Skidmore Andrew M, Bradfute Steven B
Center for Global Health, Department of Internal Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA.
Microorganisms. 2025 Mar 25;13(4):732. doi: 10.3390/microorganisms13040732.
The microbiome remains a top area of research, and it is now common to examine any organic and inorganic samples for bacterial colonization. However, due to the ubiquity of bacteria in the environment, separating the low-burden colonization of bacteria from the possible contamination of laboratory reagents remains problematic. When examining samples of expected low bacterial burden, it is common to first amplify any bacterial DNA present through PCR before sequencing. In this work, we examined nine different commercial PCR enzymes and their reaction components as possible sources of bacterial DNA contamination. We found contaminating bacterial DNA in seven of the nine reactions, and this DNA was shown to come from a variety of species. Importantly, we were able to perform these studies solely with endpoint PCR and Sanger sequencing, which are more accessible and affordable than high-throughput, short-read sequencing and real-time PCR. This work confirms that there needs to be an increased emphasis on including control reactions in microbiome studies so that contaminating DNA sequences can be identified and addressed, and that this can be achieved with minimal resources.
微生物组仍然是一个热门研究领域，现在检查任何有机和无机样本中的细菌定植情况已很常见。然而，由于环境中细菌无处不在，将低负荷细菌定植与实验室试剂可能的污染区分开来仍然存在问题。在检查预期细菌负荷较低的样本时，通常会在测序前先通过PCR扩增任何存在的细菌DNA。在这项工作中，我们检查了九种不同的商业PCR酶及其反应成分，看它们是否可能是细菌DNA污染的来源。我们在九个反应中的七个反应中发现了污染性细菌DNA，并且这种DNA被证明来自多种物种。重要的是，我们仅通过终点PCR和桑格测序就能进行这些研究，这比高通量、短读长测序和实时PCR更容易获得且成本更低。这项工作证实，在微生物组研究中需要更加重视纳入对照反应，以便能够识别和解决污染性DNA序列问题，并且这可以用最少的资源实现。
