SalivaSTAT: Direct-PCR and Pooling of Saliva Samples Collected in Healthcare and Community Setting for SARS-CoV-2 Mass Surveillance
Objectives: 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 avail...
| Main Authors: | , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-05-01
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| Series: | Diagnostics |
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| Online Access: | https://www.mdpi.com/2075-4418/11/5/904 |
| _version_ | 1797533535266930688 |
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| author | Nikhil S. Sahajpal Ashis K. Mondal Sudha Ananth Allan Njau Pankaj Ahluwalia Gary Newnam Adriana Lozoya-Colinas Nicholas V. Hud Vamsi Kota Ted M. Ross Michelle D. Reid Sadanand Fulzele Alka Chaubey Madhuri Hegde Amyn M. Rojiani Ravindra Kolhe |
| author_facet | Nikhil S. Sahajpal Ashis K. Mondal Sudha Ananth Allan Njau Pankaj Ahluwalia Gary Newnam Adriana Lozoya-Colinas Nicholas V. Hud Vamsi Kota Ted M. Ross Michelle D. Reid Sadanand Fulzele Alka Chaubey Madhuri Hegde Amyn M. Rojiani Ravindra Kolhe |
| author_sort | Nikhil S. Sahajpal |
| collection | DOAJ |
| description | Objectives: 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. Methods: 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. Results: 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. Conclusion: We have optimized an extraction-free RT-PCR assay for saliva samples that demonstrates comparable performance to FDA-EUA assay (Extraction and RT-PCR). |
| first_indexed | 2024-03-10T11:15:57Z |
| format | Article |
| id | doaj.art-35618974a0ff410f917919e1e553da3b |
| institution | Directory Open Access Journal |
| issn | 2075-4418 |
| language | English |
| last_indexed | 2024-03-10T11:15:57Z |
| publishDate | 2021-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Diagnostics |
| spelling | doaj.art-35618974a0ff410f917919e1e553da3b2023-11-21T20:23:24ZengMDPI AGDiagnostics2075-44182021-05-0111590410.3390/diagnostics11050904SalivaSTAT: Direct-PCR and Pooling of Saliva Samples Collected in Healthcare and Community Setting for SARS-CoV-2 Mass SurveillanceNikhil S. Sahajpal0Ashis K. Mondal1Sudha Ananth2Allan Njau3Pankaj Ahluwalia4Gary Newnam5Adriana Lozoya-Colinas6Nicholas V. Hud7Vamsi Kota8Ted M. Ross9Michelle D. Reid10Sadanand Fulzele11Alka Chaubey12Madhuri Hegde13Amyn M. Rojiani14Ravindra Kolhe15Department of Pathology, Medical College of Georgia, Augusta University, GA 30901, USADepartment of Pathology, Medical College of Georgia, Augusta University, GA 30901, USADepartment of Pathology, Medical College of Georgia, Augusta University, GA 30901, USADepartment of Pathology, Aga Khan University Hospital, Nairobi 30270-00100, KenyaDepartment of Pathology, Medical College of Georgia, Augusta University, GA 30901, USASchool of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USASchool of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USASchool of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USADepartment of Medicine, Medical College of Georgia, Augusta University, GA 30901, USACenter for Vaccines and Immunology, University of Georgia, GA 30602, USADepartment of Pathology, Emory University, GA 30322, USACenter for Healthy Aging, Medical College of Georgia, Augusta University, Augusta, GA 30901, USADepartment of Pathology, Medical College of Georgia, Augusta University, GA 30901, USAGlobal Laboratory Services, PerkinElmer, Waltham, MA 02451, USADepartment of Pathology, Medical College of Georgia, Augusta University, GA 30901, USADepartment of Pathology, Medical College of Georgia, Augusta University, GA 30901, USAObjectives: 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. Methods: 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. Results: 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. Conclusion: We have optimized an extraction-free RT-PCR assay for saliva samples that demonstrates comparable performance to FDA-EUA assay (Extraction and RT-PCR).https://www.mdpi.com/2075-4418/11/5/904salivaextraction-freeRT-PCRpooling |
| spellingShingle | Nikhil S. Sahajpal Ashis K. Mondal Sudha Ananth Allan Njau Pankaj Ahluwalia Gary Newnam Adriana Lozoya-Colinas Nicholas V. Hud Vamsi Kota Ted M. Ross Michelle D. Reid Sadanand Fulzele Alka Chaubey Madhuri Hegde Amyn M. Rojiani Ravindra Kolhe SalivaSTAT: Direct-PCR and Pooling of Saliva Samples Collected in Healthcare and Community Setting for SARS-CoV-2 Mass Surveillance Diagnostics saliva extraction-free RT-PCR pooling |
| title | SalivaSTAT: Direct-PCR and Pooling of Saliva Samples Collected in Healthcare and Community Setting for SARS-CoV-2 Mass Surveillance |
| title_full | SalivaSTAT: Direct-PCR and Pooling of Saliva Samples Collected in Healthcare and Community Setting for SARS-CoV-2 Mass Surveillance |
| title_fullStr | SalivaSTAT: Direct-PCR and Pooling of Saliva Samples Collected in Healthcare and Community Setting for SARS-CoV-2 Mass Surveillance |
| title_full_unstemmed | SalivaSTAT: Direct-PCR and Pooling of Saliva Samples Collected in Healthcare and Community Setting for SARS-CoV-2 Mass Surveillance |
| title_short | SalivaSTAT: Direct-PCR and Pooling of Saliva Samples Collected in Healthcare and Community Setting for SARS-CoV-2 Mass Surveillance |
| title_sort | salivastat direct pcr and pooling of saliva samples collected in healthcare and community setting for sars cov 2 mass surveillance |
| topic | saliva extraction-free RT-PCR pooling |
| url | https://www.mdpi.com/2075-4418/11/5/904 |
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