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面议MycoSEQ™ Mycoplasma Detection Kit支原体检测试剂盒
The MycoSEQ™ Mycoplasma Detection Assay, based on real-time PCR and Power SYBR® Green detection technology, is designed for the routine screening and detection of Mycoplasma and other closely related species such as Acholeplasma laidlawii and Spiroplasma citri. Through intensive bioinformatics and highly optimized multiplexed primer design, the system allows for highly sensitive, specific, and comprehensive Mycoplasma species detection. The rapid time-to-results of less than 5 hours supports in-process monitoring for the presence of Mycoplasma during cell culture manufacturing, allowing for the earliest possible detection of a contamination event, and providing protection against the spread of contamination into downstream equipment, processes, and media.
Advantages:
? Rapid time-to-results in less than 5 hours.
? Detection of more than 90 Mycoplasma species.
? Demonstrated sensitivity to detect less than 10 copies?reaction.
? Proven specificity.
? PrepSEQ™ sample preparation for high efficiency DNA recovery.
? Proprietary discriminatory positive?extraction control.
? Externally validated.
? Part of the Cell Culture Rapid Methods Program.
Mycoplasma Detection that Meets European Pharmacopoeia Guidance
Mycoplasmas, the smallest known free living organisms, are relatively common bacterial contaminants of mammalian cell cultures. Potential sources of infection include contaminated raw materials used for cell culture, exposure to contaminated cell cultures, and laboratory staff. Mycoplasmas present particular challenges because they are difficult to detect using traditional microbiological techniques. Figure 1 shows the different points in biopharmaceutical manufacturing where testing for Mycoplasma is typically performed.
Regulatory guidance requires that all products derived from mammalian cell culture be tested for the presence of Mycoplasma. In July 2007, the European Pharmacopoeia (5.8, Sec. 2.6.7) provided guidance on the validation requirements for nucleic acid amplification–based methods for detection of Mycoplasma. Applied Biosystems® designed the MycoSEQ™ Mycoplasma Detection Assay to meet these recommendations.
Detect Mycoplasma in Less than 5 Hours with an Easy-to-Run Assay
The MycoSEQ™ Mycoplasma Detection Assay has a straightforward workflow that can deliver results in less than 5 hours (Figure 2). This rapid time-to-results allows the earliest possible detection of mycoplasma contamination.
These Features Make Performing the Assay Simple:
? Variable test sample volumes, from 100 μl to 10 ml of cell culture containing up to 108 cells.
? Closed-tube, single-step detection.
? Load-and-run, walk-away automation during detection.
? No gel electrophoresis, hybridization, or washing steps.
? Minimal requirements on infrastructure and space.
? Flexible throughput.
? Optimized workflow to provide high sensitivity and specificity during routine testing.
Detect More Than 90 Mycoplasma Species
To help ensure the highest level of confidence in testing for the presence of Mycoplasma, an assay must have the ability to detect all known Mycoplasma species, not just the most common species. Intensive bioinformatics analysis was utilized to design amplification primers and reaction conditions to allow comprehensive detection of known Mycoplasma, Acholeplasma, and Spiroplasma species (Table 1), while avoiding detection of related bacterial species.
Multiparameter Analysis Using Power SYBR® Green Technology
The MycoSEQ™ assay uses highly optimized Power SYBR® Green detection technology, which utilizes multiple parameters, amplification plot (Ct), melting temperature (Tm), and derivative value (D.V.) for interpretation of results. Multiparameter analysis provides highly sensitive and specific detection of fewer than 10 Mycoplasma genome copies per reaction (Figure 3). Numerical readouts for all parameters provide objective test result interpretation.
Discriminatory Positive Control
The MycoSEQ™ Mycoplasma Assay also includes the Discriminatory Positive?Extraction Control, a large plasmid containing a Mycoplasma DNA sequence. This control was designed to behave like Mycoplasma DNA in both the sample preparation and detection portions of the assay. Additionally, the DNA sequence has been modified so that the amplicon generated from this control has a melting temperature (Tm) of approximately 84°C, which is outside the range of amplicons generated from Mycoplasma with this assay. Thus, the Tm can be used to discriminate between a positive test result from Mycoplasma and the control DNA. This novel control enables risk-free DNA spike control testing protocol design, eliminating the possibility of a false positive test result due to accidental cross-contamination of a test sample with the positive control DNA.