Multiplex PCR assay for detection of Streptococcus suis species and serotypes 2 and 1/2 in tonsils of live and dead pigs

Abstract

A PCR assay was developed for the detection of Streptococcus suis serotypes 2 and 1/2. This multiplex PCR is based on the amplification of the gene coding for 16S rRNA of S. suis and on the amplification of the cps2J gene coding for the capsule of S. suis serotypes 2 and 1/2. An internal control was constructed and added in this test to monitor the efficiency of amplification in each reaction. To evaluate the specificity of the test, 31 strains of other bacterial species related to S. suis or isolated from pigs and 42 strains of S. suis serotypes 1 and 3 to 34 were analyzed. The detection threshold of the test was 28 S. suis CFU/ml. The specificity and the sensitivity of the multiplex PCR test and the presence of an internal control allowed the analysis of biological samples without a culture step. The PCR assay was then applied to the detection of 14 S. suis serotype 1/2 strains, 88 S. suis serotype 2 strains isolated from pigs, and 25 S. suis serotype 2 strains isolated from humans. This test was also applied to analyze tonsil samples of pigs experimentally infected and carrier pigs without any symptoms.

FIG. 1.

Experimental design. Negative control animals were in unit A and were divided into two groups: group 1 (six noninfected pigs that received sterile THB) and group 2 (six noninfected pigs that were in direct contact). In unit B, six pigs were infected with S. suis strain 332 (group 3) and six pigs were in direct contact (group 4). In unit C, six pigs were infected with S. suis strain 347 (group 5) and six pigs were in direct contact (group 6). In unit D, six pigs were infected with S. suis strain 353 (group 7) and six pigs were in direct contact (group 8).

FIG. 2.

Evaluation of detection limit of the multiplex PCR. Agarose electrophoresis of the PCR products obtained from serial dilutions of S. suis S735 strain culture at a titer of 2.8 × 10⁵ CFU/ml (lane 2) and placed in tonsil biopsy specimens (1 ml of each dilution per biopsy specimen). The positions of the specific fragments of internal control, S. suis, and serotype 2 or 1/2 are indicated. Lanes: M, molecular mass marker (Smart Ladder; Eurogentec); 1, negative control of the PCR (only IPC was amplified); 3 to 8, PCR products at dilutions of 10, 10², 10³, 10⁴, 10⁵, and 10⁶.