术研究院 日本短体线虫的pcr检测试剂及试剂盒
CN105331736A (zh) * 2015-12-15 2016-02-17 林康艺 一种快速检测落选短体线虫的环等温扩增引物及其应用
CN105331735A (zh) * 2015-12-15 2016-02-17 林康艺 一种快速检测伤残短体线虫的环等温扩增引物及其应用
CN105349688A (zh) * 2015-12-15 2016-02-24 林康艺 一种快速检测穿刺短体线虫的环等温扩增引物及其应用
CN105349687A (zh) * 2015-12-15 2016-02-24 林康艺 一种快速检测桑尼短体线虫的环等温扩增引物及其应用

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