For the identification of unique bacterium, primers designed from the species-specific region on the 16S rDNA are used, based on their known 16S rDNA sequences (GenBank). The sequences of primers for real-time PCR can be designed by using Primer Express® (Applied Biosystems) and OLIGO Primer Analysis Software (Version 4.0, Molecular Biology Insights, Inc.).
One set of primer sequences for amplification of Prevotella intermedia are as follows:
Forward 5'-AATACCCGATGTTGTCCACA -3' Reverse 5'-TTAGCCGGTCCTTATTCGAA -3'
For specificity testing of primers, extracted DNAs from 107 cells of Prevotella intermedia can be examined as templates with the primers. PCR amplifications should be observed when the target strains are used as templates. There should be no increase in the fluorescence with the mismatch strains. Melting curves of all PCR products should show sharp peaks at the expected Tm of the products. These preliminary results indicate that each real-time PCR specifically amplified the target DNA.
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