I am using a genus specific qPCR for bacterial diagnosis and following up with a species specific qPCR to identify species within the genus. However, I am getting some samples that are negative with the genus specific assay that are positive with the species specific assay and they usually have a high Ct value. Is more template needed, longer elongation step, pipetting errors with small volumes?
Hi Alan,
The issue here is probably related to a sensitivity difference between the genus assay and species assay. You should look at the LOD of the genus assay and species assay using quantified DNA standards from the species that you are detecting in your experiments.
You may need to optimise the genius assay to improve sensitivity. This might include primer redesign, MgCl2 titration, annealing temp optimisation etc.
Something you should do is to use the PCR product from the species specific assay as template for the genus assay. If this is detected, then you know it is a sensitivity issue. If it is not detected, then the species assay may be detecting something non-specifically.
The last possibility I can think of is simple contamination. Your species assay reagents may be contaminated and may need to be replaced. Do you ever get a signal in your negative control?
I hope this helps.
Justin.
Hi Alan,
The issue here is probably related to a sensitivity difference between the genus assay and species assay. You should look at the LOD of the genus assay and species assay using quantified DNA standards from the species that you are detecting in your experiments.
You may need to optimise the genius assay to improve sensitivity. This might include primer redesign, MgCl2 titration, annealing temp optimisation etc.
Something you should do is to use the PCR product from the species specific assay as template for the genus assay. If this is detected, then you know it is a sensitivity issue. If it is not detected, then the species assay may be detecting something non-specifically.
The last possibility I can think of is simple contamination. Your species assay reagents may be contaminated and may need to be replaced. Do you ever get a signal in your negative control?
I hope this helps.
Justin.
Hi Justin, thanks for your comments, they are useful. I will probably try to do standard curves and troubleshoot from there. One thing is that I'm not sure if I'm following. I presume you mean as the species specific fragment is amplified if it is detected when used as template in the genus specific then it will show issues with sensitivity? However the fragment for the species specific test is from ompB while the fragment for the genus is gltA so the genus specific assay will not be targeting the amplified product. Am I right or am I missing something? Thanks for your help.
Alan
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