JCM Accepted Manuscript Posted Online 15 April 2015 J. Clin. Microbiol. doi:10.1128/JCM.00398-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Title:
2
Quick
3
Echinocandin Resistance in Candida albicans.
4
Catiana Dudiuk1, 2, Soledad Gamarra1, Cristina Jimenez-Ortigosa3,
5
Florencia
6
Guillermo Garcia-Effron1,
7
1
8
Parasitología y Micología – Facultad de Bioquímica y Ciencias
9
Biológicas
detection
of
FKS1
Leonardelli1,
Mutations
2
,
Daiana
Responsible
Macedo1,
for
David
S.
Clinical
Perlin3,
2*
.
Laboratorio de Micología y Diagnóstico Molecular - Cátedra de
10
(Argentina).
11
2
–
Universidad
Nacional
del
Litoral.
Santa
Fe
Consejo Nacional de Investigaciones Científicas y Tecnológicas
12
(CONICET). CCT-Santa Fe (Argentina).
13
3
14
Health Sciences– Newark, NJ (USA).
Public
Health
Research
Institute
–
Rutgers
Biomedical
and
15 16
Key words: Candida albicans, resistance detection, echinocandin.
17
Running title: PCR for the detection of Echinocandin resistance
18
in C. albicans.
19 20
*Corresponding author: Dr. Guillermo Garcia-Effron. Laboratorio
21
de
22
Parasitología y Micología – Facultad de Bioquímica y Ciencias
23
Biológicas – Ciudad Universitaria – Paraje el Pozo S/N – Santa
24
Fe (Santa Fe) – CP 3000 – Argentina.
25
e-mail: [email protected] Phone: +54-342-4575209 int. 135. Fax:
26
+54-342-4575216.
27 28
Micología
y
Diagnóstico
Molecular
(CONICET)
–
Cátedra
de
29
ABSTRACT
30
A
31
albicans
32
echinocandin
33
consisted of a multiplexed PCR set of 5 tubes able to detect the
34
most commonly described resistance mechanism, including FKS1 hot
35
spot 1 and hot spot 2 mutations. The performance and specificity
36
of the assay was evaluated using a double blinded panel of 50 C.
37
albicans strains. The assay showed a sensitivity of 96% and was
38
able to detect all homozygous mutants included in the collection
39
of strains demonstrating that is a robust, quick and labour
40
saving method that is suitable for a routine clinical diagnostic
41
laboratory.
42
rapid
molecular-based FKS1
gene drugs,
assay
mutations was
for
the
detection
responsible
designed
and
for
of
Candida
resistance
evaluated.
The
to
assay
43
INTRODUCTION
44
Invasive infections due to Candida albicans are a common and
45
significant
46
echinocandin drugs are becoming the antifungal of choice for the
47
management of these infections (8,22,30,32). This expanding use
48
of
49
resistance (3,10-12,15,19,20).
50
Echinocandin in vitro antifungal susceptibility testing (AST) is
51
being
52
(1,6,7,33). However, testing for antifungal resistance is not
53
routinely
54
resistance in C. albicans resulting in therapeutic failures is
clinical
echinocandins
performed
has
problem
brought
worldwide
performed
at
(5,8,21,24,34),
about
to
many
the
guide
and
emergence
therapeutic
centers.Clinical
of
the
drug
decisions
echinocandin
55
closely linked with amino acid substitutions in the hot spot
56
regions
57
complex (1,10-12,15,26,27). The detection of these mutations has
58
been proposed as the most direct and accurate way to predict
59
echinocandin clinical failure (1,4,12,27).
60
The aim of this work was to develop a molecular -based method
of
the
Fks1p
subunit
of
the
β-D-1,3-glucan
synthase
61
able to quickly and accurately detect the FKS1 mutations linked
62
with
63
performance of the proposed methodology was evaluated using a
64
blinded collection of clinical echinocandin-susceptible and -
65
resistant C. albicans isolates.
clinical
echinocandin
resistance
in
C.
albicans.
The
66 67
MATERIALS AND METHODS
68
Strains. Fifty C. albicans strains were used throughout this
69
work. All the strains were isolated from patients with proven
70
invasive fungal disease. Sixteen strains were obtained from the
71
Public Health Research Institute (PHRI; Rutgers Biomedical and
72
Health Sciences, NJ) and 34 from the Mycology and Molecular
73
Diagnostics Laboratory (LMDM) (Santa Fe, Argentina). Ten strains
74
showed homozygous FKS1 hot spot region mutations, two strains
75
showed heterozygous mutations at one of the hot spot regions and
76
one showed an homozygous mutation at the hot spot 1 of the FKS1
77
gene together with an heterozygous mutation at the hot spot 2 of
78
the FKS1 (Table 1). C. albicans ATCC 90028, ATCC 36082 and
79
SC5314 were used as the wild-type control strains to validate
80
the PCRs. C. krusei ATCC 6258 and C. parapsilosis sensu stricto
81
ATCC 22019 were used as AST control strains (6,7). The isolates
82
were
83
sequencing
84
transcribed
identified of
by
conventional
the
spacer
5.8S 1
RNA
(ITS1)
phenotypic
gene and
and
ITS2
methods
adjacent
regions
and
by
internal
(16,35).
The
85
collection of strains was assembled at the PHRI center, and
86
blinded code numbers were assigned. Also, a set of C. albicans
87
strains with known FKS1 mutations were used to develop and test
88
the proposed methodology before confirming its utility with the
89
blind study.
90
Antifungals and susceptibility testing. Caspofungin (CSF; Merck
91
& Co. Inc., Rahway, NJ), anidulafungin (ANF; Pfizer, New York,
92
NY), and micafungin (MCF; Astellas Pharma USA Inc., Deerfield,
93
IL)
94
manufacturers. Echinocandin susceptibility testing was performed
95
in
96
following the interpretive guidelines published in the M27-S4
97
document (6,7).
98
DNA isolation, primer and PCR design. C. albicans genomic DNAs
99
were extracted with phenol-chloroform method (31) or with a Q-
were
obtained
triplicate
in
as
standard
accordance
powder
with
from
CLSI
their
document
respective
M27-A3
and
100
Biogene
101
instructions.
102
numbers XM_446406 was used for primer design. Two groups of
103
primers were used throughout this work. The first primer pair,
104
named PCR control primers, consisted of two primer pairs: 1752-F
105
and
FastDNA
2232-R
C.
and
kit
(Q-Biogene)
albicans
3518-F
FKS1
and
following
gene
4266-R.
with
These
manufacturer´s
GenBank
primer
accession
pairs
were
106
designed to specifically hybridize FKS1 hot spot 1 and hot spot
107
2 regions, respectively. They were used as amplification control
108
in each of the multiplex PCR tubes. The second group of primers
109
(mutation detection primers) included five oligonucleotides that
110
were designed to detect the 8 most common mutations related with
111
echinocandin
112
sequences in Table 2). These mutation detection primers align
113
the FKS1 hot spot 1 (primers F641, S645, D648 and P649) and hot
114
spot 2 regions (primer R1361) and were added to one of the five
resistance
in
C.
albicans
(oligonucleotide
115
tubes of the PCR set, which already contained one pair of PCR
116
control primers. The hot spot 1 mutation detection primers were
117
paired with hot spot 1 PCR control primers (1752-F and 2232-R),
118
while the primer R1361 was used together with the hot spot 2
119
control primers (3518-F and 4266-R) (Figure 1). Primers were
120
designed
121
SciTools (Integrated DNA Technologies, Coralville, IA) and were
122
purchased
123
Buenos Aires, Argentina). Amplifications were carried out in a
124
25 µl volume of a mixture containing 5 mM (NH4)2SO4, 5 mM KCl,
125
10
126
albumin, 0.1% Triton X-100, 125 µM each dATP, dGTP, dCTP, and
127
dTTP
128
concentration of each of the three primers, 1.25 U of Pegasus
129
DNA polymerase (PBL, Embiotec, Buenos Aires, Argentina), and 10
130
to 25 ng of C. albicans genomic DNA. Amplification was performed
131
for one initial step of 2 min at 94°C followed by 25 cycles of
132
30 s at 94°C, 30 s at 57°C, and 1 min at 72°C and then a final
133
cycle of 10 min at 72°C in an Applied Biosystems thermocycler
134
(Tecnolab-AB, Buenos Aires, Argentina). The PCR products were
135
analyzed by electrophoresis.
136
DNA
137
(flanking the hot spot 1 and hot spot 2 regions between the nts.
138
1892 to 2232 and nts. 3904 to 4266, respectively) and 5.8s RNA
139
gene and adjacent internal transcribed spacer 1 (ITS1) and ITS2
140
regions were amplified and sequenced in both directions using
141
the primers described in Table 2. For sequencing of the FKS1
142
regions, primer pair 1752-F/2232-R and 3518-F/4266-R were used
143
for
144
subjected to sequencing using primers 1892-F and 2232-R for hot
mM
using
from
Tris-Cl
the
oligonucleotide
Integrated
(pH
8.8),
(Genbiotech,
sequencing.
PCR
DNA
1
Two
MgSO4,
Aires,
regions
amplification.
Technologies
Mm
Buenos
The
design
of
5
ng
tool
of
C.
purified
IDT
(IDT-Biodynamics,
of
bovine
Argentina),
the
the
a
0.5
albicans
fragments
serum
FKS1
were
µM
gene
then
145
spot 1 region and 3904-F and 4266-R for hot spot region 2 (Table
146
2). In Argentina, DNA sequencing was performed using a BigDye
147
Terminator
148
Biosystems,
149
manufacturer’s instructions. Sequence analysis was performed on
150
an ABI Prism 310 DNA sequencer (Applied Biosystems) using the
151
facilities
cycle
sequencing
Buenos
available
Aires,
at
ready-reaction Argentina)
Cromatida
system
according
S.A.
(Buenos
(Applied to
the
Aires,
152
Argentina). In the PHRI Center, DNA sequencing was performed
153
with
154
(Beckman Coulter, Fullerton, CA) according to the manufacturer’s
155
recommendations. Sequencing analyses were done with CEQ 8000
156
genetic analysis system software (Beckman Coulter) and with the
157
BioEdit sequence alignment editor (Ibis Therapeutics, Carlsbad,
158
CA).
159
RESULTS
160
Multiplex PCR reaction design.
161
named PCR control primers and mutation primers. These primers
162
were mixed to form the set of 5 multiplex PCR tubes. Mutation
163
primers were designed to hybridize FKS1 hot spot 1 and hot spot
164
2 regions by considering that a primer with a 3´ mismatch would
165
not hybridize under the appropriate conditions of temperature.
166
The PCR control group of primers were designed to specifically
167
hybridize C. albicans FKS1 gene hot spot flanking regions (nt.
168
1752 to 2232 and nt. 3518 to 4266). The 3´ and 5´ ends of these
169
regions
a
CEQ
dye
share
terminator
low
homology
cycle
sequencing
QuickStart
kit
We designed two sets of primers
with
C.
albicans
FKS2
and
FKS3,
170
giving
171
internal control for PCR validation (DNA quality and the absence
172
of PCR inhibitors) considering that a hot spot mutation would
173
produce
174
variables, including annealing temperature were established in
175
order to use the same PCR program regardless the used primer
176
set. For the detection of amino acid substitutions at the Fks1p
177
hot spot 1, 4 PCR tuber were used. Each of the 4 multiplex PCR
178
tubes contained a pair of control PCR primer (1752-F and 2232-R)
179
and one of the mutation detection primers named F641, S645, D648
180
and P649. PCR reactions gave one 481–bp band in all the tubes
181
(corresponding
182
primers) and a second band of 332-bp, 200-bp, 211-bp or 307-bp
183
when the template DNA was wild type at the codon that codified
184
the F641, S645, D648 or P649, respectively. Similarly, for the
185
detection of hot spot 2 substitutions at residue R1361, the last
186
PCR tube of the set included the primers 3518-F, 4266-R and
187
R1361. PCR reaction with wild type DNA gave two bands (769-bp
FKS1
a
specificity.
negative
to
PCR
the
These
result.
primer
The
amplification
pairs
multiplex
with
the
were
PCR
PCR
used
as
reaction
control
188
and 202-bp corresponding to control band and wild type band,
189
respectively). On the other hand, when mutant DNAs were used, a
190
unique PCR band was obtained in one of the tubes corresponding
191
to the control PCR (a 481-bp or 769-bp for HS1 or HS2 mutants,
192
respectively) (Figure 2).
193
Test
validation
using
a
blinded
collection
of
strains.
A
194
validation study of the 5-tube multiplex PCR assay was performed
195
using a blinded collection of 50 clinical C. albicans strains
196
that
197
different amino acid substitutions in Fks1p hot spots (Table 1).
198
The resistant strains were mostly homozygous mutants with the
199
exception of three isolates that harbor heterozygous mutations
included
13
echinocandin
resistant
strains
harboring
200
(Table 1).
201
albicans strains were considered FKS1 wild-type (echinocandin
202
susceptible) strains. The remaining 11 strain were classified as
203
echinocandin
204
substitutions at Fks1p at the following residues: F641 (n=3),
205
S645 (n=5), D648 (n=1), P649 (n=1) and R1361 (n=1). When these
206
results were compared with the AST results, 48 strains (96%)
207
were
208
resistant. Moreover, when our results were matched up with the
209
FKS1 sequencing results, 47 isolates (94%) showed equivalent
210
results. The described false negative results were all due to
211
the
212
mutations at S645S/P (strain A15) and at R1361R/H (strain M90)
213
and the third showed one hot spot 1 homozygous mutation (S645F)
214
together with one hot spot 2 heterozygous mutation at R1361R/H
215
(strain M194). This last isolate was classified as hot spot 1
216
mutant (S645) and hot spot 2 wild-type by our method, hence it
217
was considered as an echinocandin resistant strain. Similarly,
Using the described 5 tubes multiplex PCR, 39 C.
resistant
correctly
mutants
classified
heterozygous
mutants.
as
harboring
echinocandin
These
mutants
amino
susceptible
showed
acid
or
heterozygous
218
this
219
However,
220
homozygous S645F and the heterozygous mutation at R1361R/H not
221
detected by the multiplex PCR.
strain
222 223 224
DISCUSSION
by
was FKS1
considered sequencing
echinocandin this
strain
resistant
by
AST.
showed
both
the
225
It
226
antifungal therapy reduces candidemia mortality rates (13,23).
227
Multiple
228
patients risk factors to direct early empiric antifungal therapy
229
(8,17,18,25).
230
selection of a correct antifungal treatment depends on AST due
231
to
232
methods need at least 24 hours to obtain confident results for
233
therapy initiation (6,7,33). In the present work, a molecular-
234
based assay for rapid identification of echinocandin resistant
235
C. albicans strains was developed and evaluated. The described
236
methodology is able to detect the most common FKS1 mutations
237
linked with clinical echinocandin resistance in 4 hours and it
238
is based in the strict relationship between C. albicans FKS1
239
mutations and echinocandin treatment failure. The described C.
240
albicans FKS1 mutations comprise substitutions in five amino
241
acid residues, four at the hot spot 1 region (F641, S645, D648
242
and P649) and one residue at the hot spot 2 region (R1361)
is
well
the
understood
efforts
that
have
been
However,
emergence
of
rapid
it
initiation
evaluated
is
to
use
increasingly
resistance
of
(2,9,28).
appropriate
biomarkers
clear
The
that
or
the
available
AST
243
(2,28). Since C. albicans is a diploid organism, the described
244
substitutions
245
states (4,12). These mutations are genetically dominant and most
246
of
247
(2,4,12,27,28).
248
echinocandin
249
(2,10,12,28).
250
heterozygous
251
therapy and would act as an intermediate state for a stepwise
them
can
confer
be
found
in
both
cross-resistance However,
to
all
heterozygous
MIC
values
One
possible
mutants
homo-
and
would
and
heterozygotic
echinocandin
mutations
are
confer
uncommonly
explanation
is
respond
vivo
in
that
isolated
most
to
drugs lower
of
the
echinocandin
252
development
253
phenotype,
254
laboratory
255
(4,14).
256
In the blinded study, we demonstrated that the method described
257
here
258
described residues including F641S, F641L, S645P, S645Y, S645F,
259
D648Y, P649H and R1361H, which accounts for more than 98% of
260
known resistance in C. albicans. There is an important method-
261
inherent
is
of as
the
mutants
able
homozygous
described
to
and
for
detect
limitation
for
that
mutant C.
clinical
homozygous
is
the
displaying
albicans C.
in
complete
spontaneous
tropicalis
substitutions
difficulty
of
in
isolates
all
the
uncovering
262
heterozygous
263
considered a very major error when compared with sequencing or
264
AST given that our PCR method would classify as susceptible a
265
resistant strain. However, as described earlier in this section,
266
heterozygous mutants represent a tiny minority (3,4,10-12,15,26-
267
29,36).
268
susceptibility testing should be performed coupled with this
269
assay to avoid any potential false results.
270
In 2006, Balashov et al. reported an allele-specific real-time
In
mutants.
view
These
of
this
false
negative
limitation,
results
whole
cell
would
be
antifungal
271
PCR molecular-beacon assay able to identify both heterozygous
272
and homozygous mutations that alters the C. albicans Fks1p S645
273
residue (4). The method´s advantages are that it is based on a
274
more commonly available classical PCR method and that is able to
275
detect
276
residues of the hot spot regions. As any molecular-based method
277
designed
278
resistance, this method would be unable to detect non-FKS-linked
279
echinocandin resistance mechanisms or to detect newly described
280
mutations. However, the described set of PCRs is suitable to be
281
adapted
282
epidemiology changes.
283
In summary, the described PCR method proved to be a quick,
284
simple and inexpensive tool able to detect the main mechanism of
285
echinocandin resistance in C. albicans.
all
the
for
to
described
the
detect
homozygous
detection
other
of
mutations
molecular
resistance
in
the
other
mechanisms
mechanisms
of
accompanying
286 287
ACKNOWLEDGMENTS
288
This study was supported by Consejo Nacional de Investigaciones
289
Científicas y Técnicas (CONICET) grant PIP2011/331 to GGE and by
290
a Universidad Nacional del Litoral (UNL) grant (CAID-PIRCA) to
291
GGE. and SG. CD and FL have a doctoral fellowship from CONICET.
292
The
293
Health (NIH) grant AI109025 to DSP and Astellas.
Perlin
laboratory
294 295 296 297
FIGURE LEGENDS
was
funded
By
National
Institutes
of
298
Figure 1: (A) Representation of C. albicans FKS1 gene including
299
the hot spot regions (white boxes). Arrows represent the control
300
group primers (filled arrows) and the mutation detection primers
301
(dashed arrows). (B) Alignment of the mutation detection primers
302
with the wild-type (WT) FKS1. Underlined amino acids show the
303
hot
304
detected by the proposed method.
spot
regions.
Nucleotides
in
bold
show
the
mutations
305 306
Figure
307
included in each of the PCR tubes are above the photograph. Lane
308
M,
309
(echinocandin susceptible strains). Lane 4: C. albicans strain
310
M177 (Fks1p-F641S). Lane 5: Strain M119 (Fks1p-F641L). Lane 8:
311
Strain M122 (Fks1p-P649H). Lane 10: Strain A15 (Fks1p-S645S/P-
312
heterozygous mutant). Lane 11: Strain A15-10 (Fks1p-S645P). Lane
313
12:
314
D648Y).
315
mutant). Lane 18: Strain M121 (Fks1p-R1361H).
316 317
2:
Electrophoresis
molecular
Strain
marker.
M85
Lane
Lanes
gel
named
(Fks1p-S645F). 17:
Strain
(1.5%
M90
as
Lane
agarose).
WT,
15:
wild
Strain
(Fks1p-R1361R/H
The
type
M149
primers
strains
(Fks1p-
heterozygous
318 319 320
TABLE 1: Classical PCR set, DNA sequencing, and in vitro susceptibility determinations of the C. glabrata strains included in this study. Strains
a
WT (n=37) 2762 M119 M177 M205 M89 M85 M194 M149 M122 A15 d A15-10 d M121 M90
Classical PCR set results a F641 S645 D648 P649 R1361 + + + + + + + + + + +
+ + + + + + + + +
+ + + + + + + + + + + + +
+ + + + + + + + + + + + +
+ + + + + + + + + + + + +
DNA sequencing results b Fks1 Fks1 hot spot 1 hot spot 2 WT WT F641S WT F641L WT F641S WT S645P WT S645Y WT S645F WT S645F R1361R/H D648Y WT P649H WT S645S/P WT S645P WT WT R1361H WT R1361R/H
ANF
MIC (µg/ml)c CSF
MCF
0.05 (S) 0.50 (I)
0.06 (S) 4.00 (R)
0.03 (S) 1.00 (R)
0.15 0.50 1.26 2.00 1.50 4.00 4.00 0.15 0.50 2.00 0.25 0.25
2.00 4.00 8.00 4.00 2.00 4.00 4.00 2.52 4.00 8.00 2.00 1.00
0.25 1.00 4.00 4.00 2.52 2.67 2.52 0.50 0.50 4.00 0.25 0.50
(S) (I) (R) (R) (R) (R) (R) (S) (R) (R) (S) (S)
(R) (R) (R) (R) (R) (R) (R) (R) (R) (R) (R) (R)
(S) (R) (R) (R) (R) (R) (R) (I) (R) (R) (S) (R)
321 322
a
323 324
b
WT: wild type at the corresponding hot spot (Hot spot 1: 641-FLTLSLRDP-649 and hot spot 2: 1357DWIRRYTL-1364).
325 326 327
c Expressed MIC values are geometric mean of data obtained on three separate days in µg/ml (for the 37 wild type strains the presented data is the geometric mean of the MIC values of all the strains). ANF, anidulafungin. CSF, caspofungin. MCF, micafungin. Letters in parenthesis indicate that the strain is
Signs represents the presence (+) or the absence (-) of the PCR band in a electrophoresis gel.
328 329
considered echinocandin susceptible interpretative guidelines.
330 331
d Isogenic laboratory-obtained spontaneous echinocandin resistant mutants isolated by exposing strain Sc5314 to different ANF concentrations (12).
332 333
(S),
intermediate
(I)
or
resistant
(R)
using
the
CLSI
M27-S4
334
Table 2: Oligonucleotide primers used in this study. 5´ 3´ sequence
Primer orientation
FKS1 HS1 AfS and AC
CATGCCATTGGGTGGTTTAT
Sense
FKS1
FKS1 HS1 sequencing and AC
GATTTCCATTTCCGTGGTAGC
Antisense
FKS1
FKS1 HS2 AfS and AC
CTGGTGTTTTGGGTGATGTTGC
Sense
4266-R
FKS1
FKS1 HS2 sequencing and AC
GGTCAAATCAGTGAAAACCG
Antisense
F641
FKS1
Mutation detection
AATTGGTTGAATCTTATTTCTT
Sense
S645
FKS1
Mutation detection
CTAATAGGATCTCTTAAAGA
Antisense
D648
FKS1
Mutation detection
CGACAAGTTTCTAATAGGATC
Antisense
P649
FKS1
Mutation detection
GACATTGTCTTTAAGAGATCC
Sense
Oligonucleotide
Target gene
1752-F
FKS1
2232-R 3518-F
R1361
FKS1
Mutation detection
CGTTGATTGGATTAGACG
Sense
FKS1
FKS1 HS1 sequencing
CCTTGCCAAATTGGTTGAATC
Sense
3904-F
FKS1
ITS1 ITS4
a
335 a
c
1892-F
a
336
Purpose
From reference (35).
FKS1 HS2 sequencing
TACTATGGTCATCCAGGTTTCCA
Sense
r DNA
b
Molecular identification
TCCGTAGGTGAACCTGCGG
Sense
r DNA
b
Molecular identification
TCCTCCGCTTATTGATATGC
Antisense
337 338 339 340 341 342 343 344 345
b c
rDNA, ribosomal DNA AfS, amplification for subsequent sequencing; AC, amplification control; HS1, hot spot 1.
346 347 348
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349 350 351 352 353 354 355 356
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27. Perlin DS. 2007. Resistance to echinocandin-class antifungal drugs. Drug Resist. Updat. 10:p 121-130.
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529 530 531 532
35. White TJ, T.D.Bruns, S.B.Lee, J.W.Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, p 315-322. In: M.A.Innis, D.H.Gelfand, J.J.Sninsky, T.J.White (eds), PCR protocols: a
533 534
guide to methods and applications. Academic Press, Inc., San Diego, Calif.
535 536 537 538 539 540 541
36. Wiederhold NP, Grabinski JL, Garcia-Effron G, Perlin DS, Lee SA. 2008. Pyrosequencing to detect mutations in FKS1 that confer reduced echinocandin susceptibility in Candida albicans. Antimicrob. Agents Chemother. 52:p 4145-4148. doi:AAC.00959-08 [pii];10.1128/AAC.00959-08 [doi].
A)
1752-F
2232-R F641
P649 Hot Spot 1
3518-F
4266-R R1361 Hot Spot 2
S645 D648
B) WT Fks1p (HS1) WT (+) Strand WT (-) Strand F641 S645 D648 P649
K L V E S Y F F L T L S L R D P I R N L S T AAATTGGTTGAATCTTATTTCTTCTTGACATTGTCTTTAAGAGATCCTATTAGAAACTTGTCGACC TTTAACCAACTTAGAATAAAGAAGAACTGTAACAGAAATTCTCTAGGATAATCTTTGAACAGCTGG -AATTGGTTGAATCTTATTTCTT---------------------------------------------------------------------------AGAAATTCTCTAGGATAATC------------------------------------------------------CTAGGATAATCTTTGAACAGC----------------------------GACATTGTCTTTAAGAGATCC-------------------
WT Fks1p (HS2) Y N I A P A V D W I R R Y T L S I F I V F F WT (+) Strand TACAATATTGCTCCTGCCGTTGATTGGATTAGACGTTATACTTTGTCTATTTTCATTGTTTTCTTC WT (-) Strand ATGTTATAACGAGGACGGCAACTAACCTAATCTGCAATATGAAACAGATAAAAGTAACAAAAGAAG R1361 -----------------CGTTGATTGGATTAGACG-------------------------------
F641 1752-F/2232-R M
800 500 300 100
WT
F641S
F641L
P649 1752-F/2232-R WT
WT
P649H
S645 1752-F/2232-R WT
S645P/S
S645P
S645F
D648 1752-F/2232-R WT
WT
D648Y
R1361 3518-F/4266-R WT
R1361R/H R1361H
1
Title:
2
Quick
3
Echinocandin Resistance in Candida albicans.
4
Catiana Dudiuk1, 2, Soledad Gamarra1, Cristina Jimenez-Ortigosa3,
5
Florencia
6
Guillermo Garcia-Effron1,
7
1
8
Parasitología y Micología – Facultad de Bioquímica y Ciencias
9
Biológicas
detection
of
FKS1
Leonardelli1,
Mutations
2
,
Daiana
Responsible
Macedo1,
for
David
S.
Clinical
Perlin3,
2*
.
Laboratorio de Micología y Diagnóstico Molecular - Cátedra de
10
(Argentina).
11
2
–
Universidad
Nacional
del
Litoral.
Santa
Fe
Consejo Nacional de Investigaciones Científicas y Tecnológicas
12
(CONICET). CCT-Santa Fe (Argentina).
13
3
14
Health Sciences– Newark, NJ (USA).
Public
Health
Research
Institute
–
Rutgers
Biomedical
and
15 16
Key words: Candida albicans, resistance detection, echinocandin.
17
Running title: PCR for the detection of Echinocandin resistance
18
in C. albicans.
19 20
*Corresponding author: Dr. Guillermo Garcia-Effron. Laboratorio
21
de
22
Parasitología y Micología – Facultad de Bioquímica y Ciencias
23
Biológicas – Ciudad Universitaria – Paraje el Pozo S/N – Santa
24
Fe (Santa Fe) – CP 3000 – Argentina.
25
e-mail: [email protected] Phone: +54-342-4575209 int. 135. Fax:
26
+54-342-4575216.
27 28
Micología
y
Diagnóstico
Molecular
(CONICET)
–
Cátedra
de
29
ABSTRACT
30
A
31
albicans
32
echinocandin
33
consisted of a multiplexed PCR set of 5 tubes able to detect the
34
most commonly described resistance mechanism, including FKS1 hot
35
spot 1 and hot spot 2 mutations. The performance and specificity
36
of the assay was evaluated using a double blinded panel of 50 C.
37
albicans strains. The assay showed a sensitivity of 96% and was
38
able to detect all homozygous mutants included in the collection
39
of strains demonstrating that is a robust, quick and labour
40
saving method that is suitable for a routine clinical diagnostic
41
laboratory.
42
rapid
molecular-based FKS1
gene drugs,
assay
mutations was
for
the
detection
responsible
designed
and
for
of
Candida
resistance
evaluated.
The
to
assay
43
INTRODUCTION
44
Invasive infections due to Candida albicans are a common and
45
significant
46
echinocandin drugs are becoming the antifungal of choice for the
47
management of these infections (8,22,30,32). This expanding use
48
of
49
resistance (3,10-12,15,19,20).
50
Echinocandin in vitro antifungal susceptibility testing (AST) is
51
being
52
(1,6,7,33). However, testing for antifungal resistance is not
53
routinely
54
resistance in C. albicans resulting in therapeutic failures is
clinical
echinocandins
performed
has
problem
brought
worldwide
performed
at
(5,8,21,24,34),
about
to
many
the
guide
and
emergence
therapeutic
centers.Clinical
of
the
drug
decisions
echinocandin
55
closely linked with amino acid substitutions in the hot spot
56
regions
57
complex (1,10-12,15,26,27). The detection of these mutations has
58
been proposed as the most direct and accurate way to predict
59
echinocandin clinical failure (1,4,12,27).
60
The aim of this work was to develop a molecular -based method
of
the
Fks1p
subunit
of
the
β-D-1,3-glucan
synthase
61
able to quickly and accurately detect the FKS1 mutations linked
62
with
63
performance of the proposed methodology was evaluated using a
64
blinded collection of clinical echinocandin-susceptible and -
65
resistant C. albicans isolates.
clinical
echinocandin
resistance
in
C.
albicans.
The
66 67
MATERIALS AND METHODS
68
Strains. Fifty C. albicans strains were used throughout this
69
work. All the strains were isolated from patients with proven
70
invasive fungal disease. Sixteen strains were obtained from the
71
Public Health Research Institute (PHRI; Rutgers Biomedical and
72
Health Sciences, NJ) and 34 from the Mycology and Molecular
73
Diagnostics Laboratory (LMDM) (Santa Fe, Argentina). Ten strains
74
showed homozygous FKS1 hot spot region mutations, two strains
75
showed heterozygous mutations at one of the hot spot regions and
76
one showed an homozygous mutation at the hot spot 1 of the FKS1
77
gene together with an heterozygous mutation at the hot spot 2 of
78
the FKS1 (Table 1). C. albicans ATCC 90028, ATCC 36082 and
79
SC5314 were used as the wild-type control strains to validate
80
the PCRs. C. krusei ATCC 6258 and C. parapsilosis sensu stricto
81
ATCC 22019 were used as AST control strains (6,7). The isolates
82
were
83
sequencing
84
transcribed
identified of
by
conventional
the
spacer
5.8S 1
RNA
(ITS1)
phenotypic
gene and
and
ITS2
methods
adjacent
regions
and
by
internal
(16,35).
The
85
collection of strains was assembled at the PHRI center, and
86
blinded code numbers were assigned. Also, a set of C. albicans
87
strains with known FKS1 mutations were used to develop and test
88
the proposed methodology before confirming its utility with the
89
blind study.
90
Antifungals and susceptibility testing. Caspofungin (CSF; Merck
91
& Co. Inc., Rahway, NJ), anidulafungin (ANF; Pfizer, New York,
92
NY), and micafungin (MCF; Astellas Pharma USA Inc., Deerfield,
93
IL)
94
manufacturers. Echinocandin susceptibility testing was performed
95
in
96
following the interpretive guidelines published in the M27-S4
97
document (6,7).
98
DNA isolation, primer and PCR design. C. albicans genomic DNAs
99
were extracted with phenol-chloroform method (31) or with a Q-
were
obtained
triplicate
in
as
standard
accordance
powder
with
from
CLSI
their
document
respective
M27-A3
and
100
Biogene
101
instructions.
102
numbers XM_446406 was used for primer design. Two groups of
103
primers were used throughout this work. The first primer pair,
104
named PCR control primers, consisted of two primer pairs: 1752-F
105
and
FastDNA
2232-R
C.
and
kit
(Q-Biogene)
albicans
3518-F
FKS1
and
following
gene
4266-R.
with
These
manufacturer´s
GenBank
primer
accession
pairs
were
106
designed to specifically hybridize FKS1 hot spot 1 and hot spot
107
2 regions, respectively. They were used as amplification control
108
in each of the multiplex PCR tubes. The second group of primers
109
(mutation detection primers) included five oligonucleotides that
110
were designed to detect the 8 most common mutations related with
111
echinocandin
112
sequences in Table 2). These mutation detection primers align
113
the FKS1 hot spot 1 (primers F641, S645, D648 and P649) and hot
114
spot 2 regions (primer R1361) and were added to one of the five
resistance
in
C.
albicans
(oligonucleotide
115
tubes of the PCR set, which already contained one pair of PCR
116
control primers. The hot spot 1 mutation detection primers were
117
paired with hot spot 1 PCR control primers (1752-F and 2232-R),
118
while the primer R1361 was used together with the hot spot 2
119
control primers (3518-F and 4266-R) (Figure 1). Primers were
120
designed
121
SciTools (Integrated DNA Technologies, Coralville, IA) and were
122
purchased
123
Buenos Aires, Argentina). Amplifications were carried out in a
124
25 µl volume of a mixture containing 5 mM (NH4)2SO4, 5 mM KCl,
125
10
126
albumin, 0.1% Triton X-100, 125 µM each dATP, dGTP, dCTP, and
127
dTTP
128
concentration of each of the three primers, 1.25 U of Pegasus
129
DNA polymerase (PBL, Embiotec, Buenos Aires, Argentina), and 10
130
to 25 ng of C. albicans genomic DNA. Amplification was performed
131
for one initial step of 2 min at 94°C followed by 25 cycles of
132
30 s at 94°C, 30 s at 57°C, and 1 min at 72°C and then a final
133
cycle of 10 min at 72°C in an Applied Biosystems thermocycler
134
(Tecnolab-AB, Buenos Aires, Argentina). The PCR products were
135
analyzed by electrophoresis.
136
DNA
137
(flanking the hot spot 1 and hot spot 2 regions between the nts.
138
1892 to 2232 and nts. 3904 to 4266, respectively) and 5.8s RNA
139
gene and adjacent internal transcribed spacer 1 (ITS1) and ITS2
140
regions were amplified and sequenced in both directions using
141
the primers described in Table 2. For sequencing of the FKS1
142
regions, primer pair 1752-F/2232-R and 3518-F/4266-R were used
143
for
144
subjected to sequencing using primers 1892-F and 2232-R for hot
mM
using
from
Tris-Cl
the
oligonucleotide
Integrated
(pH
8.8),
(Genbiotech,
sequencing.
PCR
DNA
1
Two
MgSO4,
Aires,
regions
amplification.
Technologies
Mm
Buenos
The
design
of
5
ng
tool
of
C.
purified
IDT
(IDT-Biodynamics,
of
bovine
Argentina),
the
the
a
0.5
albicans
fragments
serum
FKS1
were
µM
gene
then
145
spot 1 region and 3904-F and 4266-R for hot spot region 2 (Table
146
2). In Argentina, DNA sequencing was performed using a BigDye
147
Terminator
148
Biosystems,
149
manufacturer’s instructions. Sequence analysis was performed on
150
an ABI Prism 310 DNA sequencer (Applied Biosystems) using the
151
facilities
cycle
sequencing
Buenos
available
Aires,
at
ready-reaction Argentina)
Cromatida
system
according
S.A.
(Buenos
(Applied to
the
Aires,
152
Argentina). In the PHRI Center, DNA sequencing was performed
153
with
154
(Beckman Coulter, Fullerton, CA) according to the manufacturer’s
155
recommendations. Sequencing analyses were done with CEQ 8000
156
genetic analysis system software (Beckman Coulter) and with the
157
BioEdit sequence alignment editor (Ibis Therapeutics, Carlsbad,
158
CA).
159
RESULTS
160
Multiplex PCR reaction design.
161
named PCR control primers and mutation primers. These primers
162
were mixed to form the set of 5 multiplex PCR tubes. Mutation
163
primers were designed to hybridize FKS1 hot spot 1 and hot spot
164
2 regions by considering that a primer with a 3´ mismatch would
165
not hybridize under the appropriate conditions of temperature.
166
The PCR control group of primers were designed to specifically
167
hybridize C. albicans FKS1 gene hot spot flanking regions (nt.
168
1752 to 2232 and nt. 3518 to 4266). The 3´ and 5´ ends of these
169
regions
a
CEQ
dye
share
terminator
low
homology
cycle
sequencing
QuickStart
kit
We designed two sets of primers
with
C.
albicans
FKS2
and
FKS3,
170
giving
171
internal control for PCR validation (DNA quality and the absence
172
of PCR inhibitors) considering that a hot spot mutation would
173
produce
174
variables, including annealing temperature were established in
175
order to use the same PCR program regardless the used primer
176
set. For the detection of amino acid substitutions at the Fks1p
177
hot spot 1, 4 PCR tuber were used. Each of the 4 multiplex PCR
178
tubes contained a pair of control PCR primer (1752-F and 2232-R)
179
and one of the mutation detection primers named F641, S645, D648
180
and P649. PCR reactions gave one 481–bp band in all the tubes
181
(corresponding
182
primers) and a second band of 332-bp, 200-bp, 211-bp or 307-bp
183
when the template DNA was wild type at the codon that codified
184
the F641, S645, D648 or P649, respectively. Similarly, for the
185
detection of hot spot 2 substitutions at residue R1361, the last
186
PCR tube of the set included the primers 3518-F, 4266-R and
187
R1361. PCR reaction with wild type DNA gave two bands (769-bp
FKS1
a
specificity.
negative
to
PCR
the
These
result.
primer
The
amplification
pairs
multiplex
with
the
were
PCR
PCR
used
as
reaction
control
188
and 202-bp corresponding to control band and wild type band,
189
respectively). On the other hand, when mutant DNAs were used, a
190
unique PCR band was obtained in one of the tubes corresponding
191
to the control PCR (a 481-bp or 769-bp for HS1 or HS2 mutants,
192
respectively) (Figure 2).
193
Test
validation
using
a
blinded
collection
of
strains.
A
194
validation study of the 5-tube multiplex PCR assay was performed
195
using a blinded collection of 50 clinical C. albicans strains
196
that
197
different amino acid substitutions in Fks1p hot spots (Table 1).
198
The resistant strains were mostly homozygous mutants with the
199
exception of three isolates that harbor heterozygous mutations
included
13
echinocandin
resistant
strains
harboring
200
(Table 1).
201
albicans strains were considered FKS1 wild-type (echinocandin
202
susceptible) strains. The remaining 11 strain were classified as
203
echinocandin
204
substitutions at Fks1p at the following residues: F641 (n=3),
205
S645 (n=5), D648 (n=1), P649 (n=1) and R1361 (n=1). When these
206
results were compared with the AST results, 48 strains (96%)
207
were
208
resistant. Moreover, when our results were matched up with the
209
FKS1 sequencing results, 47 isolates (94%) showed equivalent
210
results. The described false negative results were all due to
211
the
212
mutations at S645S/P (strain A15) and at R1361R/H (strain M90)
213
and the third showed one hot spot 1 homozygous mutation (S645F)
214
together with one hot spot 2 heterozygous mutation at R1361R/H
215
(strain M194). This last isolate was classified as hot spot 1
216
mutant (S645) and hot spot 2 wild-type by our method, hence it
217
was considered as an echinocandin resistant strain. Similarly,
Using the described 5 tubes multiplex PCR, 39 C.
resistant
correctly
mutants
classified
heterozygous
mutants.
as
harboring
echinocandin
These
mutants
amino
susceptible
showed
acid
or
heterozygous
218
this
219
However,
220
homozygous S645F and the heterozygous mutation at R1361R/H not
221
detected by the multiplex PCR.
strain
222 223 224
DISCUSSION
by
was FKS1
considered sequencing
echinocandin this
strain
resistant
by
AST.
showed
both
the
225
It
226
antifungal therapy reduces candidemia mortality rates (13,23).
227
Multiple
228
patients risk factors to direct early empiric antifungal therapy
229
(8,17,18,25).
230
selection of a correct antifungal treatment depends on AST due
231
to
232
methods need at least 24 hours to obtain confident results for
233
therapy initiation (6,7,33). In the present work, a molecular-
234
based assay for rapid identification of echinocandin resistant
235
C. albicans strains was developed and evaluated. The described
236
methodology is able to detect the most common FKS1 mutations
237
linked with clinical echinocandin resistance in 4 hours and it
238
is based in the strict relationship between C. albicans FKS1
239
mutations and echinocandin treatment failure. The described C.
240
albicans FKS1 mutations comprise substitutions in five amino
241
acid residues, four at the hot spot 1 region (F641, S645, D648
242
and P649) and one residue at the hot spot 2 region (R1361)
is
well
the
understood
efforts
that
have
been
However,
emergence
of
rapid
it
initiation
evaluated
is
to
use
increasingly
resistance
of
(2,9,28).
appropriate
biomarkers
clear
The
that
or
the
available
AST
243
(2,28). Since C. albicans is a diploid organism, the described
244
substitutions
245
states (4,12). These mutations are genetically dominant and most
246
of
247
(2,4,12,27,28).
248
echinocandin
249
(2,10,12,28).
250
heterozygous
251
therapy and would act as an intermediate state for a stepwise
them
can
confer
be
found
in
both
cross-resistance However,
to
all
heterozygous
MIC
values
One
possible
mutants
homo-
and
would
and
heterozygotic
echinocandin
mutations
are
confer
uncommonly
explanation
is
respond
vivo
in
that
isolated
most
to
drugs lower
of
the
echinocandin
252
development
253
phenotype,
254
laboratory
255
(4,14).
256
In the blinded study, we demonstrated that the method described
257
here
258
described residues including F641S, F641L, S645P, S645Y, S645F,
259
D648Y, P649H and R1361H, which accounts for more than 98% of
260
known resistance in C. albicans. There is an important method-
261
inherent
is
of as
the
mutants
able
homozygous
described
to
and
for
detect
limitation
for
that
mutant C.
clinical
homozygous
is
the
displaying
albicans C.
in
complete
spontaneous
tropicalis
substitutions
difficulty
of
in
isolates
all
the
uncovering
262
heterozygous
263
considered a very major error when compared with sequencing or
264
AST given that our PCR method would classify as susceptible a
265
resistant strain. However, as described earlier in this section,
266
heterozygous mutants represent a tiny minority (3,4,10-12,15,26-
267
29,36).
268
susceptibility testing should be performed coupled with this
269
assay to avoid any potential false results.
270
In 2006, Balashov et al. reported an allele-specific real-time
In
mutants.
view
These
of
this
false
negative
limitation,
results
whole
cell
would
be
antifungal
271
PCR molecular-beacon assay able to identify both heterozygous
272
and homozygous mutations that alters the C. albicans Fks1p S645
273
residue (4). The method´s advantages are that it is based on a
274
more commonly available classical PCR method and that is able to
275
detect
276
residues of the hot spot regions. As any molecular-based method
277
designed
278
resistance, this method would be unable to detect non-FKS-linked
279
echinocandin resistance mechanisms or to detect newly described
280
mutations. However, the described set of PCRs is suitable to be
281
adapted
282
epidemiology changes.
283
In summary, the described PCR method proved to be a quick,
284
simple and inexpensive tool able to detect the main mechanism of
285
echinocandin resistance in C. albicans.
all
the
for
to
described
the
detect
homozygous
detection
other
of
mutations
molecular
resistance
in
the
other
mechanisms
mechanisms
of
accompanying
286 287
ACKNOWLEDGMENTS
288
This study was supported by Consejo Nacional de Investigaciones
289
Científicas y Técnicas (CONICET) grant PIP2011/331 to GGE and by
290
a Universidad Nacional del Litoral (UNL) grant (CAID-PIRCA) to
291
GGE. and SG. CD and FL have a doctoral fellowship from CONICET.
292
The
293
Health (NIH) grant AI109025 to DSP and Astellas.
Perlin
laboratory
294 295 296 297
FIGURE LEGENDS
was
funded
By
National
Institutes
of
298
Figure 1: (A) Representation of C. albicans FKS1 gene including
299
the hot spot regions (white boxes). Arrows represent the control
300
group primers (filled arrows) and the mutation detection primers
301
(dashed arrows). (B) Alignment of the mutation detection primers
302
with the wild-type (WT) FKS1. Underlined amino acids show the
303
hot
304
detected by the proposed method.
spot
regions.
Nucleotides
in
bold
show
the
mutations
305 306
Figure
307
included in each of the PCR tubes are above the photograph. Lane
308
M,
309
(echinocandin susceptible strains). Lane 4: C. albicans strain
310
M177 (Fks1p-F641S). Lane 5: Strain M119 (Fks1p-F641L). Lane 8:
311
Strain M122 (Fks1p-P649H). Lane 10: Strain A15 (Fks1p-S645S/P-
312
heterozygous mutant). Lane 11: Strain A15-10 (Fks1p-S645P). Lane
313
12:
314
D648Y).
315
mutant). Lane 18: Strain M121 (Fks1p-R1361H).
316 317
2:
Electrophoresis
molecular
Strain
marker.
M85
Lane
Lanes
gel
named
(Fks1p-S645F). 17:
Strain
(1.5%
M90
as
Lane
agarose).
WT,
15:
wild
Strain
(Fks1p-R1361R/H
The
type
M149
primers
strains
(Fks1p-
heterozygous
318 319 320
TABLE 1: Classical PCR set, DNA sequencing, and in vitro susceptibility determinations of the C. glabrata strains included in this study. Strains
a
WT (n=37) 2762 M119 M177 M205 M89 M85 M194 M149 M122 A15 d A15-10 d M121 M90
Classical PCR set results a F641 S645 D648 P649 R1361 + + + + + + + + + + +
+ + + + + + + + +
+ + + + + + + + + + + + +
+ + + + + + + + + + + + +
+ + + + + + + + + + + + +
DNA sequencing results b Fks1 Fks1 hot spot 1 hot spot 2 WT WT F641S WT F641L WT F641S WT S645P WT S645Y WT S645F WT S645F R1361R/H D648Y WT P649H WT S645S/P WT S645P WT WT R1361H WT R1361R/H
ANF
MIC (µg/ml)c CSF
MCF
0.05 (S) 0.50 (I)
0.06 (S) 4.00 (R)
0.03 (S) 1.00 (R)
0.15 0.50 1.26 2.00 1.50 4.00 4.00 0.15 0.50 2.00 0.25 0.25
2.00 4.00 8.00 4.00 2.00 4.00 4.00 2.52 4.00 8.00 2.00 1.00
0.25 1.00 4.00 4.00 2.52 2.67 2.52 0.50 0.50 4.00 0.25 0.50
(S) (I) (R) (R) (R) (R) (R) (S) (R) (R) (S) (S)
(R) (R) (R) (R) (R) (R) (R) (R) (R) (R) (R) (R)
(S) (R) (R) (R) (R) (R) (R) (I) (R) (R) (S) (R)
321 322
a
323 324
b
WT: wild type at the corresponding hot spot (Hot spot 1: 641-FLTLSLRDP-649 and hot spot 2: 1357DWIRRYTL-1364).
325 326 327
c Expressed MIC values are geometric mean of data obtained on three separate days in µg/ml (for the 37 wild type strains the presented data is the geometric mean of the MIC values of all the strains). ANF, anidulafungin. CSF, caspofungin. MCF, micafungin. Letters in parenthesis indicate that the strain is
Signs represents the presence (+) or the absence (-) of the PCR band in a electrophoresis gel.
328 329
considered echinocandin susceptible interpretative guidelines.
330 331
d Isogenic laboratory-obtained spontaneous echinocandin resistant mutants isolated by exposing strain Sc5314 to different ANF concentrations (12).
332 333
(S),
intermediate
(I)
or
resistant
(R)
using
the
CLSI
M27-S4
334
Table 2: Oligonucleotide primers used in this study. 5´ 3´ sequence
Primer orientation
FKS1 HS1 AfS and AC
CATGCCATTGGGTGGTTTAT
Sense
FKS1
FKS1 HS1 sequencing and AC
GATTTCCATTTCCGTGGTAGC
Antisense
FKS1
FKS1 HS2 AfS and AC
CTGGTGTTTTGGGTGATGTTGC
Sense
4266-R
FKS1
FKS1 HS2 sequencing and AC
GGTCAAATCAGTGAAAACCG
Antisense
F641
FKS1
Mutation detection
AATTGGTTGAATCTTATTTCTT
Sense
S645
FKS1
Mutation detection
CTAATAGGATCTCTTAAAGA
Antisense
D648
FKS1
Mutation detection
CGACAAGTTTCTAATAGGATC
Antisense
P649
FKS1
Mutation detection
GACATTGTCTTTAAGAGATCC
Sense
Oligonucleotide
Target gene
1752-F
FKS1
2232-R 3518-F
R1361
FKS1
Mutation detection
CGTTGATTGGATTAGACG
Sense
FKS1
FKS1 HS1 sequencing
CCTTGCCAAATTGGTTGAATC
Sense
3904-F
FKS1
ITS1 ITS4
a
335 a
c
1892-F
a
336
Purpose
From reference (35).
FKS1 HS2 sequencing
TACTATGGTCATCCAGGTTTCCA
Sense
r DNA
b
Molecular identification
TCCGTAGGTGAACCTGCGG
Sense
r DNA
b
Molecular identification
TCCTCCGCTTATTGATATGC
Antisense
337 338 339 340 341 342 343 344 345
b c
rDNA, ribosomal DNA AfS, amplification for subsequent sequencing; AC, amplification control; HS1, hot spot 1.
346 347 348
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A)
1752-F
2232-R F641
P649 Hot Spot 1
3518-F
4266-R R1361 Hot Spot 2
S645 D648
B) WT Fks1p (HS1) WT (+) Strand WT (-) Strand F641 S645 D648 P649
K L V E S Y F F L T L S L R D P I R N L S T AAATTGGTTGAATCTTATTTCTTCTTGACATTGTCTTTAAGAGATCCTATTAGAAACTTGTCGACC TTTAACCAACTTAGAATAAAGAAGAACTGTAACAGAAATTCTCTAGGATAATCTTTGAACAGCTGG -AATTGGTTGAATCTTATTTCTT---------------------------------------------------------------------------AGAAATTCTCTAGGATAATC------------------------------------------------------CTAGGATAATCTTTGAACAGC----------------------------GACATTGTCTTTAAGAGATCC-------------------
WT Fks1p (HS2) Y N I A P A V D W I R R Y T L S I F I V F F WT (+) Strand TACAATATTGCTCCTGCCGTTGATTGGATTAGACGTTATACTTTGTCTATTTTCATTGTTTTCTTC WT (-) Strand ATGTTATAACGAGGACGGCAACTAACCTAATCTGCAATATGAAACAGATAAAAGTAACAAAAGAAG R1361 -----------------CGTTGATTGGATTAGACG-------------------------------
F641 1752-F/2232-R M
800 500 300 100
WT
F641S
F641L
P649 1752-F/2232-R WT
WT
P649H
S645 1752-F/2232-R WT
S645P/S
S645P
S645F
D648 1752-F/2232-R WT
WT
D648Y
R1361 3518-F/4266-R WT
R1361R/H R1361H
