Construction of multiplex RT-PCR to determine the expression of ZO-1, Claudin-1, and Occludin genes in pig’s intestine

Tight junction (TJ) proteins play a critical function in forming a strong intestinal barrier that protects against ingested pathogens and harmful agents. This study aimed to utilize multiplex RT PCR (mRT-PCR) to assess the expression of ZO-1, Claudin-1, and Occludin genes at mRNA level in the intestines of pigs using specific primer pairs yielding amplicons of 167 bp, 500 bp, and 235 bp, respectively. The mRT-PCR protocol was optimized for annealing temperature and primer concentrations, including primer specificity, and determining the limit of detection. Subsequently, the optimized mRT-PCR was applied to detect these genes in 48 pig intestinal samples, including duodenum, jejunum and ileum. The mRT-PCR demonstrated specificity for these genes with the annealing temperature at 58°C. The primer pair ratio for ZO-1, Claudin-1, and Occludin was 0.4 µM:0.4 µM:0.4 µM (2:2:2). The detection rate for ZO-1, Claudin-1, and Occludin genes were 83.33% (40/48), 29.17% (14/48) and 4.17% (2/48) respectively. Intriguingly, one sample tested positive for all three mRNA, while negative results were observed in 12.5% of the samples. In conclusion, in the present study, the mRT-PCR was successfully established to detect ZO-1, Claudin-1, and Occludin expression in pig intestinal tissues.