Detection of five common cell culture contaminant Molllcutes using rolling circle amplification

VernacularTitle:用滚环扩增技术检测污染细胞的5种支原体
Author: Hui WANG ; Xiaoyong ZHOU ; Fanrong KONG ; Weizhen WANG ; Yiqun DUAN
Publication Type:Journal Article
Keywords: Rolling circle amplification; Padlock probe
From: Chinese Journal of Microbiology and Immunology 2010;30(10):949-952
CountryChina
Language:Chinese
Abstract: Objective To develop a novel padlock probe and rolling circle amplification(RCA) to detect 5 common cell culture contaminant Mollicute( Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycomplasma orale, and Acholeplasma laidlawii ). Methods "Universal" primers ( SPS1, SPA2 and SPS2, SPA1) were used to amplify the Mollicute 16S-23S rRNA intergenic spacer region.Amplicon was ligate Mollicute specific padlock probe. Probe amplified and monitored using a Corbett RotorGeneTM 6000 machine. Results Five reference Mollicute strains were correctly detected by RCA method.There was no cross-reaction. RCA method can sensitive detect 10 copies templates and show strong positive signal. Sixty-two cell culture specimens were detected. Thirty-seven samples were contained single specie Mollicute and 14 samples contained two Mollicutes. Eleven samples did not contained Mollicute. RCA detection results were concordant with previously species-specific PCR. Conclusion RCA can rapid, sensitive and specific detect contamination Mollicute.