Detection of mycoplasma in contaminated mammalian cell culture using FTIR microspectroscopy

Abstract

Mycoplasma contamination represents a significant problem to the culture of mammalian cells used for research as it can cause disastrous effects on eukaryotic cells by altering cellular parameters leading to unreliable experimental results. Mycoplasma cells are very small bacteria therefore they cannot be detected by visual inspection using a visible light microscope and, thus, can remain unnoticed in the cell cultures for long periods. The detection techniques used nowadays to reveal mycoplasma contamination are time consuming and expensive with each having significant drawbacks. The ideal detection should be simple to perform with minimal preparation time, rapid, inexpensive, and sensitive. To our knowledge, for the first time, we employed Fourier transform infrared (FTIR) microspectroscopy to investigate whether we can differentiate between control cells and the same cells which have been infected with mycoplasmas during the culturing process. Chemometric methods such as HCA and PCA were used for the data analysis in order to detect spectral differences between control and intentionally infected cells, and spectral markers were revealed even at low contamination level. The preliminary results showed that FTIR has the potential to be used in the future as a reliable complementary detection technique for mycoplasma-infected cells. Graphical abstractFTIR microspectroscopy is able to differentiate between mycoplasma infected cells (LC for low contamination and HC for high contamination) and control non-infected cells (CN).

Aptamer Cocktail to Detect Multiple Species of Mycoplasma in Cell Culture

“…Some are based on immunological principles, such as enzyme-linked immunosorbent assays [24,25], and some are based on microscopic techniques, including electron microscopy and fluorescence microscopy [26][27][28]. Other detection methods include Fourier transform infrared microspectroscopy [29], biochemical tests [30], and Gaussia luciferase-based assays [31]. All Next, we determined whether the sepharose-adherent mycoplasma could be detected by A15-1, A16-1Y, or #1J aptamers.…”

Direct qPCR is a sensitive approach to detect Mycoplasma contamination in U937 cell cultures

“…While various methods exist for the detection of Mycoplasma contamination [13,14], probably the most frequently used ones are biochemical detection of Mycoplasma metabolism and PCR-based detection of Mycoplasma DNA. Though the biochemical detection of mycoplasma ATP generation (Mycoalert (Lonza, Basel, Switzerland)) is a quick protocol, it has certain disadvantages that should be mentioned, including requiring that reagents be reconstituted and brought to 22 °C before each measurement and requiring a luminometer for ATP detection.…”

Direct qPCR is a sensitive approach to detect Mycoplasma contamination in U937 cell cultures

“…While various methods exist for the detection of Mycoplasma contamination [13,14], probably the most frequently used ones are the biochemical detection of Mycoplasma metabolism and PCR-based detection of Mycoplasma DNA. Though the biochemical detection of mycoplasmal ATP generation (Mycoalert (Lonza, Basel, Switzerland)) is a quick protocol, it has certain disadvantages that should be mentioned, including the requirement of the reagents to be reconstituted and be brought to 22oC before each measurement, and the availability of a luminometer for ATP detection.…”