Development of a nested PCR assay for detection of Streptococcus equi subspecies equi in clinical equine specimens and comparison with a qPCR assay.

The study presents the development of a technique known as nested PCR (nPCR) for detecting the bacterial pathogen Streptococcus equi subspecies equi (S. equi), a cause of respiratory disease in horses. The new method is compared to the existing real-time PCR (qPCR) assay and is found to be more sensitive, potentially reducing false negatives during low bacterial shedding periods.

Objectives and Methodology

• The primary goal of this research was to improve upon existing methods for detecting the presence of S. equi in horses by developing a new nested PCR technique targeting specific genes within the bacterial pathogen.
• Two genes were targeted: the S. equi cell wall-associated M-protein (SeM), which is a key virulence factor, and an 18S rRNA internal control gene.
• The newly developed method was then compared to existing qPCR methods, utilizing the same primers and probes from the previous clinical testing assay from the Kansas State Veterinary Diagnostic Laboratory (KSVDL).

Testing and Results

• A total of 188 clinical specimens submitted to KSVDL were tested parallelly with the nPCR and qPCR assays.
• Results showed, nPCR identified 22.9% of samples as S. equi positive, whilst the qPCR assay flagged only 13.3%. This indicated a higher detection rate in nPCR method.
• None of the samples tested positive by qPCR were found to be negative by nPCR, validating the increased sensitivity of nPCR.
• All positive samples underwent DNA sequencing for further validation, with the basis of matching high nucleotide identity with the targeted SeM gene.
• A sequencing result showed that 17 out of the 18 samples that tested positive with nPCR, but not with qPCR, had over 96% nucleotide identity to the SeM gene.

Assessing Limit of Detection (LOD)

• The limit of detection (LOD) for the nPCR method was determined to be at a Ct of 37. This was favourable compared to the LOD of the qPCR (also a Ct of 37) as established by standard curve data.
• The assessment of LOD was important to establish the concentration necessary for successful sequencing and shows the ability of nPCR to confirm sequence even at low concentrations of the bacterial load.

Concluding Points

• The study concludes that the developed nPCR method is highly specific to S. equi as validated by DNA sequencing.
• The improved detection sensitivity of the nPCR method, compared to the conventional qPCR, holds the potential to reduce false-negative results, which is especially beneficial during periods of low bacterial shedding.

The research has significantly contributed to veterinary microbiology by developing a more sensitive detection method, which may greatly benefit the clinical diagnosis and treatment of equine respiratory diseases caused by S. equi bacteria.