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摘要本文报道用两步PCR法快速检测细胞培养物中污染的支原体（Mycoplasma）。两套引物选自16S rRNA和23S rRNA保守区域，外部引物PF1，PR1扩增的DNA片段在360～500bp之间，内部引物PF2，PR2扩增的DNA片段在140～220bp之间。试验表明，两步PCR法能检出污染细胞培养物七种常见的支原体。17份待检细胞培养物，两步PCR法检测，9份（52.9%）为阳性，直接培养法检测，5份（29.4%）为阳性。采用快速热循仪，使PCR反应时间大大缩短，30次循环仅需20分钟。反应液的用量也仅10μl，提高了检测效率，节省了材料，降低了试验用费。本文就两步PCR的灵敏度等特性也作了初步测试。

abstractsTwo-step PCR has been applied to detect the contamination of Mycoplasmas in the cell cultures. Two sets of universal primers were selected from the conserved regions between 16S/23S intergenic spaces of these Mycoplasma species. The first PCR produced fragments of 360 to 500bp, the second PCR products were from 140 to 220bp. The test showed that the two-step PCR could detect 9 species of Mycoplasmas, compared with the direct culture test that was able to detect 5 cell cultures positive from 17 samples, the two-step PCR could detect 9 positive. It showed that the PCR is more sensitive. We used the 1605 Air thermo-cycler, for shortening the amplification time from several hours to 20 minutes for 30 cycles, lessening the reaction volume to 10μl, and Increasing work efficiency.