During a conventional PCR amplification, an isolate was found to be positive intermediate using the primer 16S rRNA but was negative towards LipL32 primer. However when the DNA sequences was compared to the ones in BLAST, it was 100% similar to Leptospira interrogans, and none to any intermediate Leptospira species. How could this happened?
When you say " negative towards LipL32", did you perform the PCR using the positive control as well, it could simply be that your PCR condition hadn't been optimized and hence the negative result. Apart from the obvious, could you please elaborate on the nature of the 16S rRNA primers, were they full length or species specific. If they were species specific then, the blast could show only the L. interrogans. Hope this was helpful.
A conventional PCR is not enough for confirmation. Even though you have a clear band of the good size, you may have surprises. If you can, always sequence your PCR product to confirm. Also, how much times do you run your PCR? Duplicates is always better if you want to confirm results. If you are working with urine from bats, you may have low DNA loads, which means if your PCR is not sensitive enough you might have a false negative. Otherwise, optimization of your PCR might be an issue, as said by Mitesh.
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