Staphylococcus aureus Genotyping Using Novel Real-Time PCR Formats

Flavia Huygens; John Inman-Bamber; Graeme R. Nimmo; Wendy Munckhof; Jacqueline Schooneveldt; Bruce Harrison; Jennifer A. McMahon; Philip M. Giffard

doi:10.1128/JCM.00843-06

DOI: 10.1128/JCM.00843-06

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FIG. 1.
Novel real-time PCR formats. (A) DMKP. The different SNPs or templates are shown as solid and dashed lines. Mismatched primers are depicted with detached 5′ ends. (B) Single-tube kinetic PCR. A single allele-specific reaction for each SNP is compared with a universal control reaction—the “perfectly matched” reaction from a conventional kinetic PCR SNP interrogation.
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FIG. 2.
Reproducibility of STKP interrogation of the 8 high-D S. aureus SNPs. The bars represent 2.58 standard deviations of the ΔC_T for each polymorph, calculated against the tpi36 universal control reactions. This was derived from 80 STKP assays.

Tables

Figures

TABLE 1.

Primer sequences for fluorescent (LUX) and unlabeled primers

Primer name	Sequence^a
L1	FAM-caaccaCAGGGTATGATAGGCTATTGGtTG
L2	JOE-gacattgTTGGATACTTGTGGTGCAATGtC
L4	JOE-cacaaaGCCCCATCAATATCAGTTTGtG
L5	FAM-cacaaaGCCCCATCAATATCAGTTTGtG
L1A	FAM-gtatccCGGCAATGCCATTGGAtAC
L2B	JOE-gacattgTTGGATACTTGTGGTGCAATGtC
L6	FAM-caccttACGTCAAATGCGTGAAGGtG
L7	JOE-catcaaTGCGTGAAGGTGAAGTTGAtG
L8	FAM-caatccCCTTGTGAATCAAGTTCTGGAtTG
L9	JOE-caatccCCTTGTGAATCAAGTTCTGGAtTG
L10	JOE-caatcgTTGATGATTTACCAGTTCCGAtTG
L11	FAM-caatcgTTGATGATTTACCAGTTCCGAtTG
L12	JOE-cagcttCTGCGACAAGTGAATTGAAAGCtG
L13	FAM-cagcttCTGCGACAAGTGAATTGAAAGCtG
L20	JOE-gatctgTGAAGGGAATGCAGCAGAtC
L14	FAM-gtacttcaCGGGATTTGCAGAATTTGAAGtAC
L18A	JOE-cacgcTAGGTGTTGTGCCTATGCGtG
L16	FAM-cacctgTATCTCTAACGGCTTGTCAGGtG
L22	JOE-caccgAACTTCTAATGACACTGGCGGtG
U1	CGTATAAAAAGGACCAATTGGTCTG*
U2	CGTATAAAAAGGACCAATTGGTCTA*
U3	CGTATAAAAAGGACCAATTGGTCTT*
U4	GTAAATCATCAACATCTGAAGATATG
U5	GTAAATCATCAACATCTGAAGATGTG
U6	GTAAATCATCAACATCTGAAGATGTA
U7	GATCATCTTTATCTACTTCCACACGTGCA*
U8	GATCATCTTTATCTACTTCCACACGTGCT*
U9	ACCTACTAATCGCTCTCTCAAGTAA*
U10	ACCTACTAATCGCTCTCTCAAGTAT*
U11	TGCAGCACATTCAACAGAA
U12	TGCAGCACATTCAACAGAC
U13	TGCAGCACATTCAACAGAT
U14	GATGAAGAAATTAACAAAAAAGCGCCT
U15	GATGAAGAAATTAACAAAAAAGCGCCC
U16	TTGCACAATCACCAAAGATGTATTATA*
U17	TTGCACAATCACCAAAGATGTATTATT*
U18	TTGAGCTGTCTTGGTTCATTGATT
U19	AAACACTACTTGTTCCCGCTTCA
U20	GGTCAGAGCCACGTTCACCA
U21	TGCTTCAACATCCCAACCAA
U22	TGGACACCGTCTTTATCAACGTCT
S1	CAGGGTATGATAGGCTATTGGTTG
S2	GCCCCATCAATATCAGTTTGTG
S3	ACGTCAAATGCGTGAAGGTG
S4	CCTTGTGAATCAAGTTCTGGATTG
S5	TTGATGATTTACCAGTTCCGATTG
S6	CTGCGACAAGTGAATTGAAAGCTG
aroE252GF	GGTATAATACAGATGGTATCGGTTATGTG
aroE252GR	ACCTGCGCCCAAAATTAAAA
spaF	AGCACCAAAAGAGGAAGACAA
spaR	GTTTAACGACATGTACTCCGT
mecAF	GATCGCAACGTTCAATTTAATTTTG
mecAR	GCTTTGGTCTTTCTGCATTCCT

↵ a Boldface type indicates a polymorph at the SNP; lowercase type indicates hairpin loop structures of LUX primers. *, SNP is in reverse primer.

TABLE 2.

Recipe for DMKP embodiment for S. aureus genotyping using SNPs plus binary genes: distribution of fluorescent (LUX) and unlabeled primers into 11 tubes

Tube no.	LUX primer no.	Fluorescent label	Unlabeled primer name	Gene/SNP + polymorph
1	L1	FAM	U1	arcC210 C
1	L4	JOE	U4	tpi241 + 243 A+G
2	L2	JOE	U2	arcC210 T
2	L5	FAM	U5	tpi241 + 243 G+G
3	L1	FAM	U3	arcC210 A
3	L4	JOE	U6	tpi241 + 243 G+A
4	L1A	FAM	U7	arcC162 T
4	L7	JOE	U10	gmk318 A
5	L2B	JOE	U8	arcC162 A
5	L6	FAM	U9	gmk318 T
6	L8	FAM	U11	pta294 A
6	L10	JOE	U14	tpi36 T
7	L9	JOE	U12	pta294 C
7	L11	FAM	U15	tpi36 C
8	L8	FAM	U13	pta294 T
8	L12	JOE	U16	pta383 T
9	L13	FAM	U17	pta383 A
9	L18A	JOE	U20	pUB110
10	L14	FAM	U19	cna
10	L22	JOE	U22	sdrE
11	L16	FAM	U21	pvl
11	L20	JOE	U18	pT181

TABLE 3.

STKP embodiment for S. aureus genotyping using SNPs: distribution of primers into eight tubes^a

Tube no.	Locus	Possible polymorphs	Polymorph for STKP	Primer combination
1	arcC210	C,T	C	S1, U1
2	tpi241 + 243	GA,GG,AG	GA	S2, U6
3	arcC162	A,T	A	S1, U8
4	gmk318	A,T	T	S3, U9
5	pta294	A,C	A	S4, U11
6	pta383	A,T	T	S6, U16
7	tpi36*	C,T	C	S5, U15
8	tpi36*	C,T	T	S5, U14

↵ a Conventional kinetic PCR requires the same number of reactions as there are polymorphs at a particular locus. In the full S. aureus kinetic PCR genotyping method, 17 reactions are required—two for each of 7 loci, except for arcC210, tpi241 + 243, and pta294, where three polymorphs are possible and consequently three reactions are required (see third column). Single-tube kinetic PCR uses a universal control reaction—in this case tpi36 in tubes 7 and 8—to normalize cycle times obtained so that polymorphs can be deduced from a single reaction per locus. *, “universal” control reaction.

TABLE 4.

Allele-specific real-time PCR cycle times for known STs^a

Gene + polymorph^b	C_T for ST:
Gene + polymorph^b	1	30	93
arcC210GCF	19.30	28.26	31.62
arcC210GTJ	31.53	18.99	23.29
arcC210GAF	Undet	32.42	36.44
tpi241 + 243ATGJ	Undet	21.93	25.35
tpi241 + 243GTGF	26.84	16.97	20.72
tpi241 + 243GTAJ	19.11	26.60	30.13
arcC162CTF	18.44	28.28	19.07
arcC162CAJ	28.35	17.73	31.46
gmk318AAJ	23.78	21.7	24.51
gmk318ATF	Undet	20.96	20.37
tpi36TJ	30.45	21.92	20.93
tpi36CF	19.65	18.3	24.95
pta294AAF	20.75	Undet	Undet
pta294ACJ	24.38	20.07	18.88
pta294ATF	36.27	19.65	21.41
pta383CTJ	23.04	21.92	21.98
pta383CAF	32.47	19.82	21.38

↵ a Boldface type indicate reactions with perfectly matched allele-specific primers. Undet, undetermined.
↵ b 5′ bases and subterminal mismatches are indicated. F, FAM labeling; J, JOE labeling.

TABLE 5.

Clonal complexes identified from genotyping of all S. aureus isolates^a

Clonal complex	SNP profile (allele)	No. of isolates
Clonal complex	SNP profile (allele)	MSSA (pvl+)	MRSA (pvl+)
1	CGATAACT	11 (2)	19 (1)
5	CGATTACA	36 (3)	9 (0)
8	TGATACCA	10 (0)	20 (0)
9	TGATAACT	6 (0)	0 (0)
509 (or 12)	CGGTTCCA	10 (0)	2 (1)
15	CGATAACA	17 (0)	0 (0)
20	CGATTACT	13 (1)	1 (0)
22	CGGTTACA	3 (1)	4 (0)
25	CGGTAACA	4 (0)	1 (0)
30	TGGATCCA	17 (6)	17 (14)
45	CGGATCCA	10 (0)	1 (1)
72	CGATTCCA	1 (0)	0 (0)
78	TGATTACA	21 (1)	4 (0)
93	TGGTTCTA (aroE252G)	16 (12)	30 (30)
Non-93/None	TGGTTCTA (aroE252A/T)	1 (1)	2 (0)
97	TGGTAACT	2 (1)	0 (0)
239	TGAAACCA	1 (0)	72 (1)
CC1 outliers	TGATTACT	5 (0)	0 (0)
None	TGATTCCA	5 (0)	2 (1)
None	CGGATCTA	1 (1)	0 (0)
None	TGGTACCA	0 (0)	1 (0)
None	TGGTTCCA	4 (1)	7 (6)
None	TGAATCCA	0 (0)	3 (0)
Not analyzed	TGAATACA	1 (0)	0 (0)
Not analyzed	TGGAACCA	1 (0)	0 (0)

↵ a Clonal complexes are named in accordance with the clonal complex founder that corresponds to the SNP profile. STs are allocated to clonal complexes on the basis of eBURST default settings (5). The SNP profiles are indicated in the order arcC210, tpi241, tpi243, arcC162, gmk318, pta294, tpi36, pta383. Clonal complex 509 is better known as clonal complex 12, but eBURST analysis indicates that CC509 is the founder. “93” indicates that the isolates have been confirmed as ST-93 by interrogation of aroE252. “CC1 outliers” indicates that the SNP profile corresponds to a small number of outliers in clonal complex 1. “None” indicates that the profile does not correspond to a dominant clonal complex. “Not analyzed” means that the profiles do not match any ST included in the eBURST analysis (but they do match STs in the database). Complete information regarding the relationship between SNP profiles and STs is available at http://www.ihbi.qut.edu.au/research/cells_tissue/phil_giffard/ .