I am trying to amplify tomato spotted wilt virus by PCR from Chrysanthemum but sometimes the symptomatic plants doesn't amplify?
What controls are you running? What are the results of the controls? A positive control will help determine if the assay is performing properly.
How are you preparing your samples? There may be inhibitors present that are causing the PCR to not amplify.
I would suggest to use alternative TSWV host to amplify the virus and isolate the virus particle from this host. You can use the symptomatic chrysanthemum for the source of inoculation sap. Once the alternative host show iniiial systemic symptoms, you can harvest the tissue and isolate virus particle from it for PCR analysis.
It is possible when the chrysanthemum infected plant showed maximum symptoms, the virus particle in the plant tissues may actually have gone down and the virus titer have been very low in the infected plant. In such case, it may result in failure of getting virus template and result in negative PCR.
It is also true that the negative results may also be due to PCR error in any steps and the presence of inhibitors in the isolated samples, as indicated by Andrew
Thanks for both answers.
Yes I have been using human actin from hela RNA and NAD2 as controls. But the this is I have been tryin to use conventional PCR as a diagnostic method for Chrysanthemum crops. Some times the PCR amplify the TSWV particule and some other don't. actually the last time i used plants with a severe symptomatology and almost 20 plant positive for ELISA results negative by PCR.
Are you doing RT-PCR to amplify the TSWV? If you have symptoms in your plant you should have no trouble amplifying your virus. I suggest using NAD-5 as an internal control.
See Menzel, W., et al., 2002. Detection of four apple viruses by multiplex RT-PCR asssays with coamplification of plant mRNA as internal control. Journal of Virological Methods 99: 81-92
I am trying to amplify TSWV in synthomatic plants by reverse transcription and conventional PCR but not all the plants that result positive for ELISA gives me amplification.
I will suggest to mechanically sap transmit this isolate to susceptible Chilli or tomato, and check symptoms. Once you see the symptoms then put the Rt-PCR for Chilli infected samples. Also check your primer and PCR conditions. But you need to previous confirmed +ve control.
Best Luck
Did you get the answer you needed. If not let me know and tell me what you the problem is. I am not in favour of the use of Chenopodium. My device would be use Nicotiana benthamiana. I had never problems with this species. However, I noticed that in tropical countries difficulties are met. Is that a soil problem?
Thanks, I have been trying whit macerated material of tomato, chilli and chrysanthemum. I have obtained some positive controls. But the results are still some variant depending of the anatomical part of the plant used. Thanks for all the advise.
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