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Universal Screening of Congenital Cytomegalovirus using Dried Blood Spots and real-time PCR
Abstract Summary
Permanent health problems can arise when cytomegalovirus transmission occurs prenatally, resulting in congenital cytomegalovirus (cCMV). Screening for cCMV is currently not universal but reactionary to symptoms. Freshly collecting saliva, urine or blood are inadequate as symptomatic patients may no longer be infected or become infected postnatally. Dried blood spot (DBS) cards are utilized in Newborn Screening (NBS) to collect a patient sample neonatally. This makes DBS a prime sample type for universal screening of cCMV and for retrospective testing using archived samples. Two DBS extraction methods were tested using 2x 3.2mm punches and 65µL elution; the NeoMDx extraction and Thermal Shock. The eluates were directly inputted into a 15µL PCR reaction using the NeoMDx cCMV real-time PCR reagents which amplify a CMV and RPP30 target. The assay utilizes DBS controls that monitor the overall workflow from sample extraction to qPCR detection. The NeoMDx extraction takes 30 minutes, with two buffer exchanges and two incubation temperatures. The Thermal Shock method failed to amplify the CMV and RPP30 targets due to inhibition from unwashed samples. The NeoMDx-based runs reached over 95% analytical sensitivity with a commercial proficiency panel and 100% analytical specificity with known cCMV-negative DBS. The limit of detection (LoD) of the qPCR assay is 3.3 international units (IU) per reaction. The LoD of the extraction to qPCR workflow is 10IU/µL based on contrived DBS. The NeoMDx cCMV reagents can be used for different throughput labs due to its scalability and flexibility for qPCR. As hospitals and testing sites are already collecting and testing DBS, it is the easiest sample type to implement. Having a scalable and sensitive DBS-based assay is instrumental to adding cCMV to NBS, as well as retrospective testing of high-risk patients. This makes the NeoMDx cCMV real-time PCR reagents compatible with universal screening of cCMV.
Learning Objectives
- Knowledge
- Application
- Evaluation
Presentation
3440589_16114StephanieDallaire.pdf
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Presenters
Stephanie Dallaire | Primary Presenter, Co-Presenter, Author, Co-Author
stephanie.dallaire@perkinelmer.com;
Stephanie Dallaire is the Senior R&D Manager at PerkinElmer Genomics and leads the global technical support for the COVID-19 pandemic. Previously, Stephanie was a Principal Scientist in the Molecular Diagnostics division of PerkinElmer. She has led key research and product development for infectious disease diagnostics, newborn screening and prenatal testing diagnostics. In 2021, Stephanie was awarded the MVP for the Applied Genomics division of PerkinElmer and the SelectScience Scientist’s Choice award for Clinical Article of the Year for her work on COVID-19 testing strategies.
Stephanie holds a bachelor’s degree in biochemistry and molecular biology from the University of Massachusetts, Amherst, and is currently enrolled in a doctoral program at Georgia Institute of Technology.
ASHA DISCLOSURE
Financial - Receives Salary,Intellectual property rights,Ownership interest for Employment,Management position from employee.
Nonfinancial - No relevant nonfinancial relationship exists.
AAA DISCLOSURE
Financial - Receives support from PerkinElmer for All authors are employees of PerkinElmer. One or more authors are stockholders at PerkinElmer..
Eleanore Dougherty | Co-Presenter, Co-Author
eleanore.dougherty@perkinelmer.com;
Senior application scientist and Technical R&D lead for the CMV project at PerkinElmer. Across 7 years at the company, their roles have ranged from working on stem cell extraction and cryopreservation, fetal cell isolation from maternal blood draws, SARS-CoV-2 assay development and validation, clinical study oversight, and diagnostic product development. When not in the lab, they enjoy reading books and long walks on the beach.
ASHA DISCLOSURE
Financial - Receives Salary for Employment from Revvity.
Nonfinancial - No relevant nonfinancial relationship exists.
AAA DISCLOSURE
Financial - Receives support from Revvity (formally known as PerkinElmer) for Presentors are employed by the company that funds their research and development of this product..
Nidhi Nandu | Co-Presenter, Co-Author
nidhi.nandu@perkinelmer.com;
Nidhi Nandu is a senior application scientist, R&D, MDx at PerkinElmer, Inc. since April 2022. She has received her B.Sc. in Biotechnology and M.Sc. in Bioanalytical Sciences from Guru Nanak Khalsa College, Mumbai, India. She received her PhD in Chemistry in August 2021 from State University of New York, Albany. She is actively involved in developing and validating bioassays for infectious diseases.
ASHA DISCLOSURE
Financial - Receives Salary for Employment from Employee.
Nonfinancial - No relevant nonfinancial relationship exists.
AAA DISCLOSURE
Financial - Receives support from Revvity, Inc. for Employment.
Yanhong Tong | Co-Author
yanhong.tong@perkinelmer.com;
Sr. R&D Manager of MDx at PerkinElmer, Ph.D. in Molecular and Cellular Biology, with more than 15 years of experience on IVD product development. Lead a team to develop and validate MDx assays (based on multiplex real-time PCR) for newborn screening (from dried blood spot samples) and infectious diseases diagnostics, with FDA EUA approval or 510k approval. Example launched products are: NeoMDx CMV (RUO), EONIS SCID-SMA (510k), PKamp™ Respiratory SARS-CoV-2 RT-PCR Panel 1 (EUA).
ASHA DISCLOSURE
Financial -
Nonfinancial -
AAA DISCLOSURE
Financial - No relevant financial relationship exists.