TABLE 1.
Positions and sequences of oligonucleotide primers used to target 16S rRNA genes, 16S-23S rRNA intergenic spacer regions, and 5′ ends of 23S rRNA genes of several mollicutesa
| Target and primer name | Tm (°C)b | Primer sequencec |
|---|---|---|
| 16S rRNA genes | ||
| HYR+d | 71.5/96 | 182CATGATGAGTAATA GAA AGG AGC TTC ACA GCT TC216 |
| FER+d | 74.4/82 | 183TTACGGAA AAG AAG CGT TTC TTC GCT GG215 |
| ARG+d | 72.4/86 | 183GATTCCGTT GTG AAA GGA GCC CTT AAA GC210 |
| ORA5d | 75.5/84 | 189TTGTGAAA GGA GCG TTT CGT CCG CTA AG216 |
| ACH3d | 73.1/92 | 265GATGAA(/T)G(/T)CGT AGC CGG ACT GAG AGG TCT AC294 |
| GPO−1e | 76.2/96 | 326ACGGCCCAG(/A/T) ACT C(/T)CT ACG GG(/A)A GGC AGC AGT A356 |
| GPO−3f | 73.1/98 | 762GAAAGC(/T)GTG GG GAG CAA AT(/C)A GGA TTA GAT ACC CT795 |
| UNI−d | 70.5/88 | 797CC(/T)AGGGTATC TAA TCC TG(/A)TT TG CTC CCC AC768 |
| ARG−d | 75.2/84 | 853GCGTTAG CTG CGT CAG TGA ACT CTC CA828 |
| MGSOe | 71.8/86 | 1049CCA(/G) TGC ACC AT(/C)C TGT CA(/T)C(/A/T) T(/A/C)C(/A/T)T(/G/C) GT(/A)T(/A) AAC CTC1020 |
| RNA3d | 76.6/84 | 1068CGA(/G)C ACG AG(/A)C TGA CGA CAA CCA(/G) TGC AC1042 |
| ACHS | 75.7/94 | 791TGA GAA CTA AGT GTT GGG CAA AAG GTC AGT GC822 |
| MOS | 76.1/98 | 798CGC TGT AAA CGA TGA TCA TTA GTC GGT GGA AAA C831 |
| MHS | 73.4/92 | 803CGT AAA CGA TGA TCA TTA GTC GGT GGA GAATC834 |
| MAS | 75.8/100 | 803CCG TAA ACG ATG ATC ATT AGT CGG TGG AGA GTTC836 |
| MFS | 75.2/94 | 803CCC TAA ACG ATG ATC ATT AGC TGA TGG GGA A C833 |
| MHYS | 71.5/94 | 808CGT AAA CGA TGA TCA TTA GTT GGT GGA ATA ATT TC841 |
| MCGpF11g | 47.6/56 | 1383AAA CTA TGG GAG CTG GTA AT1402 |
| 16S–23S rRNA intergenic spacer regions | ||
| ACHA | 70.0/124 | 1564CTA CTT ACT AGT AGT CAT CTT GTG CTA AGT GTT AGT TAG CCT TTC1520 |
| MHA1 | 72.1/106 | 1567CAA AACT GGA TAC AAA TAT TTT TTT TCC ATA ACG TAG GTT G1529 |
| MHYA | 70.0/92 | 1585TAA AGC TCT CAA AAC TAG ACA CGA ATC GAT TAT G1553 |
| MOA | 72.4/92 | 1590CTC AAA ACT GGA TAC GAA TAT TGG GCC ATT AAC1566 |
| MHA2 | 73.3/106 | 1610AAT GTC GAT ATT CAG TTT TCA AAG AAC CGA GAG ATA AAT C1571 |
| MAA | 72.2/104 | 1613CGA TAT TCA GTT TTC AAA GAA CAA ATT GAG AGA TAG GTC1575 |
| MFA | 72.1/118 | 1634CAA AAC TAG ATA TAA AGC TTT AGA CCC ATA AAA AAG CCA CAT AAC1590 |
| SPMHYS | 70.2/114 | 1633GAT TAA CTT CAT ATT TAT TAT TTC AAC GAT CTT TTT TAT AAC CGA G1678 |
| SPMHS | 69.0/122 | 1639GAT CAA CCA TAG AAT ATT TAT ATT TTA TAA GAC AAA CAA TAG GTC ATA C1687 |
| SPACHS | 71.8/116 | 1640GAG ATC TTT GAA AAG TAG ATA AAT GAT GTC TGA AAA GAA ATA AGG1684 |
| SPMAS | 67.7/128 | 1655CAA CCT ATA GAA TAT ATC ATA CAA TAG ACA AAC AAT AGG TCT TAT ACT AC1704 |
| SPMOS | 71.0/114 | 1692GAG ACA AAT ACA AAA ACA AAA CAA TAG GTC AAA AAT ACT TAT ACG1736 |
| SPMFS | 70.8/118 | 1772CAT TGA AAT GTC TTA AAA TAC ACA TCA TAA CAA ACT ATA ACA ATA GG1818 |
| 5′-end of 23S rRNA genes | ||
| 23SS | 63.6/72 | 1820CTA AGA GCT TAT GGT GA(/G)A TGC CTT G1844 |
| R23-1Rg | 54.7/62 | 1854CTC CTA GTG CCA AGG CAT C(/T)C1835 |
| MO23A | 77.9/88 | 1954TTC AAT TCA CAG CGT GTC TCA TCA TTC GGC1924 |
| MHY23A | 74.9/88 | 1979GTT TCA ATT CGC AAC GTG TCT CGC TAA TTT G1949 |
| ACH23A | 76.5/86 | 2001CTT CGA CCG ATT TTC CCA CAT CGT TCA TC1973 |
| MH23A | 77.3/92 | 2009GTC GCA GTC CTA CAA CCC CAA CAA CAT GTG1980 |
| MA23A | 76.7/100 | 2021CTCTAA AAT GTA GTC CTA TAA CCC CAA CAA CGA ATG1986 |
| MF23A | 75.3/88 | 2132TGT AGA TCG TCC TAC AAC CCC CAT TGC TG2104 |
| 23SA1 | 75.5/80 | 2240ACA/G GA/GT TC/TC ACG TGC/T CCC GCC CTA CT2214 |
| 23SA2 | 75.1/110 | 2323CTTTTCACC TTT CCA/C TCA CG/TG TAC TA/GG TTC ACT ATC GGT2286 |
| 23SA3 | 65.7/66 | 2336CCA TCT CTG GGT TCT TTT CAC C2315 |
Specificities of primer pairs are shown in Table 2.
The first primer Tm value is that provided by the primer supplier (Sigma-Aldrich), and the second is that calculated by the formula Tm = [4 × numbers of (G+C)] + [2 × numbers of (A+T)].
Boldface numbers represent the numbered base positions at which primer sequences start and finish (position 1 corresponds to the starting point of U. parvum genome 16S rRNA genes [GenBank accession no. AE002112 and AE002127]). Underlined sequences show bases added to modify previously published primers. Letters in parentheses indicate alternative nucleotides in different Mollicutes species.
Dussurget and Roulland-Dussoix (8).
van Kuppeveld et al. (36).
van Kuppeveld et al. (37).
Harasawa et al. (10).