4.4 Inconsistencies In The Primer Performance Case Study

4.4 Inconsistencies in the Primer Performance The primers used were sourced from journals where the researchers have utilized these primers successfully. However, there were still inconsistencies in terms of the results that were obtained from this study. The cytochrome B primers all worked as expected, with results obtained mirroring the results from the journal. However, the mitochondrial cytochrome C oxidase 1 primer did not work at all. There could be several reasons why the primer did not work for this study.
Primer inconsistencies usually arise due to incorrect PCR conditions or lack of target sequence in the reaction mix. Usually, if the problem was with a reagent, preparing new stocks of samples and reagents and then setting up a
However, upon analysis of results from Primer Blast against the Bos taxa, there were hits in primer targets with expected amplicon size (Appendix 3). This is linear with the results from the first paper published on this primer (Vrijenhoek, 1994). Surprisingly, this paper did not mention about the application of this primer to the Sus taxa. More research needs to be done on Haider et al. (2012) and their conditions and how they managed to amplify the porcine genes. A limitation in this study regarding the ability to explore the usage of this primer lies in the lack of diversity of the templates used during optimization. Only porcine templates were used and thus, this primer was not able to be worked. As seen in the Primer Blast results, the template was not target sequences and so no amplifications were seen. Therefore, the specificity of this primer to porcine sources needs to be reexamined. The primer sequence needs to be redesigned to target the porcine mitochondrial cytochrome C oxidase I gene more
The bovine presence was confirmed with a second PCR analysis, which produced the same banding patterns as seen in Figure 9. Bovine DNA in Halal certified food is allowed and is not much of a concern. Sample 7 produced the strongest band at 271bp, which indicates it had the most bovine DNA presence in it. This was probably due to the presence of gelatin, as the sample was a powdered gelatinous substance. Some of the bands, including bands from sample 9 and sample 11 are very faint, which indicates that the PCR can identify even very low levels of bovine presence in the sample. According to the study done by Ilhak and Arslan (2007), it was identified that DNA concentrations of even 0.1% of the sample can be detected and amplified via PCR and can be visualized on gel