Thermocycling steps and optimization of multiplex PCR |
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Authors: | Atya Kapley Keith Lampel Hemant J Purohit |
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Institution: | (1) National Environmental Engineering Research Institute, Nagpur, 440 020, India;(2) Food and Drug Administration, 200 C Street, Washington, DC 20204, USA |
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Abstract: | 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. |
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Keywords: | enteropathogens multiplex PCR multi-step PCR thermocycling steps |
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