We received three Dog blood samples for E. canis PCR test. The DNA was extracted using QIAamp DNA blood mini kit and sujected to PCR. The template DNA was subjected to two different PCR using primer specific for E. canis and the 12S rRNA. Of the three samples all got amplified against 12S primers, but only one was found positive for E. canis.
But the lab technicians say the other two samples found negative by PCR were smear positive. What could be possible reason for a smear positive sample to benegative by PCR.
I see no problem with the DNA extraction, as the internal control (12S primers) worked properly.
Thanks
Hi. Basically, PCR method is more sensitive and more prescise than blood smear. So false negative result is not very likely. Some times it is very dificult to determine pathogen species examined only blood smear. So, if you use species specific primers and have similar pathogen to E. canis, you might not seen it on PCR.
Thanks for the answer. The primers were specific for E. canis. Could you please list out the pathogens that would look similar to E. canis upon blood smear.
Thanks
pcr is both sensitive and precise but it would only take one base changed under the 3' end of one primer to stop pcr working completely with that primer set but the physical characteristics of the organism would probably be exactly as expected Possibly also one primer has become degraded so a different pcr works but the e canis one do not work even though your dna is good quality.
Did the lab positive control sample for the e canis work in the same assay?
It could be another Ehrlichia species. Anaplasma, Rickettsia also could be very similar to E. canis throughout blood smear.
Dear paul
The E canis positive control inclued in the same run yielded PCR amplicons. Further, one of the clinical sample was also found positive. But the other two samples did not yield any amplification thought the respective DNA extract got amplified with 12S rRNA primers. Moreover the primer used in this assay were newly procured in the month of December 2019.
Then I think that Povilas Sakalauskas has the correct answer Yugendran T
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