PCR에 대한 자기 비드의 억제 효과를 어떻게 완화할 수 있습니까??

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.

하지만

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, 그리고 220015 for bead-in-PCR revealed:

아니요.	1	2	3	4	5	6	7	8	9
MB	160011（50mg/ml）			160013（50mg/ml）			220015（50mg/ml）
Content	0.5mg	0.75mg	1mg	0.5mg	0.75mg	1mg	0.5mg	0.75mg	1mg

Result as follow：

Based on electrophoresis results, amplification efficiency at a magnetic bead concentration of 0.5 mg: 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.

따라서

A special component—an enhancer—is required in this PCR system. Common enhancers include betaine, trehalose, DMSO (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.

아니요.	1	2	3	4	5	6	7	8	9
MB	160011（50mg/ml）			160013（50mg/ml）			220015（10mg/ml）
Content	0.025mg	0.05mg	0.075mg	0.025mg	0.05mg	0.075mg	0.005mg	0.01mg	0.015mg

Result as follow：

It can be observed that at 0.025 mg, 160011 그리고 160013 exhibit no difference from the control, whereas at 0.05 mg and 0.075 ~의, 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 mg, 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.：

아니요.	1	2	3	4	5	6	7	8
MB	220013（10mg/ml）				220015（10mg/ml）
Content	0.003mg	0.005mg	0.007mg	0.01mg	0.003mg	0.005mg	0.007mg	0.01mg

Result as follow：

It can be observed that the performance outcomes of 220013 그리고 22015 are indistinguishable.

The aforementioned magnetic beads 160011, 160013, 220013, 그리고 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.

공급자

상하이 링쥔 생명공학 유한회사, 주식회사에 설립되었습니다 2016 생체자성재료 및 핵산추출시약 전문제조업체입니다..

핵산 추출 및 정제 분야에서 풍부한 경험을 보유하고 있습니다., 단백질 정제, 세포 분리, 화학발광, 및 기타 기술 분야.

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