实时荧光RT-PCR与普通RT-PCR检测噬菌体MS2的差异
摘要
Objective: To compare the sensitivity and specificity by both real-time fluorescent one step reverse transcription-PCR and general two-step RT-PCR assays for detection of bacteriophageMS2, and adopt a suitable method with satisfactory sensitivity or specificity or both to detect similar bacteriophages or viruses in relative disinfectionology and microbiology tests. Methods: The real-time fluorescent one step reverse transcription-PCR and general two-step PCR assays were carried out to detect both replicase and coat protein genes segments. Then sensitivity and specificity of the two methods were compared. Results: For the replicase gene segment, the sensitivity of real-time fluorescent one step RT-PCR was up to 0.25 pg/μl, while the general two-step PCR only 2500 pg/μl. For the coat protein gene, the former was 0. 025 pg/μl in contrast to the later 250 pg/μl. In the specificity test involving ten kinds of stains, the real-time fluorescent one step RT-PCR method only detected bacteriophage f2 and F. coli. ATCC15597 (host of the bacteriophageMS2) both showing two signals of replicase and coat protein genes, while other stains were negative. However, the electrophorograms of two-step RT-PCR just presented that the bacteriophagef2 had only one band similar to the coat protein gene, meanwhile the E. coli. ATCC15597 also one to the replicase gene, but others had none. Conclusion The sensitivity of real-time fluorescent one step RT-PCR assay for bacteriophageMS2 was 104 times higher than general two-step RT-PCR, but the specificity of latter was slightly stronger.