Inactivation, storage, and PCR detection of mycoplasma on FTA® filter paper
Inactivation, storage, and PCR detection of mycoplasma on FTA® filter paper
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DOI：
10.1637/7215-060104
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发表时间：
2004-12-01
期刊：
影响因子：
1.4
通讯作者：
Meven, SH
中科院分区：
文献类型：
--
作者：
Moscoso, H;Thayer, SG;Meven, SH
我们评估了使用 Flinders Technology Associates (FTA (R)) 滤纸灭活和储存支原体 DNA 模板以及通过聚合酶链式反应 (PCR) 进行检测的可行性。 FTA (R) 纸是一种棉基纤维素膜，含有冻干化学物质，可裂解大多数类型的细菌和病毒。鸡毒支原体 (MG) 和滑液支原体 (MS) 培养物以不同体积点样在滤纸上，并在进行 PCR 之前在不同温度下保存不同时间。 MG 和 MS 在所有时间范围（1-60 天）都很容易检测到，与所用体积（1-100 μl）或储存温度（4℃-41℃）无关。 FTA (R)-PCR 的灵敏度和特异性与标准诊断 PCR 相当，可以在现场样品中检测 MG/MS，而不受非靶向支原体的干扰。通过这两种方法对 193 个现场样本进行的分析表明，与血清学和培养结果几乎 100% 一致。 DNA 在较宽温度范围内的长期稳定性使 FTA (R) 卡成为收集并同时灭活支原体的良好替代方案。它还以经济有效的方式方便地存储和运输 DNA，以进行进一步的分子分析，例如限制性酶长度多态性和核苷酸测序。
We evaluated the feasibility of using Flinders Technology Associates (FTA (R)) filter paper for the inactivation and storage of mycoplasma DNA templates and their detection by the polymerase Chain reaction (PCR). FTA (R) paper is a cotton-based cellulose membrane containing lyophilized chemicals that lyses most types of bacteria and viruses. Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) cultures were spotted at various volumes on the filter paper and stored at different temperatures for various periods of time before performing PCR. MG and MS were readily detected at all time frames (1-60 days) independent of the volume applied (1-100 mu l) or storage temperature (4 C-41 C). Sensitivity and specificity of the FTA (R)-PCR were comparable to the standard diagnostic PCR, allowing the detection of MG/MS in field samples without interference by nontargeted mycoplasma. Analysis of 193 field samples by both methods showed nearly 100% agreement with serology and culture results. The long-term DNA stability at a wide range of temperatures makes the FTA (R) cards a good alternative for collecting and simultaneously inactivating mycoplasma. It also offers the convenience of storage and transport of DNA in a cost-effective manner for further molecular analysis, such as restriction enzyme length polymorphism and nucleotide sequencing.