Consensus MLST loci to be used for strain typing within the C. neoformans/C. gattii species complex - 2009 (1)
1. W Meyer, D. M. Aanensen, T. Boekhout, M. Cogliati, M. Diaz, M. E. Esposto, M. Fisher, F. Gilgado, F. Hagen, S. Kaocharoen, A. P. Litvintseva, T. G. Mitchell, S. P. Simwami, L. Trilles, M. A. Viviani, and J. Kwon-Chung. 2009. Consensus multi-locus sequence typing scheme for Cryptococcus neoformans and Cryptococcus gattii. Med Mycol [2009 Jun 12:1-14. Epub ahead of print]
2. Fraser, J. A., S. S. Giles, E. C. Wenink, S. G. Geunes-Boyer, J. R. Wright, S. Diezmann, A. Allen, J. E. Stajich, F. S. Dietrich, J. R. Perfect and J. Heitman. 2005. Same-sex mating and the origin of the Vancouver Island Cryptococcus gattii outbreak. Nature 437:1360-1364.
3. Litvintseva, A. P., R. Thakur, R. Vilgalys, and T. G. Mitchell. 2006. Multilocus sequence typing reveals three genetic subpopulations of Cryptococcus neoformans var. grubii (serotype A), including a unique population in Botswana. Genetics 172:2223-2238.
4. D'Souza, C. A., F. Hagen, T. Boekhout, G. M. Cox, and J. Heitman. 2004. Investigation of the basis of virulence in serotype A strains of Cryptococcus neoformans from apparently immunocompetent individuals. Curr Genet 46:92-102.
5. Meyer, W., A. Castaneda, S. Jackson, M. Huynh, and E. Castaneda. 2003. Molecular typing of IberoAmerican Cryptococcus neoformans isolates. EID 9:189-195.
|
Gene Locus |
Gene Product |
Chromosome Locationa
|
Primer Name and Sequence
(If not specified differently primers listed will work for C.n. and C.g.) |
Amplification conditions |
No. of bases analysed (bp)a |
Analysed sequence fragment, start (5') and end (3') pointsa |
Ref. |
|
CAP59 |
Capsular associated protein |
1 |
CAP59F 5' CTCTACGTCGAGCAAGTCAAG 3'
CAP59R 5' TCCGCTGCACAAGTGATACCC 3'
|
94°C 3min; 35 cycles: 94oC 30s, 56oC 30s, 72oC 1min
Alternative conditions:
30 cycles: 94oC 30s, 64oC 30s, 72oC 1min
or:
30 cycles: 95°C 3min, 95oC 30s, 54oC 30s, 72oC 1min |
559
|
5' ? ACGGTACGCGCC
GGAGACAGAATG - 3'
|
2
|
|
Alternative primers:
CAP59LF 5' GTGAACAAGCTGCGGC 3'
CAP59LR 5' GGATTCAGTGTGGTGGAAGA 3' |
35 cycles: 94oC 30s, 60oC 30s, 72oC 1min |
1 |
|
GPD1 |
Glyceraldehyde-3-phosphate dehydrogenase |
7 |
GPD1F 5' CCACCGAACCCTTCTAGGATA 3'
GPD1R 5' CTTCTTGGCACCTCCCTTGAG 3'
|
94°C 3min; 35 cycles: 94oC 45s, 63oC 1min, 72oC 2min
Alternative conditions:
12 cycles; 62-56°C step-down 2°C every 2 cycles 95°C 3 min; 95°C 30 sec, 62-56°C 30 sec, 72°C 1 min; followed by 25 cycles: 95°C 30 sec, 56°C 30 sec, 72°C1 min |
543
|
5' ? GGTTTCGGTACG
GGACCCTGCCAA - 3'
|
2 |
|
LAC1 |
Laccase |
8 |
LAC1F 5' AACATGTTCCCTGGGCCTGTG 3'
LAC1R 5' ATGAGAATTGAATCGCCTTGT 3' |
94°C 3min; 30 cycles: 94oC 30s, 58oC 30s, 72oC 1min
Alternative conditions:
30 cycles: 95oC 30s, 50oC 30s, 72oC 1min |
469 |
5' ? GTAAGTATCAGC
TCAAGCTAAACA - 3' |
2 |
|
PLB1 |
Phospholipase |
12 |
PLB1F 5' CTTCAGGCGGAGAGAGGTTT 3'
PLB1R 5' GATTTGGCGTTGGTTTCAGT 3'
|
94°C 3min; 30 cycles: 94oC 45s, 61oC 45s, 72oC 1min
Alternative conditions:
12 cycles; 62-56°C step-down 2°C every 2 cycles 95°C 3 min; 95°C 30 sec, 62-56°C 30 sec, 72°C 1 min; followed by 25 cycles: 95°C 30 sec, 56°C 30 sec, 72°C1 min |
532 |
5' ? TGTTACTTGGAT
TCTGGAACATCG - 3'
|
3 |
|
SOD1 |
Cu, Zn superoxide dismutase |
5 |
Primers for C.n.
SOD1CNF 5' AAGCCTCTCATCCATATCTT 3'
SOD1CNR 5' TTCAACCACGAATATGTA 3'
Primers for C.g.
SOD1CGF 5' GATCCTCACGCCATTACG 3'
SOD1CGR 5' GAATGATGCGCTTAGTTGGA 3' |
94°C 3min; 35 cycles: 94oC 30s, 52oC 30s, 72oC 1.5min
|
700
|
5' ? CCACGTGCTCGC
ACCTGTCAATGC - 3'
|
4
|
|
Alternative primers for C.n.:
SOD1-f 5'TCTAATCGAAATGGTCAAGG 3'
SOD1-r 5' CGCAGCTGTTCGTCTGGATA 3' |
12 cycles; 62-56°C step-down 2°C every 2 cycles 95°C 3 min; 95°C 30 sec, 62-56C° 30 sec, 72C°1 min; followed by 25 cycles: 95°C 30 sec, 56°C 30 sec, 72°C1 min |
535 |
5' ? ATCGCTCACCGC
TGCCCATTGTCA - 3' |
3 |
|
URA5 |
Orotidine monophosphate pyrophosphorylase |
8 |
URA5F 5' ATGTCCTCCCAAGCCCTCGAC 3'
URA5R 5' TTAAGACCTCTGAACACCGTACTC 3' |
94°C 3min; 35 cycles: 94oC 45s, 63oC 1min, 72oC 2min
Alternative conditions:
30 cycles: 94oC 45s, 63oC 1min, 72oC 2min (C.n.)
26 cycles: 94oC 30 s, 68oC 30s, 72oC 30s (C.g.)
or:
30 cycles: 95°C 3 min; 95°C 30 sec, 63°C 30 sec, 72°C 1min |
601 |
5' ? TTTTCGGCAACT
CTTGGAAAGCTC - 3'
|
5 |
|
IGS1 |
Ribosomal RNA intergenic spacer |
2 |
IGSF 5' ATCCTTTGCAGACGACTTGA 3'
IGSR 5' GTGATCAGTGCATTGCATGA 3' |
94°C 3min; 35 cycles: 94oC 30s, 60oC 30s, 72oC 1min
Alternative conditions:
30 cycles: 94oC 30s, 56oC 30s, 72oC 1min |
723 |
5' ? TAAGCCCTTGTT
AAAGATTTATTG - 3'
|
3 |