SARS-CoV-2 RNA detection in Formalin-Fixed Paraffin-Embedded (FFPE) tissue by droplet digital PCR (ddPCR)

Ramanath Majumdar; Julie A. Vrana; Justin W. Koepplin; Dragana Milosevic; Anja C. Roden; Joaquin J. Garcia; Benjamin R. Kipp; Ann M. Moyer

doi:10.1016/j.cca.2022.05.007

SARS-CoV-2 RNA detection in Formalin-Fixed Paraffin-Embedded (FFPE) tissue by droplet digital PCR (ddPCR)

Ramanath Majumdar, Julie A. Vrana, Justin W. Koepplin, Dragana Milosevic, Anja C. Roden, Joaquin J. Garcia, Benjamin R. Kipp, Ann M. Moyer

Research output: Contribution to journal › Article › peer-review

Abstract

Background: SARS-CoV-2 is an RNA virus that primarily causes respiratory disease; however, infection of other tissue has been reported. Evaluation of SARS-CoV-2 in tissue specimens may increase understanding of SARS-CoV-2 pathobiology. Materials and Methods: A qualitative test for detection of SARS-CoV-2 in formalin-fixed paraffin-embedded (FFPE) tissues was developed and validated using droplet digital PCR (ddPCR), which has a lower limit of detection than reverse transcription (RT)-qPCR. After extraction of total RNA from unstained FFPE tissue, SARS-CoV-2 nucleocapsid (N1, N2) target sequences were amplified and quantified, along with human RPP30 as a control using the Bio-Rad SARS-CoV-2 ddPCR kit. Results: SARS-CoV-2 was detected in all 21 known positive samples and none of the 16 negative samples. As few as approximately 5 viral copies were reliably detected. Since January 2021, many tissue types have been clinically tested. Of the 195 clinical specimens, the positivity rate was 35% with placenta and fetal tissue showing the highest percentage of positive cases. Conclusion: This sensitive FFPE-based assay has broad clinical utility with applications as diverse as pregnancy loss and evaluation of liver transplant rejection. This assay will aid in understanding atypical presentations of COVID-19 as well as long-term sequelae.

Original language	English (US)
Pages (from-to)	181-187
Number of pages	7
Journal	Clinica Chimica Acta
Volume	532
DOIs	https://doi.org/10.1016/j.cca.2022.05.007
State	Published - Jul 1 2022

Keywords

COVID-19
Droplet digital PCR
Placental viral infection
SARS-CoV-2
Tissue-based viral detection
Viral detection in FFPE

ASJC Scopus subject areas

Biochemistry
Clinical Biochemistry
Biochemistry, medical

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10.1016/j.cca.2022.05.007

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title = "SARS-CoV-2 RNA detection in Formalin-Fixed Paraffin-Embedded (FFPE) tissue by droplet digital PCR (ddPCR)",

abstract = "Background: SARS-CoV-2 is an RNA virus that primarily causes respiratory disease; however, infection of other tissue has been reported. Evaluation of SARS-CoV-2 in tissue specimens may increase understanding of SARS-CoV-2 pathobiology. Materials and Methods: A qualitative test for detection of SARS-CoV-2 in formalin-fixed paraffin-embedded (FFPE) tissues was developed and validated using droplet digital PCR (ddPCR), which has a lower limit of detection than reverse transcription (RT)-qPCR. After extraction of total RNA from unstained FFPE tissue, SARS-CoV-2 nucleocapsid (N1, N2) target sequences were amplified and quantified, along with human RPP30 as a control using the Bio-Rad SARS-CoV-2 ddPCR kit. Results: SARS-CoV-2 was detected in all 21 known positive samples and none of the 16 negative samples. As few as approximately 5 viral copies were reliably detected. Since January 2021, many tissue types have been clinically tested. Of the 195 clinical specimens, the positivity rate was 35% with placenta and fetal tissue showing the highest percentage of positive cases. Conclusion: This sensitive FFPE-based assay has broad clinical utility with applications as diverse as pregnancy loss and evaluation of liver transplant rejection. This assay will aid in understanding atypical presentations of COVID-19 as well as long-term sequelae.",

keywords = "COVID-19, Droplet digital PCR, Placental viral infection, SARS-CoV-2, Tissue-based viral detection, Viral detection in FFPE",

author = "Ramanath Majumdar and Vrana, {Julie A.} and Koepplin, {Justin W.} and Dragana Milosevic and Roden, {Anja C.} and Garcia, {Joaquin J.} and Kipp, {Benjamin R.} and Moyer, {Ann M.}",

note = "Publisher Copyright: {\textcopyright} 2022",

year = "2022",

month = jul,

day = "1",

doi = "10.1016/j.cca.2022.05.007",

language = "English (US)",

volume = "532",

pages = "181--187",

journal = "Clinica Chimica Acta",

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TY - JOUR

T1 - SARS-CoV-2 RNA detection in Formalin-Fixed Paraffin-Embedded (FFPE) tissue by droplet digital PCR (ddPCR)

AU - Majumdar, Ramanath

AU - Vrana, Julie A.

AU - Koepplin, Justin W.

AU - Milosevic, Dragana

AU - Roden, Anja C.

AU - Garcia, Joaquin J.

AU - Kipp, Benjamin R.

AU - Moyer, Ann M.

PY - 2022/7/1

Y1 - 2022/7/1

N2 - Background: SARS-CoV-2 is an RNA virus that primarily causes respiratory disease; however, infection of other tissue has been reported. Evaluation of SARS-CoV-2 in tissue specimens may increase understanding of SARS-CoV-2 pathobiology. Materials and Methods: A qualitative test for detection of SARS-CoV-2 in formalin-fixed paraffin-embedded (FFPE) tissues was developed and validated using droplet digital PCR (ddPCR), which has a lower limit of detection than reverse transcription (RT)-qPCR. After extraction of total RNA from unstained FFPE tissue, SARS-CoV-2 nucleocapsid (N1, N2) target sequences were amplified and quantified, along with human RPP30 as a control using the Bio-Rad SARS-CoV-2 ddPCR kit. Results: SARS-CoV-2 was detected in all 21 known positive samples and none of the 16 negative samples. As few as approximately 5 viral copies were reliably detected. Since January 2021, many tissue types have been clinically tested. Of the 195 clinical specimens, the positivity rate was 35% with placenta and fetal tissue showing the highest percentage of positive cases. Conclusion: This sensitive FFPE-based assay has broad clinical utility with applications as diverse as pregnancy loss and evaluation of liver transplant rejection. This assay will aid in understanding atypical presentations of COVID-19 as well as long-term sequelae.

AB - Background: SARS-CoV-2 is an RNA virus that primarily causes respiratory disease; however, infection of other tissue has been reported. Evaluation of SARS-CoV-2 in tissue specimens may increase understanding of SARS-CoV-2 pathobiology. Materials and Methods: A qualitative test for detection of SARS-CoV-2 in formalin-fixed paraffin-embedded (FFPE) tissues was developed and validated using droplet digital PCR (ddPCR), which has a lower limit of detection than reverse transcription (RT)-qPCR. After extraction of total RNA from unstained FFPE tissue, SARS-CoV-2 nucleocapsid (N1, N2) target sequences were amplified and quantified, along with human RPP30 as a control using the Bio-Rad SARS-CoV-2 ddPCR kit. Results: SARS-CoV-2 was detected in all 21 known positive samples and none of the 16 negative samples. As few as approximately 5 viral copies were reliably detected. Since January 2021, many tissue types have been clinically tested. Of the 195 clinical specimens, the positivity rate was 35% with placenta and fetal tissue showing the highest percentage of positive cases. Conclusion: This sensitive FFPE-based assay has broad clinical utility with applications as diverse as pregnancy loss and evaluation of liver transplant rejection. This assay will aid in understanding atypical presentations of COVID-19 as well as long-term sequelae.

KW - COVID-19

KW - Droplet digital PCR

KW - Placental viral infection

KW - SARS-CoV-2

KW - Tissue-based viral detection

KW - Viral detection in FFPE

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SN - 0009-8981

VL - 532

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JO - Clinica Chimica Acta

JF - Clinica Chimica Acta

ER -

SARS-CoV-2 RNA detection in Formalin-Fixed Paraffin-Embedded (FFPE) tissue by droplet digital PCR (ddPCR)

Abstract

Keywords

ASJC Scopus subject areas

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