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Bacteriology

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
Flavia Huygens
1Cooperative Research Centre for Diagnostics, Institute of Health and Biomedical Innovation, QUT, Brisbane, Australia
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John Inman-Bamber
1Cooperative Research Centre for Diagnostics, Institute of Health and Biomedical Innovation, QUT, Brisbane, Australia
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Graeme R. Nimmo
2Queensland Health Pathology Service, Brisbane, Australia
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Wendy Munckhof
3Princess Alexandra Hospital and District Health Service, Brisbane, Australia
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Jacqueline Schooneveldt
2Queensland Health Pathology Service, Brisbane, Australia
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Bruce Harrison
4Corbett Life Science, Eight Mile Plains, Brisbane, Australia
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Jennifer A. McMahon
4Corbett Life Science, Eight Mile Plains, Brisbane, Australia
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Philip M. Giffard
1Cooperative Research Centre for Diagnostics, Institute of Health and Biomedical Innovation, QUT, Brisbane, Australia
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  • For correspondence: p.giffard@qut.edu.au
DOI: 10.1128/JCM.00843-06
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  • FIG. 1.
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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.

  • FIG. 2.
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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 ΔCT for each polymorph, calculated against the tpi36 universal control reactions. This was derived from 80 STKP assays.

Tables

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  • TABLE 1.

    Primer sequences for fluorescent (LUX) and unlabeled primers

    Primer nameSequencea
    L1FAM-caaccaCAGGGTATGATAGGCTATTGGtTG
    L2JOE-gacattgTTGGATACTTGTGGTGCAATGtC
    L4JOE-cacaaaGCCCCATCAATATCAGTTTGtG
    L5FAM-cacaaaGCCCCATCAATATCAGTTTGtG
    L1AFAM-gtatccCGGCAATGCCATTGGAtAC
    L2BJOE-gacattgTTGGATACTTGTGGTGCAATGtC
    L6FAM-caccttACGTCAAATGCGTGAAGGtG
    L7JOE-catcaaTGCGTGAAGGTGAAGTTGAtG
    L8FAM-caatccCCTTGTGAATCAAGTTCTGGAtTG
    L9JOE-caatccCCTTGTGAATCAAGTTCTGGAtTG
    L10JOE-caatcgTTGATGATTTACCAGTTCCGAtTG
    L11FAM-caatcgTTGATGATTTACCAGTTCCGAtTG
    L12JOE-cagcttCTGCGACAAGTGAATTGAAAGCtG
    L13FAM-cagcttCTGCGACAAGTGAATTGAAAGCtG
    L20JOE-gatctgTGAAGGGAATGCAGCAGAtC
    L14FAM-gtacttcaCGGGATTTGCAGAATTTGAAGtAC
    L18AJOE-cacgcTAGGTGTTGTGCCTATGCGtG
    L16FAM-cacctgTATCTCTAACGGCTTGTCAGGtG
    L22JOE-caccgAACTTCTAATGACACTGGCGGtG
    U1CGTATAAAAAGGACCAATTGGTCTG*
    U2CGTATAAAAAGGACCAATTGGTCTA*
    U3CGTATAAAAAGGACCAATTGGTCTT*
    U4GTAAATCATCAACATCTGAAGATATG
    U5GTAAATCATCAACATCTGAAGATGTG
    U6GTAAATCATCAACATCTGAAGATGTA
    U7GATCATCTTTATCTACTTCCACACGTGCA*
    U8GATCATCTTTATCTACTTCCACACGTGCT*
    U9ACCTACTAATCGCTCTCTCAAGTAA*
    U10ACCTACTAATCGCTCTCTCAAGTAT*
    U11TGCAGCACATTCAACAGAA
    U12TGCAGCACATTCAACAGAC
    U13TGCAGCACATTCAACAGAT
    U14GATGAAGAAATTAACAAAAAAGCGCCT
    U15GATGAAGAAATTAACAAAAAAGCGCCC
    U16TTGCACAATCACCAAAGATGTATTATA*
    U17TTGCACAATCACCAAAGATGTATTATT*
    U18TTGAGCTGTCTTGGTTCATTGATT
    U19AAACACTACTTGTTCCCGCTTCA
    U20GGTCAGAGCCACGTTCACCA
    U21TGCTTCAACATCCCAACCAA
    U22TGGACACCGTCTTTATCAACGTCT
    S1CAGGGTATGATAGGCTATTGGTTG
    S2GCCCCATCAATATCAGTTTGTG
    S3ACGTCAAATGCGTGAAGGTG
    S4CCTTGTGAATCAAGTTCTGGATTG
    S5TTGATGATTTACCAGTTCCGATTG
    S6CTGCGACAAGTGAATTGAAAGCTG
    aroE252GFGGTATAATACAGATGGTATCGGTTATGTG
    aroE252GRACCTGCGCCCAAAATTAAAA
    spaFAGCACCAAAAGAGGAAGACAA
    spaRGTTTAACGACATGTACTCCGT
    mecAFGATCGCAACGTTCAATTTAATTTTG
    mecARGCTTTGGTCTTTCTGCATTCCT
    • ↵ 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 labelUnlabeled primer nameGene/SNP + polymorph
    1L1FAMU1 arcC210 C
    1L4JOEU4 tpi241 + 243 A+G
    2L2JOEU2 arcC210 T
    2L5FAMU5 tpi241 + 243 G+G
    3L1FAMU3 arcC210 A
    3L4JOEU6 tpi241 + 243 G+A
    4L1AFAMU7 arcC162 T
    4L7JOEU10 gmk318 A
    5L2BJOEU8 arcC162 A
    5L6FAMU9 gmk318 T
    6L8FAMU11 pta294 A
    6L10JOEU14 tpi36 T
    7L9JOEU12 pta294 C
    7L11FAMU15 tpi36 C
    8L8FAMU13 pta294 T
    8L12JOEU16 pta383 T
    9L13FAMU17 pta383 A
    9L18AJOEU20pUB110
    10L14FAMU19 cna
    10L22JOEU22 sdrE
    11L16FAMU21 pvl
    11L20JOEU18pT181
  • TABLE 3.

    STKP embodiment for S. aureus genotyping using SNPs: distribution of primers into eight tubesa

    Tube no.LocusPossible polymorphsPolymorph for STKPPrimer combination
    1 arcC210C,TCS1, U1
    2 tpi241 + 243GA,GG,AGGAS2, U6
    3 arcC162A,TAS1, U8
    4 gmk318A,TTS3, U9
    5 pta294A,CAS4, U11
    6 pta383A,TTS6, U16
    7 tpi36*C,TCS5, U15
    8 tpi36*C,TTS5, 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 STsa

    Gene + polymorphb CT for ST:
    13093
    arcC210GCF 19.30 28.2631.62
    arcC210GTJ31.53 18.99 23.29
    arcC210GAFUndet32.4236.44
    tpi241 + 243ATGJUndet21.9325.35
    tpi241 + 243GTGF26.84 16.97 20.72
    tpi241 + 243GTAJ 19.11 26.6030.13
    arcC162CTF 18.44 28.28 19.07
    arcC162CAJ28.35 17.73 31.46
    gmk318AAJ 23.78 21.724.51
    gmk318ATFUndet 20.96 20.37
    tpi36TJ30.4521.92 20.93
    tpi36CF 19.65 18.3 24.95
    pta294AAF 20.75 UndetUndet
    pta294ACJ24.38 20.07 18.88
    pta294ATF36.2719.6521.41
    pta383CTJ 23.04 21.9221.98
    pta383CAF32.47 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 isolatesa

    Clonal complexSNP profile (allele)No. of isolates
    MSSA (pvl+)MRSA (pvl+)
    1CGATAACT11 (2)19 (1)
    5CGATTACA36 (3)9 (0)
    8TGATACCA10 (0)20 (0)
    9TGATAACT6 (0)0 (0)
    509 (or 12)CGGTTCCA10 (0)2 (1)
    15CGATAACA17 (0)0 (0)
    20CGATTACT13 (1)1 (0)
    22CGGTTACA3 (1)4 (0)
    25CGGTAACA4 (0)1 (0)
    30TGGATCCA17 (6)17 (14)
    45CGGATCCA10 (0)1 (1)
    72CGATTCCA1 (0)0 (0)
    78TGATTACA21 (1)4 (0)
    93TGGTTCTA (aroE252G)16 (12)30 (30)
    Non-93/NoneTGGTTCTA (aroE252A/T)1 (1)2 (0)
    97TGGTAACT2 (1)0 (0)
    239TGAAACCA1 (0)72 (1)
    CC1 outliersTGATTACT5 (0)0 (0)
    NoneTGATTCCA5 (0)2 (1)
    NoneCGGATCTA1 (1)0 (0)
    NoneTGGTACCA0 (0)1 (0)
    NoneTGGTTCCA4 (1)7 (6)
    NoneTGAATCCA0 (0)3 (0)
    Not analyzedTGAATACA1 (0)0 (0)
    Not analyzedTGGAACCA1 (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/ .

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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
Journal of Clinical Microbiology Oct 2006, 44 (10) 3712-3719; DOI: 10.1128/JCM.00843-06

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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
Journal of Clinical Microbiology Oct 2006, 44 (10) 3712-3719; DOI: 10.1128/JCM.00843-06
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KEYWORDS

Bacterial Typing Techniques
polymerase chain reaction
Staphylococcus aureus

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