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This paper presents a new miniature reverse-transcription polymerase chain-reaction (RT-PCR) system integrating a sample pretreatment device for fast nucleic-acid amplification and diagnosis of viruses and bacteria. In the system, a two-way serpentine-shape (s-shape) pneumatic micropump and a magnetic bioseparator were developed for separation and enrichment of viruses and bacteria. This new bioseparator can also be used as microheating chambers to perform RT-PCR. Taking advantage of the specific interaction between the antibodies on the surface of magnetic beads and the surface antigens on viruses or bacteria, the target virus and bacteria were recognized and further separated and purified from the biosamples by a magnetic field generated by the bioseparator. The target purified virus/bacteria was then lysed to release ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) for the subsequent RT-PCR processes. Experimental results showed that the target virus/bacteria was successfully separated and enriched by the high specificity and selectivity of antibody-conjugated magnetic beads, and the subsequent amplification of RNA/DNA was automatically completed by utilizing the on-chip microheaters and the micro temperature sensor. The high mixing efficiency of the two-way s-shape pump and the rapid heating/cooling rate of the microheating chambers can significantly shorten the pretreatment and diagnosis processes. As a whole, the developed system may provide a powerful platform for sample pretreatment and fast disease diagnosis.