Adaptable, quantitative CRISPR/Cas12a-based assay for cytomegalovirus DNA in infant saliva

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Authors and Affiliations

1. Department of Microbiology and Immunology, Tulane University School of Medicine, 1430 Tulane Avenue, SL-38, New Orleans, LA, 70112, USA

   Karissa Chao, Eva G. Gutt & Robert F. Garry

2. Division of Pediatric Infectious Diseases, Department of Pediatrics, Tulane University, New Orleans, LA, USA

   Monika L. Dietrich, Samantha C. Covey, Mambu Momoh & Robert J. Samuels

3. Kenema Government Hospital, Ministry of Health and Sanitation, Kenema, Sierra Leone

   Mambu Momoh, John Demby Sandi, Mohamed Saio Kamara, Ibrahim Umaru Fofanah, Maariam Manjia Rogers, Tiangay Mariama Patience Sallay Kallon, Robert J. Samuels & Donald S. Grant

4. Eastern Technical University of Sierra Leone, Kenema, Sierra Leone

   Mambu Momoh

5. Sierra Leone, College of Medicine and Allied Health Sciences, University of Sierra Leone, Freetown, Sierra Leone

   Donald S. Grant

6. Broad Institute of Massachusetts Institute of Technology and Harvard, Boston, MA, USA

   Pardis C. Sabeti

7. Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA

   Pardis C. Sabeti

8. Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA

   Pardis C. Sabeti

9. Department of Immunology and Infectious Disease, Harvard T.H. Chan School of Public Health, Boston, MA, USA

   Pardis C. Sabeti

10. Howard Hughes Medical Institute, Chevy Chase, MD, USA

    Pardis C. Sabeti

11. Zalgen Labs, LLC, Frederick, MD, USA

    Robert F. Garry

12. Global viral Network, Tampa, FL, USA

    Robert F. Garry

Contributions

KC, MLD, PCS, and RFG conceived and designed the study. MLD developed and optimized PCR protocol. SCC and MM completed spin-column DNA extractions and performed PCR assays. KC developed, and optimized RPA-Cas12a protocols for all read-out methods. EGG optimized, validated, and performed HUDSON DNA extractions. JDS, MSK, IUF, MMR, TSK, and KC optimized and validated RPA-Cas12a assay in Sierra Leone. MSK, IUF, and MMR performed assay robustness experiments in Sierra Leone. MLD, RJS, and DSG obtained Sierra Leonean infant samples and optimized PCR protocols in Sierra Leone. KC conducted clinical validation of RPA-Cas12a assay. KC, MLD, PCS, and RFG analyzed the data. KC and MLD wrote the paper. RFG and PCS edited the paper. KC and RFG prepared figures and tables. All authors had access to all the data and approved the final version of the manuscript.

Corresponding author

Correspondence to Robert F. Garry.

Competing interests

P.C.S. is a co-founder of, shareholder in, and consultant to Sherlock Biosciences and Delve Bio, as well as a board member of and shareholder in Danaher Corporation. RFG is a co-founder of Zalgen Labs. RFG does not receive compensation from Zalgen Labs. The other authors declare no competing interests.

Ethics approval

This study was approved by the Tulane Institutional Review Board and the Sierra Leone Ethics and Scientific Review Committee.

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