The Applied Biosystems ViralSEQ Mouse Minute Virus (MMV) Detection Assay is part of an integrated DNA purification and real-time PCR-based system for rapid, sensitive detection of MMV from a wide range of sample types. Viral DNA is isolated from a sample using the PrepSEQ 1-2-3 Nucleic Acid Extraction Kit (available separately). Highly sensitive and specific detection of the viral DNA is then performed using TaqMan real-time PCR technology. The assay includes a proprietary positive control molecule that eliminates the possibility of a false positive test result arising from inadvertent contamination of the test sample with positive control.

High specificity and rapid time-to-results
The ViralSEQ MMV assay detects the four known strains of MMV: i, p, m, and c, including seven genomic sequences reported in the NCBI database. Table 1 shows the specificity of the ViralSEQ MMV assay and lack of cross-reactivity with unrelated DNA. The assay workflow is simple and delivers results in just under 4 hours (Figure 1).

Streamlined process for surveillance and early detection
The ViralSEQ MMV Detection Assay is part of the Cell Culture Rapid Methods Program, designed to streamline the detection of three common contaminants of mammalian cell culture-based biopharmaceutical manufacturing (Figure 2). The program sets new workflow standards in efficiency and product quality, combining one sample preparation step with real-time PCR-based assays for the detection of mycoplasma, vesivirus, and MMV on one instrument platform.

Reliable results
The ViralSEQ MMV Detection Assay includes an MMV Discriminatory Positive/Extraction Control, which is a plasmid DNA containing the MMV DNA sequence that is recognized by the MMV detection assay (FAM probe). This control (Figure 3) was designed to behave like MMV DNA in both the sample preparation and detection portions of the process. In addition, the control was engineered with an additional VIC probe binding sequence. The presence of both FAM and VIC signal in a PCR reaction can be used to discriminate a positive test result between MMV (FAM) and the control DNA (FAM and VIC). This novel control enables risk-free DNA spike control testing, eliminating the possibility of a false positive test result due to accidental cross-contamination of a test sample with the positive control DNA.

Low-level detection
The ViralSEQ MMV Detection Assay uses proven TaqMan technology for highly sensitivity detection down to 10 copies of MMV genomic DNA per PCR reaction (viral particles equivalent to 0.002 TCID50). Analysis of 10-fold serial dilutions of MMV genomic DNA demonstrates the broad dynamic range and efficiency of the assay (Figures 4 and 5).

Superior sample preparation method
The PrepSEQ 1-2-3 Nucleic Acid Extraction Kit (available separately) helps enable consistent and high percentage recovery of MMV genomic DNA from complex sample types that include CHO cells and bovine serum. Figure 6 shows that the Ct values of DNA extracted from a spiked sample were comparable to an unspiked control that was amplified in the real-time PCR reaction, demonstrating the highly efficient DNA recovery using the PrepSEQ 1-2-3 kit.