When I checked the presence of NDM gene in river water I received un specific bands amplified with NDM 621 primer. The band size was larges than the positive control. The positive control was located in the exact same location. I did the sequencing. Genes for some hydrolysis enzymes of Pseudomonas.Spp are present. The gene for the msba was also present. I repeated the experiment four times as well. Can any one help me to solve me this issue. I feel there should be mutation of NDM gene. Thank You
So you are trying to amplify the NDM gene of which species? Have you checked the specificity of your primer against other species? Are you sure the species with your NDM gene is present in the sample? How many liters of water did you filter/centrifuge to generate your sample?
Don't think you are using correct PCR protocol and electrophoresis protocol. What is unspecific? What is the ladder size? What is expected?
You need to discuss things with seniors to save time and resources.
Robert Adolf Brinzer
1. For Ralstonia insidiosa strain
I conducted sequencing too and I could find closea phylogenetic relationship with NDM20
2.Yes. I have conducted PCR with the same primers to detect NDM in other species
3. Yes. I conducted a sequncing and I could find Ralstonia insidiosa strain is present in the sample
4.I filtered 1.00l of water and inoculated 200 microliters of water to the agar plate containing ampicillin 20mg/L
Thank you for your reply. Robert Adolf Brinzer
Abhijeet Singh Yes. I have done PCR with the same primers for other species before and have conducted electrophoresis. Even I repeated the experiment three times using two different DNA extraction kits.
Unspecificity is the DNA is amplified with NDM 621 primer, but band sizes are larger than the positive control.
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