Table 2.
PCR primer sequences, EvaGreen qPCR recipe, and qPCR cycling conditions for the detection of Chlamydia trachomatis 16S rDNA sequence.
| Primer name | Sequence |
|---|---|
| CT16S.Fwd | 5’-TAGTGGCGGAAGGGTTAG-3’ |
| CT16S.Rev | 5’-CGTCATAGCCTTGGTAGG-3’ |
| C. trachomatis 16S rDNA PCR amplicon sequence | 5’-TAGTGGCGGAAGGGTTAGTAATGCATAGATAATTTGTCCTTAACTTGGGAATAACGGTTGGAAACGGCCGCTAATACCGAATGTGGCGATATTTGGGCATCCGAGTAACGTTAAAGAAGGGGATCTTAGGACCTTTCGGTTAAGGGAGAGTCTATGTGATATCAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCTATGACG-3’ |
| EvaGreen qPCR recipe | |
| Component | Volume (μL) per 25 μL reaction |
| 10X ThermoPol buffer | 2.5 |
| Template DNAa | 3 |
| 10 μM CT16S.Fwd | 1.25 |
| 10 μM CT16S.Rev | 1.25 |
| 4 mM dNTP mix | 2.5 |
| 20X EvaGreen | 1.25 |
| 2 × 107 Taq DNA polymerase dried bacteria / 3 μL | 3 |
| Water | Up to 25 μL |
| PCR cycling conditions | |
| Step 1: 95 °C | 10 min |
| Step 2: 95 °C | 10 sec |
| Step 3: 55 °C | 15 sec |
| Step 4: 72 °C | 30 sec |
a
A 10-fold dilution series of plasmid or gBlock™ (IDT) templates starting from 2 × 106 copies/μL to 2 × 102 copies/μL may be used. No template controls should receive an equal amount of water or non-specific templates.
Set the qPCR machine to perform 45 cycles of amplification (Steps 2 to 4, with EvaGreen fluorescence measured in Step 4 of each cycle) followed by melt curve analysis of the PCR amplicons.