恶性疟原虫体外培养污染皮氏罗尔斯顿菌的检测

Detection of blood contamination caused by Ralstonia pickettii during in vitro culture of Plasmodium falciparum

目的探讨恶性疟原虫体外培养过程中皮氏罗尔斯顿菌污染的检测及预防。方法体外培养恶性疟原虫,涂片、染色后镜检观察;提取被污染虫血的总DNA,采用真菌、细菌通用引物对保守序列进行PCR扩增和克隆测序,将测序结果在Gen Bank中比对。结果导致恶性疟原虫体外培养失败的污染物单个细胞和污染物聚集体在油镜下的形态分别与恶性疟原虫环状体和裂殖体相似,但其胞质内无空泡或无明显细胞核,且位于红细胞外侧;测序比对结果显示污染物与皮氏罗尔斯顿菌相似度>99%。结论恶性疟原虫体外培养过程中污染的皮氏罗尔斯顿菌形态与恶性疟原虫环状体或裂殖体相似,可导致镜检误判甚至使培养失败。因此,应严格无菌操作以防疟原虫体外培养污染。

Objective To detect and prevent bacterial contamination caused by Ralstonia pickettii during in vitro culture of Plasmodium falciparum. Methods in vitro culture of a P.falciparumstrain(3D7)was performed as usual.Thick and thin blood films were prepared to monitor culturing and were then stained with Giemsa stain and examined under a microscope.Genomic DNA of the contaminated blood culture was extracted using a Biamp DNA blood kit according to the manufacturer’s instructions.Primers specific to the conserved regions of bacteria and fungus were respectively selected for amplification with PCR.The PCR products were subjected to cloning and sequencing.The sequencing results were further compared using the Basic Local Alignment Sequence Tool(BLAST)in GenBank.Results Giemsa slides revealed a mixture of parasite and contaminant morphology.Single cells and clumps of the contaminant were quite similar to the ring and schizont stages of P.falciparum.However,the contaminant cells were usually located outside of red blood cells,with no vacuoles in the cytoplasm and no obvious nuclei.Blast results indicated that the contaminant was highly consistent with R.pickettii,with a similarity greater than 99%. Conclusion The morphology of R.pickettii in blood culture is similar to P.falciparumin the ring and schizont stages.R.pickettii contamination can lead to misjudgment of the growth stage of P.falciparumand even result in failure to culture this parasite.Therefore,more stringent sterile procedure is required to avoid contamination during P.falciparumcultivation in vitro.