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Development of a One-Step Immunocapture Real-Time RT-PCR Assay for Detection of Tobacco Mosaic Virus in Soil
Open Project Program of Key Laboratory of Tobacco Pest Monitoring Controlling & Integrated Management, Tobacco Research Institute of CAAS, Qingdao 266101, China
Shandong Weifang Tobacco Co., Ltd., Weifang 250100, China
* Author to whom correspondence should be addressed.
Received: 30 October 2012; in revised form: 13 November 2012 / Accepted: 14 November 2012 / Published: 4 December 2012
Abstract: Tobacco mosaic virus (TMV) causes significant losses in many economically important crops. Contaminated soils may play roles as reservoirs and sources of transmission for TMV. In this study we report the development of an immunocapture real-time RT-PCR (IC-real-time RT-PCR) assay for direct detection of TMV in soils without RNA isolation. A series of TMV infected leaf sap dilutions of 1:101, 1:102, 1:103, 1:104, 1:105 and 1:106 (w/v, g/mL) were added to one gram of soil. The reactivity of DAS-ELISA and conventional RT-PCR was in the range of 1:102 and 1:103 dilution in TMV-infested soils, respectively. Meanwhile, the detection limit of IC-real-time RT-PCR sensitivity was up to 1:106 dilution. However, in plant sap infected by TMV, both IC-real-time RT-PCR and real-time RT-PCR were up to 1:106 dilution, DAS-ELISA could detect at least 1:103 dilution. IC-real-time RT-PCR method can use either plant sample extracts or cultivated soils, and show higher sensitivity than RT-PCR and DAS-ELISA for detection of TMV in soils. Therefore, the proposed IC-real-time RT-PCR assay provides an alternative for quick and very sensitive detection of TMV in soils, with the advantage of not requiring a concentration or RNA purification steps while still allowing detection of TMV for disease control.
Keywords: Tobacco mosaic virus; immunocapture qRT-PCR; detection; soil
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MDPI and ACS Style
Yang, J.-G.; Wang, F.-L.; Chen, D.-X.; Shen, L.-L.; Qian, Y.-M.; Liang, Z.-Y.; Zhou, W.-C.; Yan, T.-H. Development of a One-Step Immunocapture Real-Time RT-PCR Assay for Detection of Tobacco Mosaic Virus in Soil. Sensors 2012, 12, 16685-16694.
Yang J-G, Wang F-L, Chen D-X, Shen L-L, Qian Y-M, Liang Z-Y, Zhou W-C, Yan T-H. Development of a One-Step Immunocapture Real-Time RT-PCR Assay for Detection of Tobacco Mosaic Virus in Soil. Sensors. 2012; 12(12):16685-16694.
Yang, Jin-Guang; Wang, Feng-Long; Chen, De-Xin; Shen, Li-Li; Qian, Yu-Mei; Liang, Zhi-Yong; Zhou, Wen-Chang; Yan, Tai-He. 2012. "Development of a One-Step Immunocapture Real-Time RT-PCR Assay for Detection of Tobacco Mosaic Virus in Soil." Sensors 12, no. 12: 16685-16694.