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Volume 31, Supplement—May 2025
SUPPLEMENT ISSUE
Supplement

Establishing Methods to Monitor Influenza (A)H5N1 Virus in Dairy Cattle Milk, Massachusetts, USA

Elyse StachlerComments to Author , Andreas Gnirke, Kyle McMahon, Michael Gomez, Liam Stenson, Charelisse Guevara-Reyes, Hannah Knoll, Toni Hill, Sellers Hill, Katelyn S. Messer, Jon Arizti-Sanz, Fatinah Albeez, Elizabeth Curtis, Pedram Samani, Natalia Wewior, David H. O’Connor, William Vuyk, Sophia E. Khoury, Matthew K. Schnizlein, Nicole C. Rockey, Zachariah Broemmel, Michael Mina, Lawrence C. Madoff, Shirlee Wohl, Lorraine O’Connor, Catherine M. Brown, Al Ozonoff, Daniel J. Park, Bronwyn L. MacInnis1, and Pardis C. Sabeti1
Author affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA (E. Stachler, A. Gnirke, K. McMahon, M. Gomez, L. Stenson, C. Guevara-Reyes, H. Knoll, T. Hill, S. Hill, K.S. Messer, J. Arizti-Sanz, F. Albeez, E. Curtis, P. Samani, N. Wewior, A. Ozonoff, D.J. Park, B.L. MacInnis, P.C. Sabeti); University of Puerto Rico-Rio Piedras, San Juan, Puerto Rico, USA (C. Guevara-Reyes); Harvard University, Cambridge (P. Samani, B.L. MacInnis, P.C. Sabeti); University College London, London, UK (P. Samani); University of Wisconsin-Madison, Madison, Wisconsin, USA (D.H. O’Connor, W. Vuyk); The University of Texas at Austin, Austin, Texas, USA (S.E. Khoury); Michigan State University, East Lansing, Michigan, USA (M.K. Schnizlein); Duke University, Durham, North Carolina, USA (N.C. Rockey, Z. Broemmel); Immune Observatory, Boston, Massachusetts, USA (M. Mina); University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA (L.C. Madoff); Massachusetts Department of Public Health, Boston (L.C. Madoff, S. Wohl, C.M. Brown); Brigham and Women’s Hospital, Boston (S. Wohl); Massachusetts Department of Agricultural Resources, Boston (L. O’Connor); Boston Children’s Hospital, Boston (A. Ozonoff); Harvard Medical School, Boston (A. Ozonoff); Howard Hughes Medical Institute, Chevy Chase, Maryland, USA (P.C. Sabeti)

Main Article

Figure 1

Validation and characterization of dPCR and qPCR on synthetic spike-in samples in study of methods to monitor influenza A(H5N1) virus in dairy cattle milk, Massachusetts, USA. A, B) Limit of detection analysis for correlation of dPCR concentrations with qPCR Ct values (A) and measured concentrations compared to expected concentrations for both qPCR and dPCR (B). C, D) Detection of dPCR (H5_Taq and RP_Bov) (C) and qPCR (H5_Taq) (D) assays using serial dilutions of synthetic H5N1 RNA standard material. For qPCR data, we combined and jointly analyzed all standard curve data from runs during retail milk testing. Fitted lines in panels A and D represent simple linear regression lines of best fit. Error bars indicate +1 SD. Ct, cycle threshold; dPCR, digital PCR; qPCR, quantitative PCR; R2, coefficient of determination.

Figure 1. Validation and characterization of dPCR and qPCR on synthetic spike-in samples in study of methods to monitor influenza A(H5N1) virus in dairy cattle milk, Massachusetts, USA. A, B) Limit of detection analysis for correlation of dPCR concentrations with qPCR Ct values (A) and measured concentrations compared to expected concentrations for both qPCR and dPCR (B). C, D) Detection of dPCR (H5_Taq and RP_Bov) (C) and qPCR (H5_Taq) (D) assays using serial dilutions of synthetic H5N1 RNA standard material. For qPCR data, we combined and jointly analyzed all standard curve data from runs during retail milk testing. Fitted lines in panels A and D represent simple linear regression lines of best fit. Error bars indicate +1 SD. Ct, cycle threshold; dPCR, digital PCR; qPCR, quantitative PCR; R2, coefficient of determination.

Main Article

1These senior authors contributed equally to this article.

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