QPCR amplification was performed in a total volume of 20 μl, including the 10 μl of Probe qPCR Mix (with UNG) (Takara), 500 nM target/reference primers, 250 nM target/reference probes, and 9 μl of each template DNA. The qPCR conditions were as follows: 25 °C for 10 min, followed by 45 cycles of 15 s at 95 °C and 34 s at 60 °C. Fluorescent accumulation data were analysed using ABI 7500 Software Version 2.3 (Applied Biosystems). Threshold cycle (CT) values of < 40 were defined as a positive result for the U. parvum and U. urealyticum, respectively. In addition, the quality of PCR extraction was monitored by amplifying human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cervical swabs. CT values of > 37 suggest the failure of DNA extraction or qPCR amplification. Standard curves were generated by plotting the CT of the qPCR performed on a ten fold dilution series of purified DNA from U. parvumand U. urealyticum.
The primers and probes were designed based on the ParC gene conserved regions of U. parvum and U. urealyticum, respectively (Additional file1 ), which have previously been used for qPCR [28 ].
U. parvum, forward: 5′-TAGTTGCTCATAAAATCAC-3′,
Reverse: 5′-CGTTCCATATATAAACAGCTATAAC-3′,
Probe: 5′- FAM-CTATGCGTGAAAAGATG-BHQ1–3′;
U. urealyticum, forward: 5′- TAAGTGTTTTAGTATTAGTGAGC-3′;
Reverse: 5′- TGCTGCTAAAACGCTTTGTGC-3’C;
Probe: 5′- FAM-CACCATCACCACTTTATT-BHQ1–3′.
The primers and probes were designed based on the ParC gene conserved regions of U. parvum and U. urealyticum, respectively (Additional file
U. parvum, forward: 5′-TAGTTGCTCATAAAATCAC-3′,
Reverse: 5′-CGTTCCATATATAAACAGCTATAAC-3′,
Probe: 5′- FAM-CTATGCGTGAAAAGATG-BHQ1–3′;
U. urealyticum, forward: 5′- TAAGTGTTTTAGTATTAGTGAGC-3′;
Reverse: 5′- TGCTGCTAAAACGCTTTGTGC-3’C;
Probe: 5′- FAM-CACCATCACCACTTTATT-BHQ1–3′.