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, 12 (12), 16685-94

Development of a One-Step Immunocapture Real-Time RT-PCR Assay for Detection of Tobacco Mosaic Virus in Soil

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Development of a One-Step Immunocapture Real-Time RT-PCR Assay for Detection of Tobacco Mosaic Virus in Soil

Jin-Guang Yang et al. Sensors (Basel).

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.

Figures

Figure 1.
Figure 1.
The detection of TMV in soil by the assays of DAS-ELISA (A), RT-PCR (A) and IC-real-time RT-PCR (B). Lanes 1 to 6 are the detections of TMV in one gram soil samples artificially blended with different dilutions naturally TMV-infected leaf at 1:101, 1:102, 1:103, 1:104, 1:105 and 1:106 (1 mL, w/v, g/mL), respectively, and, Lanes 7 to 9 are the detection of TMV from ToMV-contaminated soil, TMV-free soil, and healthy leaf extract, respectively. “+” represents positive result of DAS-ELISA for detecting TMV, “−” represents negative result of DAS-ELISA for detecting TMV. (B) Sensitivity of the IC-real-time RT-PCR assay as monitored by amplified curve. Shown from left to right are the curves of soil contamined with decreasing TMV-infected leaf saps at 1:101, 1:102, 1:103, 1:104, 1:105 and 1:106 (1 mL, w/v, per g soil) dilutions. The curves roughly parallel to the X axis are TMV from ToMV-contaminated soil, TMV-free soil, and healthy leaf extract, respectively.
Figure 2.
Figure 2.
The detection of TMV in plant by the assays of DAS-ELISA (A), RT-PCR (A) and IC-real-time RT-PCR (B). Lanes 1 to 6 are dilutions at 1:101, 1:102, 1:103, 1:104, 1:105 and 1:106 (1 mL, w/v, per g soil) of naturally TMV-infected leaf saps, respectively, and, Lanes 7 to 9 are the template of ToMV-contaminated soil, TMV-free soil, and healthy leaf extract, respectively. “+” represents positive result of DAS-ELISA for detecting TMV, “−” represents negative result of DAS-ELISA for detecting TMV. (B) Sensitivity of the IC-real-time RT-PCR assay as monitored by amplified curve. Shown from left to right are the curves of decreasing TMV-infected leaf saps at 1:101, 1:102, 1:103, 1:104, 1:105 and 1:106 (1 mL, w/v, per g soil) dilutions. The curves roughly parallel to the X axis are TMV from ToMV-contaminated soil, TMV-free soil, and healthy leaf extract, respectively.
Figure 3.
Figure 3.
The specificity of IC-real-time RT-PCR for the detection of TMV in soil. The specificity of the IC-real-time RT-PCR assay as monitored by amplified curve (A) and melting curves (B) for TMV CP gene. (A) Amplified curve shown from left to right are the curves of TMV Fumeng isolate, TMV Chuxiong-1 isolate, ToMV and TMV-free soil. (B) Melting curves for TMV CP gene with single peak from three replicates of two different templates including TMV leaves and viruliferous soils.
Figure 4.
Figure 4.
The standard curve obtained for the quantitation of purified TMV. The plot indicates the correlation between the Ct values and the log of the TMV particle units (Log GU). The equation of the regression line shown is y = −0.37x + 34.97; R2 = 0.99.

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