Multiplex PCR Assays for Simultaneous Detection of Six Major Serotypes and Two Virulence-Associated Phenotypes ofStreptococcus suisin Tonsillar Specimens from Pigs

Abstract

Multiplex PCR assays for the detection and identification of various Streptococcus suis strains in tonsillar specimens from pigs were developed and evaluated. In two separate reactions, five distinct DNA targets were amplified. Three targets, based on the S. suis capsular polysaccharide (cps) genes specific for serotypes 1 (and 14), 7, and 9, were amplified in multiplex PCR I. Two other targets, based on the serotype 2-(and 1/2-) specific cps gene and the epf gene, encoding the EF proteins of virulent serotype 2 and highly virulent serotype 1 strains, were amplified in multiplex PCR II. To identify false-negative results, firefly luciferase (luc) DNA and primers based on the luc gene were included in the assay. The multiplex PCR assays were evaluated with tonsillar specimens from pigs infected with S. suis strains. The results obtained with the PCR assays were compared with the results obtained with a bacteriological examination. Most (94%) of the results obtained with multiplex PCR assays were confirmed by the bacteriological examination. The PCR method seems to be more sensitive compared to the bacteriological method, since the remaining 6% of the samples were positive by PCR and negative by bacteriological examination. These results indicate that the PCR method is highly specific for the detection of S. suis strains most frequently involved in clinical disease in infected pig herds. The serotypes found by PCR in tonsillar specimens from diseased pigs were compared with the serotypes of the strains isolated from the affected tissues of the same pigs. The results showed that there is significant association between carriership and clinical illness for S. suis serotype 9 and EF-positive serotype 2 strains and not for serotype 7 and EF-negative serotype 2 (or 1/2) strains.

Prevalência de Streptococcus suis tipo 2 por meio da técnica de reação em cadeia da polimerase em suínos abatidos no Estado do Mato Grosso

“…Essa maior prevalência pode ser explicada devido à dificuldade no isolamento da bactéria estudada, pois o local de colonização do S. suis é coinfectado com diversos microrganismos (MAROIS et al, 2007). Outros fatores seriam a alta sensibilidade e a especificidade da técnica de PCR realizada em tonsilas de suínos (WISSELINK, et al, 2002), em comparação com o isolamento bacteriano na mesma amostra, além do fato de ser muito mais rápida de executar (WISSELINK, et al, 1999). SWILDENS et al (2005), no estudo de suínos abatidos na Holanda, detectaram 18% de amostras positivas para S. suis tipo 2 por meio da PCR.…”

Rapid identification of Gram-positive anaerobic coccal species originally classified in the genus Peptostreptococcus by multiplex PCR assays using genus- and species-specific primers

“…It has the potential to yield considerable savings of time and effort in the laboratory and to identify bacteria more accurately. Since its introduction, multiplex PCR has been successfully applied to the identification of many bacteria (Tran & Rudney, 1999;Song et al, 2000;Yeboah-Manu et al, 2001;Wisselink et al, 2002). In the present study, based on accurate 16S rDNA sequence data of GPAC species obtained in a previous study (Song et al, 2003), a two-step multiplex PCR identification scheme was established to rapidly and accurately identify 14 valid GPAC species originally classified in the genus Peptostreptococcus.…”

Fifteen Streptococcus suis serotypes identified by multiplex PCR

“…This single assay correctly identified S. suis at the species level and differentiated its serotypes using the same system, so this multiplex PCR has advantages over previously reported PCR systems that detect only a limited range of serotypes (Kerdsin et al, 2009;Marois et al, 2004;Okwumabua et al, 2003;Smith et al, 1999a, b;Silva et al, 2006;Wang et al, 2011a;Wisselink et al, 2002). Our multiplex PCR could not differentiate serotype 2 from serotype 1/2, or serotype 14 from serotype 1, so antisera are required to distinguish these serotypes.…”

“…Thus, PCR is a more attractive alternative because of its rapid analytical capacity and low cost. PCR-based identification of S. suis serotypes 1, 14, 1/2, 2, 7, 9 and 16 has been reported previously (Kerdsin et al, 2009;Marois et al, 2004;Okwumabua et al 2003;Smith et al, 1999a, b;Silva et al, 2006;Wang et al, 2011a;Wisselink et al, 2002). However, the genetic sequences of 15 S. suis capsule loci are available in GenBank (Wang et al, 2011b), so we developed a multiplex PCR to identify these 15 S. suis serotypes using two reactions.…”