Multicenter validation of the analytical accuracy of Salmonella PCR: towards an international standard

Abstract

As part of a major international project for the validation and standardization of PCR for detection of five major food-borne pathogens, four primer sets specific for Salmonella species were evaluated in-house for their analytical accuracy (selectivity and detection limit) in identifying 43 Salmonella spp. and 47 non-Salmonella strains. The most selective primer set was found to be 139-141 (K. Rahn, S. A. De Grandis, R. C. Clarke, S. A. McEwen, J. E. Galán, C. Ginocchio, R. Curtiss III, and C. L. Gyles, Mol. Cell. Probes 6:271-279, 1992), which targets the invA gene. An extended determination of selectivity by using 364 strains showed that the inclusivity was 99.6% and the exclusivity was 100% for the invA primer set. To indicate possible PCR inhibitors derived from the sample DNA, an internal amplification control (IAC), which was coamplified with the invA target gene, was constructed. In the presence of 300 DNA copies of the IAC, the detection probability for primer set 139-141 was found to be 100% when a cell suspension containing 10(4) CFU/ml was used as the template in the PCR (50 CFU per reaction). The primer set was further validated in an international collaborative study that included 16 participating laboratories. Analysis with 28 coded ("blind") DNA samples revealed an analytical accuracy of 98%. Thus, a simple PCR assay that is specific for Salmonella spp. and amplifies a chromosomal DNA fragment detected by gel electrophoresis was established through extensive validation and is proposed as an international standard. This study addresses the increasing demand of quality assurance laboratories for standard diagnostic methods and presents findings that can facilitate the international comparison and exchange of epidemiological data.

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.

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.

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.

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.