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class="site-inner"><div class="content-sidebar-wrap"><main class="content"><article class="post-2559 post type-post status-publish format-standard has-post-thumbnail category-bioprocessing tag-mycoplasma-pcr-test tag-mycoplasma-testing tag-mycoseq-plus tag-rapid-mycoplasma-test-kit tag-rapid-mycoplasma-testing entry" aria-label="Accelerate your Mycoplasma Testing"><header class="entry-header"><h1 class="entry-title">Accelerate your Mycoplasma Testing</h1> <span class="post-info-author"> By <a href="https://www.thermofisher.com/blog/author/lifeinthelabstaff/" title="Posts by Life in the Lab Staff" rel="author">Life in the Lab Staff</a> </span> <span class="post-info-date"> 02.07.2024 </span> </header><div class="entry-content"><p><img decoding="async" class="alignnone wp-image-2562 size-full" src="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/02/mycoplasma-header.jpg" alt="Mycoplasma" width="1569" height="1024" srcset="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/02/mycoplasma-header.jpg 1569w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/02/mycoplasma-header-300x196.jpg 300w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/02/mycoplasma-header-1024x668.jpg 1024w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/02/mycoplasma-header-768x501.jpg 768w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/02/mycoplasma-header-1536x1002.jpg 1536w" sizes="(max-width: 1569px) 100vw, 1569px" /></p> <h2>Explore key elements of rapid mycoplasma detection</h2> <h2></h2> <h2>Introduction</h2> <p>In the vast realm of microbiological contaminants, there exists mycoplasmas, a group of microorganisms that are often overlooked and underestimated. However, those who work with mammalian cell cultures will whole-heartedly agree that mycoplasmas are insidious pathogens capable of causing considerable damage. In the field of cell therapy manufacturing, ensuring the absence of mycoplasma contamination is of utmost importance. To help this, different kits and controls can accurately detect a true mycoplasma contamination. In this blog, we will delve into some features of the Applied Biosystems MycoSEQ Mycoplasma Detection Kits. Additionally, we will address assay sensitivity and provide recommendations for test materials useful for qualification purposes.</p> <h2>Background</h2> <p>MycoSEQ Mycoplasma Detection Kits offer a fully integrated solution for real-time PCR–based mycoplasma detection. Used throughout the bioproduction workflow, MycoSEQ kits are an alternative to costly, time-consuming culture-based tests, facilitating accurate and actionable results in about five hours. Backed by technical and regulatory experts and a successful track-record of regulatory acceptance, MycoSEQ Detection Kits can help simplify mycoplasma detection for both in-process and lot-release testing.</p> <p>The MycoSEQ Plus Mycoplasma Detection Kit is a probe-based quantitative PCR (qPCR) assay kit featuring gold-standard TaqMan chemistry. Developed specifically for the detection of mycoplasma in cell therapy and complex bioproduction samples, TaqMan-based assays enable performance that meets or exceeds the common regulatory sensitivity requirement of 10 CFU/mL (or equivalent 10 GC/mL) LOD, the high level of performance needed to support product lot release testing.</p> <h2>Composition of the Discriminatory Positive Control (DPC)</h2> <p>The DPC plays a crucial role in confirming the accuracy and reliability of mycoplasma detection kits. In the MycoSEQ Plus kit, the DPC is a plasmid control. It has its dedicated vial and is not formulated with any other reagents. What sets the DPC in the MycoSEQ Plus kit apart is the inclusion of both Applied Biosystems FAM and VIC reporters, enhancing the detection capabilities of the kit. The FAM reporter is also the channel used for reporting actual mycoplasma. This control feature gives confidence that the reporter used for mycoplasma detection is functioning as expected. VIC is included to provide a unique signal to the DPC, which allows the discrimination of an accidental control contamination from a true positive result.</p> <h2>Benefits of the Internal Positive Control (IPC)</h2> <p>The Internal Positive Control (IPC) plays a crucial role in this assay by validating the integrity of the test run. It helps detect any inhibitory factors or errors during the extraction or amplification process that might lead to false-negative results. By incorporating an IPC, it can help ensure the accuracy and reliability of the test results, providing a higher level of confidence in the data produced. If the IPC fails to amplify, it indicates that there may have been a problem with the procedure, prompting a retest and thereby preventing incorrect interpretation of results. The IPC acts as a safety net, helping to ensure the precision of the molecular diagnostic process.</p> <h2>Assay sensitivity: MycoSEQ Plus Kit vs. SYBR assay</h2> <p>When it comes to assay sensitivity, both the MycoSEQ Plus kit and the SYBR assay deliver comparable results. When used with a suitable sample preparation method, both kits can detect mycoplasma at levels below 10 GC/mL, which meets the required sensitivity threshold for lot release testing. It is important to note that when comparing Ct values, direct comparisons across different types of kits are not recommended. Ct values are relative measures that depend on assay designs, chemistries, and analysis settings. The key factor is the ability of a kit to consistently meet the sensitivity threshold requirement.</p> <h2>Using live mycoplasma for qualification</h2> <p>Live mycoplasma is not needed for qualification and validation of either MycoSEQ kit. Both kits detect the presence of genomic DNA, regardless of if it is from live mycoplasma or a stock of purified gDNA. While the MycoSEQ Plus kit is designed to detect the presence of mycoplasma gDNA, it is not recommended to bring live mycoplasma into the facility for qualification purposes. To mitigate risks associated with live mycoplasma, alternative methods can be employed. One possibility is to use purified mycoplasma gDNA from a reputable source. A second option is to use inactivated mycoplasma cells. Alternatively, customers can collaborate with a mycoplasma reference lab that specializes in live mycoplasma spiking studies.</p> <h2>Conclusion</h2> <p>The MycoSEQ Plus kit, with its comprehensive control design and enhanced detection capabilities, offers an effective solution for mycoplasma detection in cell therapy manufacturing. The comparable assay sensitivity of both the MycoSEQ Plus kit and the SYBR assay helps ensure either kit can provide reliable results for lot release testing. By following recommended sample preparation methods, such as avoiding the use of live mycoplasma, laboratories can support the integrity of their cell therapy production processes and support the safety of their products.</p> <p>Remember, when it comes to mycoplasma detection, accuracy, and reliability are paramount. Choose the right tools and protocols to safeguard your cell therapy manufacturing workflow. Visit our <a href="https://www.thermofisher.com/us/en/home/life-science/bioproduction/contaminant-and-impurity-testing/mycoplasma-detection.html">mycoplasma detection solution page</a> to learn more.</p> <h2>Recommended resources</h2> <ul> <li><strong>Product bulletin: </strong><a href="https://assets.thermofisher.com/TFS-Assets/BPD/Reference-Materials/mycoseq-mycoplasma-detection-product-bulletin.pdf" target="_blank" rel="noopener">MycoSEQ Mycoplasma Detection Kits</a></li> <li><strong>Infographic:</strong> <a href="https://assets.thermofisher.com/TFS-Assets/BPD/Reference-Materials/rapid-mycoplasma-testing-infographic.pdf" target="_blank" rel="noopener">Rapid Mycoplasma testing: Meeting regulatory requirements with confidence</a></li> <li><strong>Webinars: </strong><a href="https://www.thermofisher.com/us/en/home/global/forms/bioprocessing/pharma-analytics-mycoplasma-detection.html">On-demand presentations about mycoplasma testing solutions</a></li> </ul> <aside 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