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Food Microbiology

Multicenter Validation of the Analytical Accuracy of Salmonella PCR: towards an International Standard

Burkhard Malorny, Jeffrey Hoorfar, Cornelia Bunge, Reiner Helmuth
Burkhard Malorny
1Federal Institute for Health Protection of Consumers and Veterinary Medicine, D-12277 Berlin, Germany
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Jeffrey Hoorfar
2Danish Veterinary Institute, DK-1790 Copenhagen, Denmark
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Cornelia Bunge
1Federal Institute for Health Protection of Consumers and Veterinary Medicine, D-12277 Berlin, Germany
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Reiner Helmuth
1Federal Institute for Health Protection of Consumers and Veterinary Medicine, D-12277 Berlin, Germany
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  • For correspondence: r.helmuth@bgvv.de
DOI: 10.1128/AEM.69.1.290-296.2003
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  • FIG. 1.
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    FIG. 1.

    Cloned artificial 253-bp sequence of the IAC. For the invA PCR assay, a 157-bp IAC was amplified by using primer set 139-141. The underlined italic sequence is the binding sequence of primer 139, and the underlined roman sequence is the binding sequence of primer 141.

  • FIG. 2.
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    FIG. 2.

    Features of a two-row by two-column contingency table with respect to the reference culture method and the alternative PCR method. The derived formulas used for analysis in the validation study are shown below the table. a, number of samples that are both reference positive and PCR positive (true positive); b, number of samples that are reference negative but PCR positive (false positive); c, number of samples that are reference positive but PCR negative (false negative); d, number of samples that are both reference and PCR negative (true negative).

  • FIG. 3.
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    FIG. 3.

    Coamplification of different copy numbers of the IAC (157-bp fragment) and Salmonella genomic DNA (284-bp fragment) by using primer set 139-141. The initial numbers of Salmonella DNA and IAC copies per reaction tube are indicated above the gels. The sizes of the PCR products are indicated at left. Marker X (Roche Diagnostics) was used as the molecular weight standard (MW). Ten microliters of the 25-μl PCR mixture was loaded per well.

  • FIG. 4.
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    FIG. 4.

    Detection probability of the Salmonella PCR assay using primer set 139-141 at serially 10-fold-diluted cell concentrations of serotype Typhimurium phage type DT104 reference strain 51K61. The detection probability was established in the presence of 30 or 300 copies of IAC DNA. Five microliters of 10-fold-diluted cell suspensions (100 to 106 CFU/ml) were used as the template in the PCR. The graph shows a sigmoidal fit of data points generated by 30 repetitive PCRs from six independent experiments.

Tables

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  • TABLE 1.

    Salmonella strains used for inclusivity PCR tests and results of preliminary screening

    Serotype (subspecies)SerogroupTotal no. of strainsNo. of strains selected for prescreeningNo. of strains testing positive with primer set:
    P1-P2S18-S19ST11-ST15139-141
    Enteritidis (I)D160101091010
    Typhimurium (I)B601010101010
    Hadar (I)C2-C3511111
    Virchow (I)C1511111
    Infantis (I)C1511111
    Heidelberg (I)B511111
    Newport (I)C2-C3511111
    Brandenburg (I)B511111
    Saintpaul (I)B2011111
    Agona (I)B511111
    Blockley (I)C2-C3511111
    Bovismorbificans (I)C2-C3511111
    Bredeney (I)B511111
    Derby (I)B511111
    Dublin (I)D1511111
    Livingstone (I)C1511111
    Montevideo (I)C1511111
    Paratyphi B (I)B511111
    Senftenberg (I)E42—b————
    Litchfield (I)C2-C32—————
    Typhi (I)D12—————
    42:r:- (II)T211111
    9,12:z:z39 (II)D111111
    48:d:z6 (II)Y1—————
    42:b:e,n,x,z15 (II)T1—————
    30:1,z28,z6 (II)N1—————
    21:g,z51:- (IIIa)L111101
    47:r:- (IIIa)X1—————
    18:z4,z32:- (IIIa)K1—————
    50:z:z52 (IIIb)Z11111a1
    47:1,v:z (IIIb)X1—————
    18:i,v:z (IIIb)K1—————
    16:z4,z32:- (IV)I111111
    48:g,z51:- (IV)Y1—————
    11:z4,z23:- (IV)F1—————
    44:r:- (V)V11001a1
    66:z65:- (V)1—————
    48:z35:- (V)Y1—————
    45:a:e,n,x (VI)W111111
    1,6,14,25:a:e,n,x (VI)H1—————
    41:b:1,7 (VI)S1—————
    Total2424342414243
    • ↵ a Only faint fragments were obtained but included as positive response.

    • ↵ b —, not included.

  • TABLE 2.

    Salmonella-related strains used for exclusivity PCR tests

    OrganismNo. of strainsNo. of strains selected for preliminary screening
    Citrobacter brakii 10
    Citrobacter diversis 11
    Citrobacter freundii 195
    Escherichia coli 167
    Escherichia fergusonii 10
    Erwinia herbicola 10
    Enterobacter aerogenes 41
    Enterobacter agglomerans 21
    Enterobacter amnigenus 10
    Enterobacter asbunae 10
    Enterobacter asburiae 10
    Enterobacter cloacae 21
    Enterobacter gergoviae 20
    Enterobacter sakazakii 10
    Enterobacter tarda 10
    Enterobacter taylorae 20
    Enterococcus faecalis 33
    Ewingella americana 10
    Hafnia alvei 31
    Klebsiella oxytoca 31
    Klebsiella pneumoniae 54
    Kluyvera ascorbata 10
    Listeria inucua 22
    Listeria ivanovii 21
    Listeria monocytogenes 83
    Micrococcus kristinae 10
    Morganella morganii 41
    Obesumbacterium proteus 10
    Proteus agglomerans 10
    Proteus mirabilis 41
    Proteus vulgaris 32
    Providencia heimbachae 10
    Providencia stuartii 10
    Pseudomonas aeruginosa 20
    Pseudomonas alcaligenes 10
    Rhanella aquatilis 10
    Serratia marcescens 31
    Serratia odorifera 20
    Shigella boydii 33
    Shigella flexneri 11
    Shigella sonnei 10
    Staphylococcus aureus 44
    Yersinia enterocolitica 41
    Total12247
  • TABLE 3.

    Selected primer sets for selectivity tests

    TargetPrimerSequenceSize of PCR product (bp)Annealing temp (°C)Reference
    oriC P1TTATTAGGATCGCGCCAGGC1635525
    P2AAAGAATAACCGTTGTTCAC
    ompC S18ACCGCTAACGCTCGCCTGTAT1595816
    S19AGAGGTGGACGGGTTGCTGCCGTT
    Random fragmentST11AGCCAACCATTGCTAAATTGGCGCA429601
    ST15GGTAGAAATTCCCAGCGGGTACTG
    invA 139GTGAAATTATCGCCACGTTCGGGCAA2846421
    141TCATCGCACCGTCAAAGGAACC
  • TABLE 4.

    Results of the collaborative study validating the reproducibility of the analytical accuracy using primer set 139-141

    Laboratory no.Inclusivity (%)Exclusivity (%)Positive predictivity (%)Negative predictivity (%)Analytical accuracy (%)Kappa indexa
    11001001001001001.000
    29410010092960.926
    396979894960.927
    41001001001001001.000
    498929497950.903
    6b94676594770.561
    71001001001001001.000
    81001001001001001.000
    91001001001001001.000
    109110010088950.889
    111001001001001001.000
    129610010094980.951
    1394929492930.854
    141001001001001001.000
    158710010081920.826
    161001001001001001.000
    All97969796960.926
    All but laboratory 697999996980.952
    • ↵ a Kappa values of <0.01 indicate no concordance, 0.1 to 0.4 indicate weak concordance, 0.41 to 0.60 indicate clear concordance, 0.61 to 0.80 indicate strong concordance, and 0.81 to 1.00 indicate nearly complete concordance.

    • ↵ b Laboratory 6 was excluded from the final statistical analysis due to a markedly lower Kappa index compared with those of the other laboratories.

  • TABLE 5.

    Results of classification of strains used in the interlaboratory study (laboratory 6 excluded)

    Strain and designationCorrect classification of strain (%)
    Non-Salmonella strains
        Citrobacter diversis EU-NS25100.0
        Hafnia alvei EU-NS3593.3
        Staphylococcus aureus EU-NS22100.0
        Citrobacter freundii EU-NS24100.0
        Enterobacter cloacae EU-NS32100.0
        Enterococcus faecalis EU-NS3100.0
        Escherichia coli ATCC 2592297.7
        Escherichia coli O128 EU-NS10100.0
        Escherichia coli O157 EU-NS11100.0
        Klebsiella oxytoca EU-NS1893.3
        Klebsiella pneumoniae EU-NS17100.0
        Proteus vulgaris EU-NS28100.0
        Shigella boydii EU-NS38100.0
        Shigella flexneri 2a EU-NS27100.0
        Yersinia enterocolitica EU-NS41100.0
        Serratia marcescens EU-NS36100.0
    Salmonella serotypesa
        Hadar 99-601100.0
        Enteritidis 00-490.9
        Bredeney 00-215100.0
        Dublin 98-44369.7
        Blockley 98-1262100.0
        Typhimurium 00-510100.0
        42:b:e,n,x,z15 (II) 98-130890.9
        18:z4,z32:- (IIIa) 99-155697.0
        47:1,v:z (IIIb) 00-269100.0
        48:g,z51:- (IV) 98-3722100.0
        66:z65:- (V) K-135497.0
        1,6,14,25:a:e,n,x (VI) 94-2957100.0
    Controls
        Negative (TE buffer)100.0
        Positive (serotype Typhimurium 51K61)100.0
    • ↵ a Numerals in parentheses indicate subspecies.

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Multicenter Validation of the Analytical Accuracy of Salmonella PCR: towards an International Standard
Burkhard Malorny, Jeffrey Hoorfar, Cornelia Bunge, Reiner Helmuth
Applied and Environmental Microbiology Jan 2003, 69 (1) 290-296; DOI: 10.1128/AEM.69.1.290-296.2003

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Multicenter Validation of the Analytical Accuracy of Salmonella PCR: towards an International Standard
Burkhard Malorny, Jeffrey Hoorfar, Cornelia Bunge, Reiner Helmuth
Applied and Environmental Microbiology Jan 2003, 69 (1) 290-296; DOI: 10.1128/AEM.69.1.290-296.2003
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KEYWORDS

Polymerase Chain Reaction
Salmonella

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