DEVELOPMENT OF DUPLEX TAQMAN REAL-TIME PCR TO DETECT CTX AND TDH

[Objective]To develop a duplex TaqMan real-time PCR for the detection of ctx and tdh.[Methods]The conserved region of TDH and CTX gene were used to design primers and probes,and the duplex TaqMan real-time PCR system of detecting ctx and tdh was established.The sensitivity and specificity of duplex TaqMan real-time PCR system was evaluated.[Results]The duplex TaqMan real-time PCR system detecting ctx and tdh was established.In the optimized reaction system of ctx and tdh duplex TaqMan real-time PCR,the concentrations of ctx primers and probe were 200 nmol/L and 100 nmol/L,respectively;the concentrations of tdh primers and probe were 200 nmol/L and 100 nmol/L,respectively.The sensitivity and specificity of this reaction system were both 100%.The detection limits of the optimized reaction system to two plasmids were both 1.0×102 copies/μl,and the amplification efficiency was 94% and 97.7%,respectively.[Conclusion]This study established a duplex TaqMan real-time PCR for the detection of ctx and tdh.This method had satisfying sensitivity and specificity approved by experiment results.The detection limit can be 1.0×102 copies/μl,which is 100times higher than traditional PCR method.Further,duplex real-time PCR can detect two pathogenic bacteria or two virulent genes in the same reaction system at the same time.This method acts as an alternative to the traditional method which is time-consuming and laborious.