A sandwich duplex immuno PCR for rapid and sensitive identification of Clostridium perfringens alpha and enterotoxin

The prevalence and lethality associated with C. perfringens alpha (CPA) and enterotoxin (CPE) toxaemia necessitate the need for rapid and definitive detection systems to initiate management measures. In the present study, a sandwich duplex immuno-capture PCR (SD-IPCR) was developed by employing IgY antibodies against a bivalent protein r-Cpae derived from CPA and CPE for antigen capture and reporter antibodies against truncated CPA or CPE conjugated to oligomers of distinguishable size for antigen revealing and signal amplification. The avian immunoglobulin's (IgY) were devoid of reactivity with S. aureus protein A (SpA), a commensal that often co-exists with C. perfringens. The assay was specific, had a detection limit (LOD) of 1 pg/ml for both CPA and CPE in PBS and improved the LOD by 10⁴ folds compared to an analogous sandwich ELISA with same set of antibodies. In spiking studies, a ten-fold reduction in LOD was observed in case of intestinal tissue samples (10 pg/ml) however, no change in LOD was observed when SD-IPCR was applied on to faecal, serum or muscle tissue samples. Of the 136 natural samples examined, the SD-IPCR could detect CPA and CPE in 29.4% and 35.3% samples, while the sandwich ELISAs could detect the same in 25.7% and 25% samples respectively owing to the relatively lesser sensitivity. The LOD and specificity of the SD-IPCR demonstrates its applicability as an efficient and rapid platform for direct detection CPA and CPE from diverse samples matrices in clinical microbiological and meat testing laboratories.