Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (22): 4366-4374.doi: 10.3864/j.issn.0578-1752.2016.22.009

• PLANT PROTECTION • Previous Articles     Next Articles

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

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

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