Development and Evaluation of a TaqMan Real-Time PCR Assay for the Rapid Detection of Cross-Contamination of RD (Human) and L20B (Mouse) Cell Lines Used in Poliovirus Surveillance

Abstract

Background: The cross-contamination of cell lines in culture is a persistent problem. Genetically modified L20B (Mouse) and RD (Human Rhabdomyosarcoma) cell lines are commonly used in poliovirus research, surveillance, and diagnostics. Cross-contamination between these cell lines leads to unreproducible results and unreliable surveillance data, negatively affecting public health. The gold standard method for cell authentication is Short Tandem Repeats analysis, which is time-consuming and expensive. The disadvantage of STR is limited detection of interspecies contamination.

Methods: This assay targets the mitochondrial cytochrome c oxidase subunit I (MTCO1) gene, a highly conserved and emergent DNA barcode region for detection of cross-contamination in RD and L20B cell lines. The MagNA Pure Compact instrument and ABI 7500 Fast Dx Real-time PCR systems were used for DNA extraction and to perform real-time PCR respectively.

Results: The newly developed assay is very sensitive with a limit of detection of 100 RD cells/1 million L20B/mL. The amplification efficiency and R²-value were 102.26% and 0.9969 respectively. We evaluated specificity of the assay with five human and four mouse cell lines, as well as monkey and rat cell lines. The assay showed no cross-reactivity with genomic DNA from human, mouse, rat, or monkey cell lines. The analytical sensitivity was also evaluated by spiking varying amounts of RD cells (0.001% - 10%) into L20B cells. There was no difference in C_T values when running single-plex or duplex PCR reactions with similar experimental conditions.

Conclusions: We have developed and validated a TaqMan real-time PCR assay, a sensitive method for the detection of cross-contamination of RD and L20B cell lines.

Keywords: Cell lines cross-contamination; L20B cells; RD cells; TaqMan Real-time PCR.

Figure 1:

Evaluation of Analytical Sensitivity and Specificity TaqMan real-time PCR assay. The equal number of RD and L20B cells were mixed and DNA was extracted. Five dilutions (From 10,000pg/μL to 1pg/μL) were prepared for the evaluation of the instrument response. The real-time PCR assay amplification curve and linear regression data for RD cells are shown in Figure 1a. With RD cells, the instrument showed a 3.56-fold (Standard deviation: 0.32) increase in C_T value with every 10-fold dilution. The real-time PCR assay amplification curve and linear regression data for L20B cells are shown in Figure 1b. With L20B cells, the instrument showed a 3.60-fold (Standard deviation: 0.26 increase in C_T value with every 10-fold dilution.

Figure 1:

Evaluation of Analytical Sensitivity and Specificity TaqMan real-time PCR assay. The equal number of RD and L20B cells were mixed and DNA was extracted. Five dilutions (From 10,000pg/μL to 1pg/μL) were prepared for the evaluation of the instrument response. The real-time PCR assay amplification curve and linear regression data for RD cells are shown in Figure 1a. With RD cells, the instrument showed a 3.56-fold (Standard deviation: 0.32) increase in C_T value with every 10-fold dilution. The real-time PCR assay amplification curve and linear regression data for L20B cells are shown in Figure 1b. With L20B cells, the instrument showed a 3.60-fold (Standard deviation: 0.26 increase in C_T value with every 10-fold dilution.

Figure 2:

Evaluation of analytical sensitivity of TaqMan real-time PCR assay. For Analytical sensitivity, five different amount of RD cells (0.001%, 0.01%, 0.1%, 1.0% to 10%) were mixed with different L20B cells (90% to 100%) and DNA was extracted (100% L20B cells equal to 10⁴ cells/PCR reaction). The limit of detection of cross-contamination was 100 RD cells/1million L20B cells/mL (0.01% RD:100% L20B mixed cells).