Hi everyone, I'm doing PCR for mycoplasma detection I'm using the primers GPO-3 and MGSO; the denaturation, annealing, and elongation temperatures and times were 95oC for 2min, 95oC for 30s, 57oC for 45s, 72oC for 1min, 72oC for 7min, for 40 cycles. The results were analized by gel electrophoresis using 1.5% agarose.
A band in 270pb is a positive result, but in some samples a band is amplified in 200pb. I was suggested that this band could be interpreted as a positive result for a different mycoplasma genus.
Hello
Do you check the specifity of the primers by primer blast on other tools?
Moreover, in my opinion the set time for amplification a 270bp amplicon, is relatively long that could be result to start new round of amplification and leads to a shorter band.
Sequencing this band would identify what it is but another possibility is that 40 cycles is huge amplification ( every 10cycles can be 1000 times more product)
so this middle band could be a snp or short indel in the strong bottom band forming a heteroduplex with the bottom band and this heteroduplex will run slowly so will look larger than the bottom band.
You could test this idea by purifying the bottom band and heating it and cooling slowly and if the middle band appears again on an agarose gel then it is likely to be a heteroduplex from 2 amplimers running at the same size forming the bottom band
When correctly checked before ordering, a primer should ideally not amplify non-specific regions. You must verify the primer specificity in your situation. In your scenario, 40 PCR cycles is a larger amount that increases the likelihood of producing non-specific or low-abundance products. As a result, minimizing the quantity of PCR cycles or improving PCR conditions could lessen the likelihood that non-specific bands will emerge.
The primers GPO3-MGSO used for mycoplasma detection could potentially amplify a double band. The search results indicate that PCR assays using these primers have been employed for mycoplasma detection. However, it is noted that nonspecific bands can be present in PCR products, which might result in the amplification of a double band. This highlights the importance of careful interpretation of PCR results, especially when using primers like GPO3-MGSO for mycoplasma detection.
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