Sahajpal Nikhil S, Mondal Ashis K, Ananth Sudha, Njau Allan, Ahluwalia Pankaj, Newnam Gary, Lozoya-Colinas Adriana, Hud Nicholas V, Kota Vamsi, Ross Ted M, Reid Michelle D, Fulzele Sadanand, Chaubey Alka, Hegde Madhuri, Rojiani Amyn M, Kolhe Ravindra
Department of Pathology, Medical College of Georgia, Augusta University, GA 30901, USA.
Department of Pathology, Aga Khan University Hospital, Nairobi 30270-00100, Kenya.
Diagnostics (Basel). 2021 May 19;11(5):904. doi: 10.3390/diagnostics11050904.
Limitations of widespread current COVID-19 diagnostic testing exist in both the pre-analytical and analytical stages. To alleviate these limitations, we developed a universal saliva processing protocol (SalivaSTAT) that would enable an extraction-free RT-PCR test using commercially available RT-PCR kits.
We optimized saliva collection devices, heat-shock treatment, and homogenization. Saliva samples (879) previously tested using the FDA-EUA method were reevaluated with the optimized SalivaSTAT protocol using two widely available commercial RT-PCR kits. A five-sample pooling strategy was evaluated as per FDA guidelines.
Saliva collection (done without any media) showed performance comparable to that of the FDA-EUA method. The SalivaSTAT protocol was optimized by incubating saliva samples at 95 °C for 30-min and homogenization, followed by RT-PCR assay. The clinical sample evaluation of 630 saliva samples using the SalivaSTAT protocol with PerkinElmer (600-samples) and CDC (30-samples) RT-PCR assay achieved positive (PPA) and negative percent agreements (NPAs) of 95.0% and 100%, respectively. The LoD was established as ~60-180 copies/mL by absolute quantification. Furthermore, a five-sample-pooling evaluation using 250 saliva samples achieved a PPA and NPA of 92% and 100%, respectively.
We have optimized an extraction-free RT-PCR assay for saliva samples that demonstrates comparable performance to FDA-EUA assay (Extraction and RT-PCR).
当前广泛使用的新冠病毒诊断检测在分析前和分析阶段均存在局限性。为了缓解这些局限性，我们开发了一种通用唾液处理方案（SalivaSTAT），该方案能够使用市售的逆转录聚合酶链反应（RT-PCR）试剂盒进行无需提取的RT-PCR检测。
我们优化了唾液收集装置、热休克处理和匀浆过程。使用两种广泛使用的商业RT-PCR试剂盒，采用优化后的SalivaSTAT方案对先前使用美国食品药品监督管理局（FDA）紧急使用授权（EUA）方法检测的879份唾液样本进行重新评估。按照FDA指南评估了五样本混合策略。
唾液收集（无需任何培养基）显示出与FDA-EUA方法相当的性能。通过将唾液样本在95°C孵育30分钟并进行匀浆，然后进行RT-PCR检测，对SalivaSTAT方案进行了优化。使用PerkinElmer（600份样本）和美国疾病控制与预防中心（CDC）（30份样本）的RT-PCR检测，采用SalivaSTAT方案对630份唾液样本进行临床样本评估，阳性百分一致率（PPA）和阴性百分一致率（NPA）分别达到95.0%和100%。通过绝对定量确定检测限为~60-180拷贝/毫升。此外，使用250份唾液样本进行的五样本混合评估的PPA和NPA分别为92%和100%。
我们优化了一种用于唾液样本的无需提取的RT-PCR检测方法，其性能与FDA-EUA检测方法（提取和RT-PCR）相当。
