The Matrix Effect in the RT-PCR Detection of SARS-CoV-2 Using Saliva without RNA Extraction
The present work focuses on the detection of SARS-CoV-2 in saliva, contributing to understanding the inhibition effect of the matrix and its influence on the results. Detection of viral genes ORF1ab, N, and E was performed by RT-PCR using saliva directly in the reaction without RNA extraction. Diffe...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-06-01
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| Series: | Diagnostics |
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| Online Access: | https://www.mdpi.com/2075-4418/12/7/1547 |
| _version_ | 1797406879274500096 |
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| author | Orlando Morais Manuel Rui Alves Carla Ramos Fernando Ferreira Paulo Fernandes |
| author_facet | Orlando Morais Manuel Rui Alves Carla Ramos Fernando Ferreira Paulo Fernandes |
| author_sort | Orlando Morais |
| collection | DOAJ |
| description | The present work focuses on the detection of SARS-CoV-2 in saliva, contributing to understanding the inhibition effect of the matrix and its influence on the results. Detection of viral genes ORF1ab, N, and E was performed by RT-PCR using saliva directly in the reaction without RNA extraction. Different amounts of saliva were spiked with increasing amounts of viral RNA from COVID-19 patients and subjected to RT-PCR detection. In parallel, 64 saliva samples from confirmed COVID-19 patients were used in two different amounts directly in the RT-PCR reaction and their results compared. The presence of saliva in the RT-PCR always causes a positive shift of the Ct values, but a very high between-person variability of its magnitude was obtained, with increases ranging from 0.93 to 11.36. Viral targets are also affected differently depending on the initial number of viral particles. Due to inhibitors present in saliva, the duplication of sample volume causes only 48 to 61% of the expected Ct value decrease depending on the viral target gene. The use of saliva has advantages, but also limitations, due to potential inhibitors present in the matrix. However, the choice of the target and the right amount of sample may significantly influence the results. |
| first_indexed | 2024-03-09T03:33:03Z |
| format | Article |
| id | doaj.art-fd2ef1b53784433db28603a940755470 |
| institution | Directory Open Access Journal |
| issn | 2075-4418 |
| language | English |
| last_indexed | 2024-03-09T03:33:03Z |
| publishDate | 2022-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Diagnostics |
| spelling | doaj.art-fd2ef1b53784433db28603a9407554702023-12-03T14:53:33ZengMDPI AGDiagnostics2075-44182022-06-01127154710.3390/diagnostics12071547The Matrix Effect in the RT-PCR Detection of SARS-CoV-2 Using Saliva without RNA ExtractionOrlando Morais0Manuel Rui Alves1Carla Ramos2Fernando Ferreira3Paulo Fernandes4Escola Superior de Tecnologia e Gestão, Instituto Politécnico de Viana do Castelo, Rua Escola Industrial e Comercial de Nun’Álvares, 4900-347 Viana do Castelo, PortugalEscola Superior de Tecnologia e Gestão, Instituto Politécnico de Viana do Castelo, Rua Escola Industrial e Comercial de Nun’Álvares, 4900-347 Viana do Castelo, PortugalEscola Superior de Tecnologia e Gestão, Instituto Politécnico de Viana do Castelo, Rua Escola Industrial e Comercial de Nun’Álvares, 4900-347 Viana do Castelo, PortugalACES Cávado III Barcelos/Esposende, Rua Dr. Abel Varzim, 4750-253 Barcelos, PortugalEscola Superior de Tecnologia e Gestão, Instituto Politécnico de Viana do Castelo, Rua Escola Industrial e Comercial de Nun’Álvares, 4900-347 Viana do Castelo, PortugalThe present work focuses on the detection of SARS-CoV-2 in saliva, contributing to understanding the inhibition effect of the matrix and its influence on the results. Detection of viral genes ORF1ab, N, and E was performed by RT-PCR using saliva directly in the reaction without RNA extraction. Different amounts of saliva were spiked with increasing amounts of viral RNA from COVID-19 patients and subjected to RT-PCR detection. In parallel, 64 saliva samples from confirmed COVID-19 patients were used in two different amounts directly in the RT-PCR reaction and their results compared. The presence of saliva in the RT-PCR always causes a positive shift of the Ct values, but a very high between-person variability of its magnitude was obtained, with increases ranging from 0.93 to 11.36. Viral targets are also affected differently depending on the initial number of viral particles. Due to inhibitors present in saliva, the duplication of sample volume causes only 48 to 61% of the expected Ct value decrease depending on the viral target gene. The use of saliva has advantages, but also limitations, due to potential inhibitors present in the matrix. However, the choice of the target and the right amount of sample may significantly influence the results.https://www.mdpi.com/2075-4418/12/7/1547SARS-CoV-2salivaRT-PCRCt valuessensitivitydetection |
| spellingShingle | Orlando Morais Manuel Rui Alves Carla Ramos Fernando Ferreira Paulo Fernandes The Matrix Effect in the RT-PCR Detection of SARS-CoV-2 Using Saliva without RNA Extraction Diagnostics SARS-CoV-2 saliva RT-PCR Ct values sensitivity detection |
| title | The Matrix Effect in the RT-PCR Detection of SARS-CoV-2 Using Saliva without RNA Extraction |
| title_full | The Matrix Effect in the RT-PCR Detection of SARS-CoV-2 Using Saliva without RNA Extraction |
| title_fullStr | The Matrix Effect in the RT-PCR Detection of SARS-CoV-2 Using Saliva without RNA Extraction |
| title_full_unstemmed | The Matrix Effect in the RT-PCR Detection of SARS-CoV-2 Using Saliva without RNA Extraction |
| title_short | The Matrix Effect in the RT-PCR Detection of SARS-CoV-2 Using Saliva without RNA Extraction |
| title_sort | matrix effect in the rt pcr detection of sars cov 2 using saliva without rna extraction |
| topic | SARS-CoV-2 saliva RT-PCR Ct values sensitivity detection |
| url | https://www.mdpi.com/2075-4418/12/7/1547 |
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