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Multiplex PCR (M-PCR), a method that detects more than two target loci in a single reaction, relies on the variables which influence single template specific PCR. We describe here the role of temperature cycles in ensuring the efficiency of detection. We have designed a multi-step protocol, which uses gradients between the temperature steps. This has facilitated the target specific annealing in the developed M-PCR. We have examined various thermocycling steps and optimized the M-PCR protocol using 105 to 101 cells of Escherichia coli, Salmonella typhi, and Vibrio cholera as template in a single reaction. The sensitivity of the detection observed was 102 cells of each pathogen used in the study.