المهنية في صناعة الخرز المغناطيسي الحيوي
كيف يمكن التخفيف من التأثير المثبط للخرزات المغناطيسية على تفاعل البوليميراز المتسلسل?
With the advancement of
precision medicine and the reduction in NGS sequencing costs,magnetic bead-based nucleic acid extraction has become standard practice in molecular diagnostics laboratories. To achieve higher recovery rates and avoid errors caused by incomplete elution, an increasing number of researchers opt to add magnetic beads capturing nucleic acids directly into the PCR system for amplification.
لكن
While this “PCR with Magnetic beads” approach streamlines workflows, it has introduced insidious frustrations for many practitioners: PCR amplification failures, elevated Ct values, or even absent amplification curves. The culprit behind these issues is often the magnetic beads themselves, which inhibit the PCR system.
Many intuitively assume magnetic beads are merely inert solid carriers that should not interfere with amplification. Yet experimental data consistently demonstrates widespread bead-induced PCR inhibition.
Preliminary experiments employing our magnetic bead products 160011, 160013, و 220015 for bead-in-PCR revealed:
| لا. | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| MB | 160011（50mg/ml） | 160013（50mg/ml） | 220015（50mg/ml） | ||||||
| Content | 0.5ملغ | 0.75ملغ | 1ملغ | 0.5ملغ | 0.75ملغ | 1ملغ | 0.5ملغ | 0.75ملغ | 1ملغ |
Result as follow：
Based on electrophoresis results, amplification efficiency at a magnetic bead concentration of 0.5 ملغ: 220015 < 160013 < 160011; Given the known magnetic bead particle sizes: 220015 < 160013 < 160011, and considering their specific surface areas: 160011 < 160013 < 220015, the binding sites per bead under identical conditions are thus: 160011＜160013＜220015; This may explain why PCR inhibition becomes more pronounced as magnetic bead concentration increases.
Consequently
A special component—an enhancer—is required in this PCR system. Common enhancers include betaine, trehalose, [دمس] (dimethyl sulfoxide), glycerol, and BSA (bovine serum albumin). Among these, BSA acts as a sacrificial substrate, preferentially binding to magnetic beads to shield the polymerase from inhibitor interference.
So we sought to determine whether reducing magnetic bead concentration (0.025 mg–0.075 mg) supplemented with 100 μg/ml BSA as a PCR enhancer would yield more favourable results.
| لا. | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| MB | 160011（50mg/ml） | 160013（50mg/ml） | 220015（10mg/ml） | ||||||
| Content | 0.025ملغ | 0.05ملغ | 0.075ملغ | 0.025ملغ | 0.05ملغ | 0.075ملغ | 0.005ملغ | 0.01ملغ | 0.015ملغ |
Result as follow：
It can be observed that at 0.025 ملغ, 160011 و 160013 exhibit no difference from the control, whereas at 0.05 mg and 0.075 ل, their efficacy diminishes progressively. 220015, consistent with previous analyses, not only exhibits excellent suspension properties but also possesses a large specific surface area with numerous binding sites. This enables effective adsorption of Taq polymerase, thereby inhibiting amplification. When the magnetic bead concentration was reduced to 0.005 ملغ, the band intensity reached only half that of the control.
Following the foundational optimisation under these conditions, our 220013 product shall be selected for efficacy validation.：
| لا. | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| MB | 220013（10mg/ml） | 220015（10mg/ml） | ||||||
| Content | 0.003ملغ | 0.005ملغ | 0.007ملغ | 0.01ملغ | 0.003ملغ | 0.005ملغ | 0.007ملغ | 0.01ملغ |
Result as follow：
It can be observed that the performance outcomes of 220013 و 22015 are indistinguishable.
The aforementioned magnetic beads 160011, 160013, 220013, و 220015 can all achieve optimal results in PCR through system optimisation. They are suitable for application in POCT rapid testing, pathogenic microorganism detection, automated extraction, and similar scenarios.
مزود
شركة شنغهاي لينغجون للتكنولوجيا الحيوية, المحدودة.تأسست في 2016 وهي شركة متخصصة في تصنيع المواد المغناطيسية الحيوية وكواشف استخلاص الحمض النووي.
لدينا خبرة غنية في استخراج وتنقية الحمض النووي, تنقية البروتين, فصل الخلايا, التألق الكيميائي, وغيرها من المجالات التقنية.
منتجاتنا تستخدم على نطاق واسع في العديد من المجالات, مثل الفحوصات الطبية, الاختبارات الجينية, البحوث الجامعية, التكاثر الوراثي, وهكذا. نحن لا نقدم المنتجات فحسب، بل يمكننا أيضًا إجراء تصنيع المعدات الأصلية, أوديإم, وغيرها من الاحتياجات. إذا كان لديك حاجة ذات صلة, لا تتردد في الاتصال بنا .
