Liu Weina, Zhu Yong, Stellacci Francesco
Institute of Materials, École Polytechnique Fédérale de Lausanne, Station 12, Lausanne 1015, Switzerland.
Institute of Bioengineering, École Polytechnique Fédérale de Lausanne, Station 12, Lausanne 1015, Switzerland.
ACS Sustain Chem Eng. 2023 Mar 24;11(14):5524-5536. doi: 10.1021/acssuschemeng.2c07309. eCollection 2023 Apr 10.
Polymerase chain reaction (PCR) kits have been used as common diagnosing tools during the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, with daily worldwide usage in the millions. It is well known that at the beginning of the pandemic, there was a shortage of PCR kits. So far, the ecosystem of a PCR kit is linear use; that is, kits are produced, used once, and disposed of as biolab waste. Here, we show that to mitigate the risk of future shortages, it is possible to envision recyclable PCR kits based on a more sustainable use of nucleic acid resources. A PCR kit is mainly composed of primers, nucleotides, and enzymes. In the case of a positive test, the free nucleotides are polymerized onto the primers to form longer DNA strands. Our approach depolymerizes such strands, keeping the primers and regenerating the nucleotides, i.e., returning the nucleic acid materials to the original state. The polymerized long DNA strands are hydrolyzed into nucleotide monophosphates that are then phosphorylated into triphosphates using a method that is developed from a recent publication. We used oligonucleotides with a 3'-terminal phosphorothioate (PS) backbone modification as nonhydrolyzable PCR primers, which are able to undergo the recycling process unchanged. The nuclease resistance of oligonucleotides with a ribose sugar modification was also evaluated, which showed worse recycling efficiency than PS-modified oligonucleotides. We successfully recycled both PCR primers and nucleotide monomers (∼75% yield). We demonstrate that the method allows for the direct reuse of PCR kits. We also show that the recycled primers can be isolated and then added to endpoint or quantitative PCR. This recycling approach provides a new path for circularly reusing nucleic acid materials in PCR kits.
在严重急性呼吸综合征冠状病毒2（SARS-CoV-2）大流行期间，聚合酶链反应（PCR）试剂盒一直被用作常见的诊断工具，全球每日使用量达数百万份。众所周知，在大流行初期，PCR试剂盒短缺。到目前为止，PCR试剂盒的使用模式是线性的；也就是说，试剂盒生产出来后只使用一次，然后作为生物实验室废物处理。在此，我们表明，为降低未来短缺的风险，可以设想基于更可持续地利用核酸资源的可回收PCR试剂盒。一个PCR试剂盒主要由引物、核苷酸和酶组成。在检测呈阳性的情况下，游离核苷酸会聚合到引物上，形成更长的DNA链。我们的方法是将这些链解聚，保留引物并再生核苷酸，即将核酸材料恢复到原始状态。聚合的长DNA链被水解成单磷酸核苷酸，然后使用从最近一篇文献中开发的方法将其磷酸化为三磷酸核苷酸。我们使用具有3'-末端硫代磷酸酯（PS）骨架修饰的寡核苷酸作为不可水解的PCR引物，它们能够在回收过程中保持不变。还评估了具有核糖修饰的寡核苷酸的核酸酶抗性，结果表明其回收效率比PS修饰的寡核苷酸差。我们成功回收了PCR引物和核苷酸单体（产率约75%）。我们证明了该方法可以直接重复使用PCR试剂盒。我们还表明，回收的引物可以分离出来，然后添加到终点或定量PCR中。这种回收方法为PCR试剂盒中核酸材料的循环再利用提供了一条新途径。
