Development of a multiplex reverse transcription-PCR assay for simultaneous detection of pepper viruses

PENG MIAO; WANG PEI; XIONG XING-YAO; DAI LIANG-YING; HU XIN-XI

doi:10.56093/ijas.v88i5.80030

Authors

PENG MIAO Hunan Agricultural University, Changsha, Hunan, China 410 128
WANG PEI College of Horticulture and Landscape, Hunan Agricultural University, Changsha, Hunan, China 410 128
XIONG XING-YAO Hunan Agricultural University, Changsha, Hunan, China 410 128
DAI LIANG-YING College of Plant Protection, Hunan Agricultural University, Changsha, Hunan, China 410 128
HU XIN-XI Hunan Provincial Engineering Research Center for Potatoes, Hunan Agricultural University, Changsha, Hunan, China 410 128

https://doi.org/10.56093/ijas.v88i5.80030

Keywords:

Multiplex RT-PCR, Pepper, Virus

Abstract

The aim of the present study was to establish a fourplex RT-PCR method for examining the infecting peppers to improve the efficiency of pepper virus detection, which includes Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Broad bean wilt virus- 2 (BBWV-2) and Potato virus Y (PVY). According to the genome sequences of TMV, CMV, BBWV-2 and PVY published by GenBank, four specific pairs of primers were designed to verify the specificity and sensitivity of detection system by simplex and multiplex RT-PCR method. In addition, using set multiplex RT-PCR method pepper plant samples of two geographic locations in China were tested and verified by simplex RT-PCR. The anticipated amplicon sizes of four primer pairs targeting TMV, CMV, BBWV-2 and PVY in both simplex and multiplex RT-PCR were 237 bp, 323 bp, 480 bp and 1057 bp, respectively, and established fourplex RT-PCR system was able to check out all sorts of viruses accurately from the pepper leaves of two geographic locations in China, which was 37.21%, 81.4%, 23.26%, and 2.33% respectively, and two or more mixed infections viruses sample accounted for 48.83% of the pepper samples. The research results show a detection technique of multiplex RT-PCR method for major peppers' viruses, which is a rapid, sensitive and reliable method and examines CMV, TMV, PVY and BBWV-2.

Downloads

Download data is not yet available.

Author Biography

XIONG XING-YAO, Hunan Agricultural University, Changsha, Hunan, China 410 128

References

Ben K M, Simon V, and Marrakchi M. 2009. Contribution of host plant resistance and geographic distance to the structure of Potato virus Y(PVY) populations in pepper in northern Tunisia. Plant Pathology 58: 763–72. DOI: https://doi.org/10.1111/j.1365-3059.2009.02064.x

Bolou B A Bi B Moury K A et al. 2015. First report of pepper vein yellows virus in filed-grown pepper in Ivory coast. Journal of Plant Pathology 97: 67–77.

Daniels J, and Campbell R H. 1992. Characterization of cucumber mosaic virus isolates from California. Plant Disease 76: 1245–0. DOI: https://doi.org/10.1094/PD-76-1245

David Gaytán and Francisco Benita. 2014. On the competitiveness of Mexico’S dry chili production. Economics of Agriculture 2: 307–17. DOI: https://doi.org/10.5937/ekoPolj1402307G

Guang jun-Guo, Diao wei-Ping, Jin bing-Liu et al. 2014. Research progress of pepper mosaic virus resistance. North China Agricultural University 29: 77–84.

Hu X, Lei Yan, Wang P, Tang L, He C, Song Y, Xiong X and Nie X. 2015. Development of a multiplex reverse transcription-PCR assay for simultaneous detection of garlic viruses. Journal of Integrative Agriculture 14(5): 900–8. DOI: https://doi.org/10.1016/S2095-3119(14)60892-3

Jian hua-Wang, Shao ting-Zhang, Dian-Gong. 2012. Establishment and application of molecular detection method of Chrysanthemum. Journal of Tropical Crops 33(2): 342–5.

Jian yun-Zhang. 2013. Molecular identification of two kinds of viruses in xinjiang pepper and eggplant. Shihezi University, Xinjiang.

Jie-Liang, Jian hua-Wang, Shao yan-Zhang et al. 2015. Discovery and detection of Sweet Potato Spotted Virus (PVMV) in Hainan Province. Journal of Tropical Crops 36(5): 966–71.

Jing ze-Guo, Xing hong-Li, Fang-Liao et al. 2008. Preliminary study on real-time fluorescent RT-PCR for detecting pepper light mottle virus. Plant Protection 2: 117-20.

Klisewicz J M. 1965. Identity of viruses from safflower affected with necrosis. Plant Disease Reporter 49(6): 541–5.

Li hao-Wang, Zheng hai-Zhang, Sheng li-Mao et al. 2016. Germplasm innovation of sweet potato resistance to tomato spotted wilt virus. Chinese Vegetables 1(2): 19–23.

Li shuang-Wang, Wen-Chen, Qing qun-Tan et al. 2015. Detection of chrysanthemum virus disease in Guizhou Province. Guizhou Agricultural Sciences 43(8): 99–101.

Nie X and Singh R P. 2001. A novel usage of random primers for multiplex RT-PCR detection of virus and viroid in aphids, leaves, and tubers. Journal of Virological Methods 91: 37–49. DOI: https://doi.org/10.1016/S0166-0934(00)00242-1

Olawale A, Olusegun S, Balogun K and Titilope K. 2012. Occurrence and distribution of pepper veinal mottle virus and cucumber mosaic virus in pepper in Ibadan, Nigeria. Virology Journal 9: 79. DOI: https://doi.org/10.1186/1743-422X-9-79

Palukaitis P, and Garcia A F. 2003. Cucumber viruses. Adv Virus Res 62: 241–323. DOI: https://doi.org/10.1016/S0065-3527(03)62005-1

Pei yong-Peng. 2016. Advances in tobacco resistance to tobacco mosaic virus related genes. Subtropical Plant Science 45(1): 95–100.

Qiang-Zhang, Chun zhu-Zhang, Ke mei-Li et al. 2014. Identification and pathogenicity analysis of isolated pepper isolates of Xinjiang pepper light mottle virus (PMMoV) in Xinjiang. Chinese Agricultural Science Bulletin 30(25): 296–302.

Qiang-Zhang, Ming-Luo, Ke mei-Li et al. 2014. Detection and identification of several processed pepper seed viruses in Southwest Agricultural University 27(6): 2385–92.

Si yao-Guo, Yan-Tong, Ya-Huang et al. 2015. Preliminary identification and analysis of the pathogen of chili virus in Chongqing. Gardening Journal 42(2): 263–70.

Uga H and Tsuda S. 2005. A one-step reverse transcription polymerase chain reaction system for the simultaneous detection and identification of multiple topovirus infections. Phytopathology 95: 166–71. DOI: https://doi.org/10.1094/PHYTO-95-0166

Wei-Gao, Yong-Wang, Chun xiang-Zhang et al. 2016. Investigation of Pepper Virus disease and preliminary identification of toxicants in. Shandong Agricultural Sciences 3: 123–7.

Xing hua-Zheng, Kun-Hong, Li chang-Yang et al. 2013. Molecular identification of Guizhou chilli light mottle virus isolates. Guizhou Agricultural Sciences 41(5): 30–2.

Xu Z and Barnett O W. 1983. Identification of a cucumber mosaic virus strain from naturally infected peanut in China. Plant Disease 68: 386–9. DOI: https://doi.org/10.1094/PD-68-386

Ya-Huang. 2015. Pathogen identification of chili virus disease in chongqing and construction of Multiple RT-PCR detection system. Southwest University, Chongqing.

Yan chun-Lin, Ming yun-Luo, Jiang-Lin, et al. Study on occurrence regularity and control technique of pepper virus disease [J]. Hubei Agricultural Sciences 9(48): 2142–4.

Yiyi-Liu, Wen bo-Xu, Sheng xue-Liu et al. 2012. Molecular identification of Broad Bean Wilt virus 2 and analysis of 5 - terminal nucleotide sequence of RNA1. Journal of Shihezi University (Natural Science Edition): 30(6): 695–9.

Yu rong-Yao, Guo hua-Chen, Lan xiang-Feng et al. 2013. Molecular detection of genotypes of Pepper Virus disease in Beiyuan Vegetable Base. Chinese Vegetables 10: 84–9.

Zun lian-Zhao, Lian lian-Shi, Gen tang-Tan et al. 2004. Identification and distribution of genotypes of chilli virus pathogens in major capsicum in gansu province. Chinese Agricultural Sciences 37(11): 1738–42.