Currently, one of the most reliable methods for viral infection detection are polymerase chain reaction (PCR) based assays. This process is time and resource heavy, requiring multiple steps of lysis, extraction, purification, and amplification procedures. Herein, we have developed a method to detect virus off swabs using solely shaker-mill based mechanical lysis and the transfer of the viral lysate directly to a PCR assay for virus detection, bypassing the substantial reagent and time investments required for extraction and purification steps. Using Human Coronavirus 229E (HCoV-229E) as a model system, we spiked swabs in vitro for proof-of-concept testing. Swabs were spiked in serial dilutions from 1.2 × 106 to 1.2 × 101 copies/mL and then placed in 2 mL tubes with viral transport media (VTM) to mimic the specimen collection procedures in the clinic prior to processing via shaker-mill homogenization. After homogenization, 1 μL of lysate was processed using RT-qPCR for amplification of the nucleocapsid (N) gene, qualifying viral detection. HCoV-229E in vitro spiked swabs were processed in a novel two-step, direct-to-PCR methodology for viral detection. After running 54 swabs, we confidently determined our limit of detection to be 1.2 × 103 viral copies/mL with 96.30% sensitivity. We have proven that the shaker-mill homogenization-based two-step, direct-to-PCR procedures provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in PCR for the detection of HCoV-229E. This finding allows for reductions in the time and resources required for PCR based virus detection in comparison to the traditional extraction-to-PCR methodology.

中文翻译：

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一种使用人类冠状病毒229E进行拭子病毒检测的新颖的两步直接PCR方法。

当前，用于病毒感染检测的最可靠的方法之一是基于聚合酶链反应（PCR）的检测方法。该过程耗时且耗费资源，需要多个步骤的裂解，提取，纯化和扩增程序。本文中，我们开发了一种仅使用基于振动筛的机械裂解法将拭子中的病毒检测出来的方法，并将病毒裂解液直接转移到PCR检测病毒中，从而绕开了提取和纯化步骤所需的大量试剂和时间。使用人类冠状病毒229E（HCoV-229E）作为模型系统，我们在体外掺入了拭子以进行概念验证测试。将棉签以1.2×106的连续稀释加标至1。2×101拷贝/ mL，然后将其置于装有病毒传输介质（VTM）的2 mL试管中，以模拟通过摇磨机匀浆处理之前在临床中的标本采集程序。匀浆后，使用RT-qPCR处理1μL裂解物以扩增核衣壳（N）基因，从而鉴定病毒。HCoV-229E体外加标棉签采用新颖的两步法直接PCR方法进行病毒检测。运行54个棉签后，我们有把握地将检测限确定为1.2×103病毒拷贝/ mL，灵敏度为96.30％。我们已经证明，基于振动筛均质化的两步直接PCR程序可提供足够的病毒拭子裂解，其中所得的裂解物可直接用于PCR检测HCoV-229E。