Mycoplasma contamination threatens both the safety of biologics produced in cell substrates as well as the quality of scientific results based on cell-culture observations. Methods currently used to detect contamination of cells include culture, enzymatic activity, immunofluorescence and PCR but suffer from some limitations. High throughput sequencing (HTS) can be used to identify microbes like mycoplasmas in biologics since it enables an unbiased approach to detection without the need to design specific primers to pre-amplify target sequences but it does not enable the confirmation of microbial infection since this could reflect carryover of inert sequences. In order to unambiguously differentiate the presence of live or dead mycoplasmas in biological products, the present method was developed based on metabolic RNA labelling of newly synthetized mycoplasmal RNAs. HTS of labelled RNA detected A549 cell infection with Acholeplasma laidlawii in a manner similar to both PCR and culture and demonstrated that this technique can unambiguously identify bacterial species and differentiates infected cells from cells exposed to a high inoculum of heat-inactivated mycoplasmas. This method therefore combines the advantage of culture (that detects only live microorganisms) with those of molecular tests (rapidity) together with a very broad range of bacterial detection and identification.

支原体污染不仅威胁细胞底物中产生的生物制剂的安全性，而且威胁到基于细胞培养观察结果的科学结果的质量。当前用于检测细胞污染的方法包括培养，酶活性，免疫荧光和PCR，但是存在一些局限性。高通量测序（HTS）可用于鉴定生物制剂中的微生物（如支原体），因为它无需设计特定的引物即可预扩增靶序列，从而提供了一种无偏见的检测方法，但由于这样做可能无法确认微生物感染反映惰性序列的残留。为了明确区分生物产品中存在活的或死的支原体，本方法是基于新合成的支原体RNA的代谢RNA标记开发的。HTS标记的RNA检测到A549细胞感染以与PCR和培养均相似的方式，发现了无窝小球藻，并证明该技术可以明确鉴定细菌种类，并将感染的细胞与暴露于高热灭活支原体接种物的细胞区分开。因此，该方法将培养（仅检测活微生物）的优势与分子检测（快速）的优势以及细菌检测和鉴定的广泛范围相结合。