Haojie Wang# , Jianxing Chen# , Yue Sun , Tongqing An , Yue Wang , Hongyan Chen , Changqing Yu , Changyou Xia , He Zhang 

Front Cell Infect Microbiol.2024 Nov 18:14:1475878.doi: 10.3389/fcimb.2024.1475878. eCollection 2024.

Abstract

Introduction:  Streptococcus suis  (SS) is one of the most important pathogens causing major economic losses in the global pig farming industry and is a serious threat to public health safety. It has multiple serotypes, with poor cross-protection between serotypes, and effective typing methods are lacking.

Methods: In this study, a quadruplex TaqMan fluorescence quantitative PCR assay that can differentiate between  Streptococcus suis  types 2, 7 and 9 was developed using the  gdh  gene, a generic gene for  Streptococcus suis , and  cps2J ,  cps7H  and  cps9J , genes encoding podocarp-associated genes for types 2, 7 and 9, respectively, as targets.

Results: The method is specific enough to accurately type  Streptococcus suis  pigmentosus without detecting non-target pathogens ( Escherichia coli ,  Pasteurella multocida ,  Staphylococcus aureus ,  Streptococcus agalactiae, Streptococcus pneumoniae  and et al). The sensitivity was high, with a minimum lower detection line of 10 copies for P-SS and P-SS9, and 100 copies for P-SS2 and P-SS7. The standard curves generated showed good linearity with R2 of 0.999, 0.999, 0.997 and 0.998 respectively. The repeatability was good, with coefficients of variation between batch to batch and batch to batch tests ranging from 0.21% to 1.10%. Testing of 156 samples yielded 68 positive and 88 negative samples, of which the positive rate of SS was 5.77% (9/156), SS2 was 20.51% (32/156), SS7 was 8.33% (13/156) and SS9 was 9.6% (15/156), which was in line with the existing fluorescent quantitative PCR assay of 93.75%~100%, which was higher than the detection rate of conventional PCR.

Discussion: The quadruplex TaqMan fluorescence quantitative PCR method of  Streptococcus suis  generic, type 2, 7 and 9 established in this study can accurately differentiate the three serotypes of  Streptococcus suis  that currently have high prevalence and pathogenicity, which is of great importance for accurate clinical prevention and treatment, epidemiological investigation and vaccine development.

Keywords: Streptococcus suis; quadruplex TaqMan fluorescence quantitative PCR; type 2; type 7; type 9; typing.