Multiplex polymerase chain reaction for identification and differentiation of Streptococcus equi subsp. zooepidemicus and Streptococcus equi subsp. equi.

This research study investigates the use of multiplex polymerase chain reaction (PCR) in identifying and differentiating two closely related species of Streptococcus bacteria. The method entails design of oligonucleotide primers based on species-specific regions of the gene encoding superoxide dismutase A (sodA) and also employs amplification of exotoxin-encoding genes for further differentiation.

Identifying the Two Streptococcus Subspecies

• The two bacterial species involved in the study, Streptococcus equi subsp. zooepidemicus and S. equi subsp. equi, are closely related, which makes their identification quite challenging.
• One of the primary strategies used here is PCR. This is a technique typically used in molecular biology to amplify a single or few copies of a piece of DNA, allowing researchers to create thousands to millions of copies of a particular DNA sequence.
• The researchers made use of oligonucleotide primers designed according to species-specific parts of the sodA gene, which encodes the superoxide dismutase A enzyme. These primers are used to initiate the DNA synthesis in PCR and by designing them based on species-specific sequences, it aids in the identification and differentiation of the two bacteria species.

Further Differentiation of Streptococcus Subspecies

• Further differentiation between the two subspecies was accomplished by amplifying the genes seeH and seeI, which encode for the exotoxins SeeH and SeeI respectively.
• These exotoxins could be detected in the case of S. equi subsp. equi, but not for S. equi subsp. zooepidemicus, serving as a distinguishing feature between the two.
• Exotoxins are toxic substances secreted by bacteria and serve as an important facet in their pathogenicity. As such, it is crucial to identify and understand these toxin-coding genes in bacterial species.

Simplified Identification Through sodA-seeI Multiplex PCR

• The final part of their methodology involved a further simplistic approach to identification and differentiation, accomplished using sodA-seeI multiplex PCR.
• Multiplex PCR is an advanced form of PCR, which allows the amplification of multiple target sequences in a single PCR experiment – this saves time and resources spent on multiple individual PCR reactions.
• By targeting the sodA and seeI genes simultaneously in a single PCR reaction, the researchers were able to efficiently differentiate between the two Streptococcus species.