Objectives

PCR-based typing of the emm gene Streptococcus pyogenes often results in the amplification of multiple bands. This has resulted in the misclassification of strains into types based on non-emm gene sequences. We aimed to improve the specificity of the emm typing PCR reaction using a primer called CDC3, the sequence for which has been previously used to identify emm genes in silico.

Methods

The proposed primer CDC3 was validated in silico from a global database of 1688 GAS genomes and in vitro with 32 isolates. PCR reactions were performed on genomic DNA from each isolate, using the published CDC1 forward primer with the CDC2 reverse primer or the new CDC3 reverse primer. The products were examined by gel electrophoresis, and representative PCR products were sequenced.

Results

In 1688 S. pyogenes genomes, the previous CDC2 reverse primer annealed in silico in 1671 emm genes and also in 2109 non emm genes in close proximity, whereas the new CDC3 primer annealed in 1669 emm genes only. The remaining 19 genes without a CDC3 binding site were chimeric emm genes. The PCR pair CDC1+CDC3 produced a single band at appropriate molecular weight in all 32 isolates tested, while the CDC1+CDC2 pair produced more than one band in 13 of 32 isolates (40%).

Conclusions

The new CDC3 primer is more specific for emm genes than the previous CDC2 primer and represents a simple solution to reduce the potential for mistyping S. pyogenes strains.

中文翻译：

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化脓性链球菌的更新的打字方法。

目标

Emm基因化脓性链球菌的基于PCR的分型通常会导致多个条带的扩增。这导致了基于非emm基因序列的菌株错误分类。我们旨在使用称为CDC3的引物改善emm型PCR反应的特异性，该序列先前已用于在计算机中鉴定emm基因。

方法

拟议的引物CDC3在全球1688个GAS基因组数据库中进行了计算机验证，并在体外与32个分离株进行了验证。使用公开的CDC1正向引物和CDC2反向引物或新的CDC3反向引物，对每种分离物的基因组DNA进行PCR反应。通过凝胶电泳检查产物，并对代表性的PCR产物进行测序。

结果

在1688年的化脓性链球菌基因组中，先前的CDC2反向引物在计算机上对1671个emm基因和2109个非emm基因进行了近乎退火，而新的CDC3引物仅在1669个emm基因中进行了退火。其余没有CDC3结合位点的19个基因是嵌合emm基因。PCR对CDC1 + CDC3在所有测试的32个分离物中均产生了一条分子量合适的单条带，而CDC1 + CDC2对在32个分离物中的13个中产生了一条以上的条带（占40％）。

结论

与以前的CDC2引物相比，新的CDC3引物对emm基因更具特异性，并且是减少化脓性链球菌菌株潜在可能性的简单解决方案。