SARS-CoV-2: Comparative analysis of different RNA extraction methods

[Display omitted] •Alternative suitable methods to extract SARS-CoV-2 RNA should be taken into account to cope with the ongoing pandemic.•Extraction efficiencies of different viral RNA kit brands can be optimized for detection of SARS-CoV-2 genome.•The accuracy and performance of the Allplex 2019-nC...

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Veröffentlicht in:	Journal of virological methods Jg. 287; S. 114008
Hauptverfasser:	Ambrosi, Cecilia, Prezioso, Carla, Checconi, Paola, Scribano, Daniela, Sarshar, Meysam, Capannari, Maurizio, Tomino, Carlo, Fini, Massimo, Garaci, Enrico, Palamara, Anna Teresa, De Chiara, Giovanna, Limongi, Dolores
Format:	Journal Article
Sprache:	Englisch
Veröffentlicht:	Netherlands Elsevier B.V 01.01.2021 The Author(s). Published by Elsevier B.V
Schlagworte:	COVID-19 - diagnosis COVID-19 infection COVID-19 Nucleic Acid Testing - methods Diagnostic Tests, Routine etiological agents Genes, Viral - genetics Humans Limit of Detection Oro- nasopharyngeal swabs Pharynx - virology quantitative polymerase chain reaction Reproducibility of Results reverse transcription RNA RNA extraction RNA, Viral - analysis RNA, Viral - genetics RNA, Viral - isolation & purification rRT-PCR SARS-CoV-2 SARS-CoV-2 - genetics SARS-CoV-2 - isolation & purification separation Severe acute respiratory syndrome coronavirus 2 RNA extraction SARS-CoV-2 Oro- nasopharyngeal swabs rRT-PCR
ISSN:	0166-0934, 1879-0984, 1879-0984
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Abstract	[Display omitted] •Alternative suitable methods to extract SARS-CoV-2 RNA should be taken into account to cope with the ongoing pandemic.•Extraction efficiencies of different viral RNA kit brands can be optimized for detection of SARS-CoV-2 genome.•The accuracy and performance of the Allplex 2019-nCoV assay can be further improved.•New optimized primer pairs for the N gene, considering its variability and stability, are highly recommended. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. Currently, several automatic methods that include RNA extraction are available. However, due to the growing demand, a shortage in kit supplies could be experienced in several labs. For these reasons, the use of different commercial or in-house protocols for RNA extraction may increase the possibility to analyze high number of samples. Herein, we compared the efficiency of RNA extraction of three different commercial kits and an in-house extraction protocol using synthetic ssRNA standards of SARS-CoV-2 as well as in oro-nasopharyngeal swabs from six COVID-19-positive patients. It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection.
AbstractList	Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. Currently, several automatic methods that include RNA extraction are available. However, due to the growing demand, a shortage in kit supplies could be experienced in several labs. For these reasons, the use of different commercial or in-house protocols for RNA extraction may increase the possibility to analyze high number of samples. Herein, we compared the efficiency of RNA extraction of three different commercial kits and an in-house extraction protocol using synthetic ssRNA standards of SARS-CoV-2 as well as in oro-nasopharyngeal swabs from six COVID-19-positive patients. It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection. [Display omitted] •Alternative suitable methods to extract SARS-CoV-2 RNA should be taken into account to cope with the ongoing pandemic.•Extraction efficiencies of different viral RNA kit brands can be optimized for detection of SARS-CoV-2 genome.•The accuracy and performance of the Allplex 2019-nCoV assay can be further improved.•New optimized primer pairs for the N gene, considering its variability and stability, are highly recommended. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. Currently, several automatic methods that include RNA extraction are available. However, due to the growing demand, a shortage in kit supplies could be experienced in several labs. For these reasons, the use of different commercial or in-house protocols for RNA extraction may increase the possibility to analyze high number of samples. Herein, we compared the efficiency of RNA extraction of three different commercial kits and an in-house extraction protocol using synthetic ssRNA standards of SARS-CoV-2 as well as in oro-nasopharyngeal swabs from six COVID-19-positive patients. It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. Currently, several automatic methods that include RNA extraction are available. However, due to the growing demand, a shortage in kit supplies could be experienced in several labs. For these reasons, the use of different commercial or in-house protocols for RNA extraction may increase the possibility to analyze high number of samples. Herein, we compared the efficiency of RNA extraction of three different commercial kits and an in-house extraction protocol using synthetic ssRNA standards of SARS-CoV-2 as well as in oro-nasopharyngeal swabs from six COVID-19-positive patients. It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. Currently, several automatic methods that include RNA extraction are available. However, due to the growing demand, a shortage in kit supplies could be experienced in several labs. For these reasons, the use of different commercial or in-house protocols for RNA extraction may increase the possibility to analyze high number of samples. Herein, we compared the efficiency of RNA extraction of three different commercial kits and an in-house extraction protocol using synthetic ssRNA standards of SARS-CoV-2 as well as in oro-nasopharyngeal swabs from six COVID-19-positive patients. It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection. • Alternative suitable methods to extract SARS-CoV-2 RNA should be taken into account to cope with the ongoing pandemic. • Extraction efficiencies of different viral RNA kit brands can be optimized for detection of SARS-CoV-2 genome. • The accuracy and performance of the Allplex 2019-nCoV assay can be further improved. • New optimized primer pairs for the N gene, considering its variability and stability, are highly recommended. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. Currently, several automatic methods that include RNA extraction are available. However, due to the growing demand, a shortage in kit supplies could be experienced in several labs. For these reasons, the use of different commercial or in-house protocols for RNA extraction may increase the possibility to analyze high number of samples. Herein, we compared the efficiency of RNA extraction of three different commercial kits and an in-house extraction protocol using synthetic ssRNA standards of SARS-CoV-2 as well as in oro-nasopharyngeal swabs from six COVID-19-positive patients. It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection.
ArticleNumber	114008
Author	Palamara, Anna Teresa Tomino, Carlo Scribano, Daniela Fini, Massimo De Chiara, Giovanna Prezioso, Carla Checconi, Paola Sarshar, Meysam Garaci, Enrico Limongi, Dolores Ambrosi, Cecilia Capannari, Maurizio
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Keywords	RNA extraction SARS-CoV-2 Oro- nasopharyngeal swabs rRT-PCR
Language	English
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Snippet	[Display omitted] •Alternative suitable methods to extract SARS-CoV-2 RNA should be taken into account to cope with the ongoing pandemic.•Extraction... Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been... • Alternative suitable methods to extract SARS-CoV-2 RNA should be taken into account to cope with the ongoing pandemic. • Extraction efficiencies of different...
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SubjectTerms	COVID-19 - diagnosis COVID-19 infection COVID-19 Nucleic Acid Testing - methods Diagnostic Tests, Routine etiological agents Genes, Viral - genetics Humans Limit of Detection Oro- nasopharyngeal swabs Pharynx - virology quantitative polymerase chain reaction Reproducibility of Results reverse transcription RNA RNA extraction RNA, Viral - analysis RNA, Viral - genetics RNA, Viral - isolation & purification rRT-PCR SARS-CoV-2 SARS-CoV-2 - genetics SARS-CoV-2 - isolation & purification separation Severe acute respiratory syndrome coronavirus 2
Title	SARS-CoV-2: Comparative analysis of different RNA extraction methods
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