Please suggest steps for detecting amplification by loop mediated isothermal amplification (LAMP) assay using lateral flow dipstick (LFD). The FIP is biotin labelled and probe is FITC labelled since i am using LFD from Milenia.
I was able to get very faint test line by incubation of probe (Tm 67) with amplicon at 65 degree celcius for 5 minutes. But the result is non-reproducible.
I have tried various concentration of probe and different hybridization temperature based on published literature. I have tried with fresh reagents also. There is no signal on test line despite very high amplification.
Please suggest how to troubleshoot the problem or how to carry out LAMP assay with LFD.
Have you tested if the problem is in the isothermal amplification reaction or in the LFD? I mean, after isothermal amplification (I gess 65ºC during 30-60 min) have you tested (by rt-pcr or agarosa gel) if the amplification reaction is working properly?
I have same opinion as Dr Hector D De Paz. First you need to check the amplification reaction. In LAMP assay, non-targeted amplification is common problem for many researcher.
I have confirmed the amplification on agarose gel electrophoresis.The specificity has been checked by restriction enzyme digestion. The loop mediated isothermal amplification assay is well standardized and specific. Now I am trying to use LFD as detection format. Further suggestions for troubleshooting are welcomed.
I have no experience in lateral flow detection. I'm sorry. But clearly it seems that the problem is in the LFD. Have you the possibility to test the LFD with other target as a positive control?
How much of the product are you loading. First try optimizing LFD detection using a positive control.
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