中国农业科学 ›› 2016, Vol. 49 ›› Issue (22): 4366-4374.doi: 10.3864/j.issn.0578-1752.2016.22.009

• 植物保护 • 上一篇    下一篇

蓝莓休克病毒IC-RT-nested PCR检测技术

谢丽雪,郑 姗,张立杰,张小艳,李 韬   

  1. 福建省农业科学院果树研究所,福州350013
  • 收稿日期:2016-08-08 出版日期:2016-11-16 发布日期:2016-11-16
  • 通讯作者: 李韬,Tel:0591-87573907;E-mail:leetao06@163.com
  • 作者简介:谢丽雪,E-mail:xielx_faas@126.com
  • 基金资助:
    国家质检公益性行业科研专项(201410076)、福建省公益类科研院所专项(2015R1014-5,2014R1014-15)、福建省种业创新与产业化工程项目(2014S1477-22)

Development of IC-RT-nested PCR for the Detection of Blueberry shock virus

XIE Li-xue, ZHENG Shan, ZHANG Li-jie, ZHANG Xiao-yan, LI Tao   

  1. Fruit Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350013
  • Received:2016-08-08 Online:2016-11-16 Published:2016-11-16

摘要: 【目的】蓝莓休克病毒(Blueberry shock virus,BlShV)是蓝莓上主要病毒之一,侵染蓝莓后能够对其产量造成严重影响。研究旨在建立用于BlShV快速检测的IC-RT-nested PCR技术,为该病毒的检测鉴定提供可靠的技术手段。【方法】根据GenBank已报道的BlShV基因序列,设计一对外侧引物(BlShV-F/BlShV-R)和一对内侧引物(BlShV-1/BlShV-2),以感染BlShV的蓝莓病叶为材料,利用免疫IC-RT-PCR(免疫捕获RT-PCR)和nested PCR(巢式PCR)建立BlShV的IC-RT-nested PCR检测技术;分别以BlShV、蓝莓焦枯病毒(Blueberry scorch virus,BlScV)、蓝莓带化病毒(Blueberry shoestring virus,BSSV)、蓝莓叶斑驳病毒(Blueberry leaf mottle virus,BLMoV)、烟草环斑病毒(Tobacco ringspot virus,TRSV)、番茄环斑病毒(Tomato ringspot virus,ToRSV)和健康蓝莓叶片为对象,测定该检测技术的特异性;将BlShV第1轮、第2轮PCR产物分别回收纯化后连接到pMD18-T载体,连接产物转化大肠杆菌DH5α后筛选阳性克隆,进行测序和序列比对验证该检测技术的准确性;将感染BlShV的蓝莓病叶提取液用健康蓝莓叶片提取液进行10倍梯度稀释,测定该检测技术的灵敏度,并与DAS-ELISA方法相比较;利用建立的IC-RT-nested PCR对收集的中国福建、吉林、辽宁地区蓝莓叶片样品(53份)和进境美国蓝莓叶片(15份)进行实际检测,并对上述样品采用普通RT-PCR方法进行验证。【结果】建立的IC-RT-nested PCR方法成功从感染BlShV病叶提取液第1轮PCR扩增出约746 bp大小的目的片段,第2轮PCR扩增出约486 bp大小的目的片段,未从健康蓝莓叶片提取液和空白对照扩增出目的片段;特异性测定结果表明,IC-RT-nested PCR具有良好的特异性,仅从感染BlShV蓝莓病叶提取液第1轮、第2轮PCR分别扩增到目的片段,而从BlScV、BSSV、BLMoV、TRSV、ToRSV和健康蓝莓叶片提取液第1轮、第2轮均未扩增到相应的目的片段;序列分析结果显示,感染BlShV蓝莓病叶提取液第1轮、第2轮PCR产物的序列同预期大小完全一致,分别为746、486 bp,将获得的两轮PCR产物序列与GenBank数据库中的BlShV基因序列进行比对,结果显示第1轮、第2轮PCR产物的序列与已报道的BlShV核苷酸序列一致性均达99%,证实两轮PCR产物均为BlShV特异性产物,进一步验证了扩增结果的准确性;灵敏度测定结果表明,IC-RT-nested PCR的第1轮PCR能够检测到稀释102倍的BlShV病叶提取液,灵敏度与DAS-ELISA相当,经过第2轮PCR,灵敏度提高100倍,能够检测到稀释104倍的BlShV病叶提取液;蓝莓样品IC-RT-nested PCR测定结果显示,2份来自于美国的蓝莓叶片样品扩增出大小约486 bp的目的片段,BlShV检出率为13.3%,而中国福建、吉林、辽宁地区蓝莓叶片样品上均未扩增出相应的目的片段,未检测到BlShV,该检测结果与普通RT-PCR验证结果完全相符,表明建立的IC-RT-nested PCR能够用于蓝莓样品的实际检测。【结论】建立的IC-RT-nested PCR具有快速、特异、准确和灵敏的优点,适合于田间和进出境口岸蓝莓样品上BlShV的检测鉴定。

关键词: 蓝莓休克病毒, IC-RT-nested PCR, 检测

Abstract: 【Objective】 Blueberry shock virus (BlShV) is one of the major viruses infecting blueberry, which can cause serious impacts on the yield of blueberry. In order to provide a reliable technique for rapid detection and identification of BlShV, IC-RT-nested PCR assay was developed.【Method】 A primer set containing outer primers (BlShV-F/BlShV-R) and inner primers (BlShV-1/BlShV-2) was designed according to the reported BlShV gene sequences, and IC-RT-nested PCR assay was established by combining immunocapture reverse transcriptase polymerase chain reaction and nested PCR with BlShV infected leaf as material. The specificity of the IC-RT-nested PCR was determined by testing extracts of leaf tissues from BlShV, Blueberry scorch virus (BlScV), Blueberry shoestring virus (BSSV), Blueberry leaf mottle virus (BLMoV), Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV)and healthy blueberry leaf. PCR products were purified, and then ligated to pMD18-T vector. After transformed into Escherichia coli DH5α, positive clones were screened and sequenced to further confirm the validation of the IC-RT-nested PCR assay. To test the sensitivity of the IC-RT-nested PCR, serial ten-fold dilution of the extracts prepared from BlShV-infected blueberry leaves was made by addition of corresponding amount of healthy plant extracts. The solutions were subsequently subjected to the IC-RT-nested PCR assay and DAS-ELISA, respectively. A total of 68 samples of blueberry from different regions (53 samples from Fujian, Jilin and Liaoning regions in China and 15 samples from America) were collected and detected for the presence of BlShV with the established IC-RT-nested PCR, and the result was validated by conventional RT-PCR. 【Result】 The established IC-RT-nested PCR amplified fragment of about 746 and 486 bp only from BlShV infected leaf extract by the first and second round PCR, respectively. No target fragment was observed from healthy blueberry leaves extract and blank control. The IC-RT-nested PCR assay showed a good specificity to BlShV with the expected 746 and 486 bp fragments for the first and second round PCR, respectively. No cross-reaction was observed from BlScV, BSSV, BLMoV, TRSV, ToRSV and healthy blueberry leaves. Sequence analysis showed that the sequences of the first and second round PCR product (746 and 486 bp, respectively) was identical with the expected size, and shared an extremely high homology (99%) with the previously published BlShV gene sequence. The results of sequence analysis confirmed that the two PCR products were BlShV specific products, and further verified the accuracy of amplification results. Sensitivity assay showed that the first round of IC-RT-nested PCR could successfully detect BlShV from leave extracts diluted up to 102, which was as sensitive as DAS-ELISA. After the second round PCR, the sensitivity of IC-RT-nested PCR was increased by 100 times, with a limit of 104 dilution of BlShV infected leaf extract. Field sample test revealed that the target fragment (about 486 bp) was amplified from two blueberry leaf samples from America by IC-RT-nested PCR, and the detection rate was 13.3%. However, no corresponding target fragment was amplified from samples from Fujian, Jilin, Liaoning regions of China. The result of field sample test was in accord with the results of conventional RT-PCR. 【Conclusion】 The established IC-RT-nested PCR is a rapid, specific, accurate and sensitive method for the detection and identification of BlShV on blueberry samples from field and port of entry-exit.

Key words: Blueberry shock virus (BlShV), IC-RT-nested PCR, detection