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Monoclonal Antibody-Based Serological Methods for Detection of Cucumber Green Mottle Mosaic Virus

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Monoclonal Antibody-Based Serological Methods for Detection of Cucumber Green Mottle Mosaic Virus

Haili Shang et al. Virol J.

Abstract

Background: Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, can be transmitted by seeds and infects many cucurbit species, causing serious yield losses in cucumber and watermelon plants. In this paper, five serological methods including antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA), triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), Dot-immunobinding assay (DBIA), direct tissue blot immunoassay (DTBIA) and immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) were described for detection and diagnosis of CGMMV.

Results: Using the purified CGMMV particles as immunogens, six murine monoclonal antibodies (MAbs) were produced. Five serological methods were established using the MAb 4H1 and detection sensitivity was compared using purified preparations and infected-plant tissue extracts. The detection sensitivity of ACP-ELISA was 0.16 ng of purified CGMMV, whereas TAS-ELISA was more sensitive than ACP-ELISA with a minimum detection of 0.04 ng of purified CGMMV. The sensitivities of TAS-ELISA and DBIA were similar for detecting CGMMV in infected-plant tissue extracts, and were four times higher than ACP-ELISA. The IC-RT-PCR was the most sensitive method, which could detect as little as 0.1 pg of purified virus. The detection sensitivity of IC-RT-PCR for CGMMV-infected plant tissues was about 400 times higher than that of TAS-ELISA and DBIA.

Conclusions: The established ACP-ELISA, TAS-ELISA, DBIA and DTBIA are suitable for routine CGMMV detection of large-scale samples in the field survey, while IC-RT-PCR is more sensitive and suitable for acquiring information about the viral genome.

Figures

Figure 1
Figure 1
Electron micrograph of purified cucumber green mottle mosaic virus. Bar = 0.2 μm.
Figure 2
Figure 2
Cross reactivities of anti-CGMMV MAbs with four tobamoviruses by ACP-ELISA. The absorbance value was the mean value obtained from three independent assays at 30 min after adding the substrate at room temperature.
Figure 3
Figure 3
Sensitivity analyses of ACP-ELISA (A) and TAS-ELISA (B). A: The sensitivities of ACP-ELISA and TAS-ELISA in the detection of purified CGMMV. The purified virus preparation was two-fold diluted in PBS buffer. CK: healthy tissues preparation was two-fold diluted in PBS buffer. The dilution endpoint of ACP-ELISA and TAS-ELISA were 1:1024000 and 4096000, corresponding to an equivalent of 0.16 ng and 0.04 ng of purified viruses respectively. B: The sensitivities of ACP-ELISA and TAS-ELISA in the detection of CGMMV in infected leaf extracts. CGMMV-infected leaf extracts were two-fold diluted in PBS buffer. CK: healthy leaf extracts were two-fold diluted in PBS buffer. The dilution endpoint of ACP-ELISA and TAS-ELISA were 1:5120 and 1:20480 (w/v, g mL-1), respectively.
Figure 4
Figure 4
Sensitivity analyses of DBIA for detecting CGMMV in infected plants. 1-16, CGMMV-infected leaf tissue extracts two-fold diluted from 1:5 to 1:81920 (w/v, g mL-1) and the original concentration was 1 g mL-1. CK- and CK+, healthy and CGMMV-infected leaf tissue extracts diluted 1:30 (w/v, g mL-1), respectively. Brown spots indicate positive reaction and green spot indicates negative reaction.
Figure 5
Figure 5
Detection of CGMMV in infected cucumber plants by DTBIA. The imprints from left to right were prepared from a single cut surface of same plant tissues. Lane A (1-8): Young stems infected with CGMMV; Lane B (1-8): Young leaves infected with CGMMV; Lane C (1-8): Healthy young stems; Lane D (1-8): Healthy young leaves.
Figure 6
Figure 6
Sensitivity of IC-RT-PCR for the detection of purified CGMMV (A) and CGMMV-infected plants (B). A: Purified virions with two-fold diluted from 1:5000 to 1:2560000 (from left to right). The original purified virus concentration was 1.28 μg mL-1. The lane 1 was diluted at 1:5000, corresponding to 256 pg of purified virus. The lane 9 was diluted at 1:1280000, corresponding to 0.1 pg of purified virus. B: CGMMV-infected leaf tissue saps with two-fold dilution from 1:200 to 1:819200 (w/v, g mL-1) (Lanes 1-11). Lane 12 was positive control and lane 13 was negative control. The original cucumber tissue extracts concentration was 0.05 g mL-1. M: 1 kb DNA marker.

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