Strategy to Develop and Evaluate a Multiplex RT-ddPCR in Response to SARS-CoV-2 Genomic Evolution
The worldwide emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since 2019 has highlighted the importance of rapid and reliable diagnostic testing to prevent and control the viral transmission. However, inaccurate results may occur due to false negatives (FN) cause...
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2021-11-01
|
| Series: | Current Issues in Molecular Biology |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1467-3045/43/3/134 |
| _version_ | 1827673341651058688 |
|---|---|
| author | Laura A. E. Van Poelvoorde Mathieu Gand Marie-Alice Fraiture Sigrid C. J. De Keersmaecker Bavo Verhaegen Koenraad Van Hoorde Ann Brigitte Cay Nadège Balmelle Philippe Herman Nancy Roosens |
| author_facet | Laura A. E. Van Poelvoorde Mathieu Gand Marie-Alice Fraiture Sigrid C. J. De Keersmaecker Bavo Verhaegen Koenraad Van Hoorde Ann Brigitte Cay Nadège Balmelle Philippe Herman Nancy Roosens |
| author_sort | Laura A. E. Van Poelvoorde |
| collection | DOAJ |
| description | The worldwide emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since 2019 has highlighted the importance of rapid and reliable diagnostic testing to prevent and control the viral transmission. However, inaccurate results may occur due to false negatives (FN) caused by polymorphisms or point mutations related to the virus evolution and compromise the accuracy of the diagnostic tests. Therefore, PCR-based SARS-CoV-2 diagnostics should be evaluated and evolve together with the rapidly increasing number of new variants appearing around the world. However, even by using a large collection of samples, laboratories are not able to test a representative collection of samples that deals with the same level of diversity that is continuously evolving worldwide. In the present study, we proposed a methodology based on an in silico and in vitro analysis. First, we used all information offered by available whole-genome sequencing data for SARS-CoV-2 for the selection of the two PCR assays targeting two different regions in the genome, and to monitor the possible impact of virus evolution on the specificity of the primers and probes of the PCR assays during and after the development of the assays. Besides this first essential in silico evaluation, a minimal set of testing was proposed to generate experimental evidence on the method performance, such as specificity, sensitivity and applicability. Therefore, a duplex reverse-transcription droplet digital PCR (RT-ddPCR) method was evaluated in silico by using 154 489 whole-genome sequences of SARS-CoV-2 strains that were representative for the circulating strains around the world. The RT-ddPCR platform was selected as it presented several advantages to detect and quantify SARS-CoV-2 RNA in clinical samples and wastewater. Next, the assays were successfully experimentally evaluated for their sensitivity and specificity. A preliminary evaluation of the applicability of the developed method was performed using both clinical and wastewater samples. |
| first_indexed | 2024-03-10T04:23:31Z |
| format | Article |
| id | doaj.art-0d914115867441bc97a735d0778ac7a9 |
| institution | Directory Open Access Journal |
| issn | 1467-3037 1467-3045 |
| language | English |
| last_indexed | 2024-03-10T04:23:31Z |
| publishDate | 2021-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Current Issues in Molecular Biology |
| spelling | doaj.art-0d914115867441bc97a735d0778ac7a92023-11-23T07:44:09ZengMDPI AGCurrent Issues in Molecular Biology1467-30371467-30452021-11-014331937194910.3390/cimb43030134Strategy to Develop and Evaluate a Multiplex RT-ddPCR in Response to SARS-CoV-2 Genomic EvolutionLaura A. E. Van Poelvoorde0Mathieu Gand1Marie-Alice Fraiture2Sigrid C. J. De Keersmaecker3Bavo Verhaegen4Koenraad Van Hoorde5Ann Brigitte Cay6Nadège Balmelle7Philippe Herman8Nancy Roosens9Transversal Activities in Applied Genomics, Sciensano, 1050 Brussels, BelgiumTransversal Activities in Applied Genomics, Sciensano, 1050 Brussels, BelgiumTransversal Activities in Applied Genomics, Sciensano, 1050 Brussels, BelgiumTransversal Activities in Applied Genomics, Sciensano, 1050 Brussels, BelgiumFood Pathogens, Sciensano, 1050 Brussels, BelgiumFood Pathogens, Sciensano, 1050 Brussels, BelgiumEnzootic, Vector-Borne and Bee Diseases, Sciensano, 1180 Brussels, BelgiumEnzootic, Vector-Borne and Bee Diseases, Sciensano, 1180 Brussels, BelgiumExpertise and Service Provision, Sciensano, 1050 Brussels, BelgiumTransversal Activities in Applied Genomics, Sciensano, 1050 Brussels, BelgiumThe worldwide emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since 2019 has highlighted the importance of rapid and reliable diagnostic testing to prevent and control the viral transmission. However, inaccurate results may occur due to false negatives (FN) caused by polymorphisms or point mutations related to the virus evolution and compromise the accuracy of the diagnostic tests. Therefore, PCR-based SARS-CoV-2 diagnostics should be evaluated and evolve together with the rapidly increasing number of new variants appearing around the world. However, even by using a large collection of samples, laboratories are not able to test a representative collection of samples that deals with the same level of diversity that is continuously evolving worldwide. In the present study, we proposed a methodology based on an in silico and in vitro analysis. First, we used all information offered by available whole-genome sequencing data for SARS-CoV-2 for the selection of the two PCR assays targeting two different regions in the genome, and to monitor the possible impact of virus evolution on the specificity of the primers and probes of the PCR assays during and after the development of the assays. Besides this first essential in silico evaluation, a minimal set of testing was proposed to generate experimental evidence on the method performance, such as specificity, sensitivity and applicability. Therefore, a duplex reverse-transcription droplet digital PCR (RT-ddPCR) method was evaluated in silico by using 154 489 whole-genome sequences of SARS-CoV-2 strains that were representative for the circulating strains around the world. The RT-ddPCR platform was selected as it presented several advantages to detect and quantify SARS-CoV-2 RNA in clinical samples and wastewater. Next, the assays were successfully experimentally evaluated for their sensitivity and specificity. A preliminary evaluation of the applicability of the developed method was performed using both clinical and wastewater samples.https://www.mdpi.com/1467-3045/43/3/134droplet digital PCRCOVID-19SARS-CoV-2wastewaterrespiratory samplesmonitoring |
| spellingShingle | Laura A. E. Van Poelvoorde Mathieu Gand Marie-Alice Fraiture Sigrid C. J. De Keersmaecker Bavo Verhaegen Koenraad Van Hoorde Ann Brigitte Cay Nadège Balmelle Philippe Herman Nancy Roosens Strategy to Develop and Evaluate a Multiplex RT-ddPCR in Response to SARS-CoV-2 Genomic Evolution Current Issues in Molecular Biology droplet digital PCR COVID-19 SARS-CoV-2 wastewater respiratory samples monitoring |
| title | Strategy to Develop and Evaluate a Multiplex RT-ddPCR in Response to SARS-CoV-2 Genomic Evolution |
| title_full | Strategy to Develop and Evaluate a Multiplex RT-ddPCR in Response to SARS-CoV-2 Genomic Evolution |
| title_fullStr | Strategy to Develop and Evaluate a Multiplex RT-ddPCR in Response to SARS-CoV-2 Genomic Evolution |
| title_full_unstemmed | Strategy to Develop and Evaluate a Multiplex RT-ddPCR in Response to SARS-CoV-2 Genomic Evolution |
| title_short | Strategy to Develop and Evaluate a Multiplex RT-ddPCR in Response to SARS-CoV-2 Genomic Evolution |
| title_sort | strategy to develop and evaluate a multiplex rt ddpcr in response to sars cov 2 genomic evolution |
| topic | droplet digital PCR COVID-19 SARS-CoV-2 wastewater respiratory samples monitoring |
| url | https://www.mdpi.com/1467-3045/43/3/134 |
| work_keys_str_mv | AT lauraaevanpoelvoorde strategytodevelopandevaluateamultiplexrtddpcrinresponsetosarscov2genomicevolution AT mathieugand strategytodevelopandevaluateamultiplexrtddpcrinresponsetosarscov2genomicevolution AT mariealicefraiture strategytodevelopandevaluateamultiplexrtddpcrinresponsetosarscov2genomicevolution AT sigridcjdekeersmaecker strategytodevelopandevaluateamultiplexrtddpcrinresponsetosarscov2genomicevolution AT bavoverhaegen strategytodevelopandevaluateamultiplexrtddpcrinresponsetosarscov2genomicevolution AT koenraadvanhoorde strategytodevelopandevaluateamultiplexrtddpcrinresponsetosarscov2genomicevolution AT annbrigittecay strategytodevelopandevaluateamultiplexrtddpcrinresponsetosarscov2genomicevolution AT nadegebalmelle strategytodevelopandevaluateamultiplexrtddpcrinresponsetosarscov2genomicevolution AT philippeherman strategytodevelopandevaluateamultiplexrtddpcrinresponsetosarscov2genomicevolution AT nancyroosens strategytodevelopandevaluateamultiplexrtddpcrinresponsetosarscov2genomicevolution |