Comparison of Extraction Methods for the Detection of Avian Influenza Virus RNA in Cattle Milk

Abstract

Since early 2024, a multistate outbreak of highly pathogenic avian influenza H5N1 has been affecting dairy cattle in the USA. The influenza viral RNA concentrations in milk make it an ideal matrix for surveillance purposes. However, viral RNA detection in multi-component fluids such as milk can be complex, and optimization of influenza detection methods is thus required. Raw bulk tank milk and mastitis milk samples were artificially contaminated with an avian influenza strain and subjected to five extraction methods. HCoV-229E and synthetic RNA were included as exogenous internal process controls. Given the high viral load usually observed in individual raw milk samples, four out of five tested methods would enable influenza detection in milk with normal texture, over a time window of at least 2 weeks post-onset of clinical signs. Nevertheless, sample dilution 1:3 in molecular transport medium prior to RNA extraction provided the best results for dilution of inhibitory substances and a good recovery rate of influenza RNA, that reached 12.5 ± 1.2% and 10.4 ± 3.8% in two independent experiments in bulk milk and 11.2 ± 3.6% and 10.0 ± 2.9% on two cohorts of mastitis milk samples. We have also shown compatibility of an influenza RT-qPCR system with synthetic RNA detection for simultaneous validation of the RNA extraction and RT-qPCR processes.

Keywords: H5N1; PCR; RNA extraction; dairy cattle; highly pathogenic avian influenza; milk.

Figure 1

Schematic illustration of the five methods compared for viral RNA extraction of IAV in cattle milk. Method M1 corresponds to the manufacturer’s instructions with RNA extraction directly from 140 µL of medium or milk. For methods M2 or M3, milk samples were diluted 1:1 (method M2) or 1:3 (method M3) in molecular transport medium prior to RNA extraction from the diluted sample. For method M4, a swab was dipped into undiluted milk prior to being discharged in molecular transport medium. RNA was extracted from the molecular transport medium. Finally, for method M5, milk was diluted 1:3 in molecular transport medium prior to RNA extraction omitting adding lysis into AVL and directly loading on the silica-based membrane spin column.

Figure 2

IAV and IPC detection in virus transport medium (VTM) and raw bulk tank milk. Triplicate VTM and bulk milk samples were artificially contaminated with four ten-fold dilutions of IAV and extracted with methods M1 to M4. Constant quantities of HCoV-229E and commercial IPC were added at the lysis step. (A) Cq values obtained with IAV-1 RT-qPCR (y-axis) corresponding to final IAV concentrations ranging from 8.2 × 10³ to 8.2 × 10⁶ gc/mL of milk (x-axis). Mean values and standard deviations from three technical replicates are shown. (B,C) Cq values from RT-qPCR detecting both hCoV-229E (B) and commercial IPC (C) in a duplex format. The results of samples contaminated with four IAV dilutions in three technical replicates each are shown. In the absence of inhibition, Cq values for HCoV-229E or commercial IPC are expected to be similar across RNA extraction methods.

Figure 3

Assessment of compatibility of IAV and commercial IPC detection in IAV-2 duplex RT-qPCR system. (A) Tenfold dilutions of A/Cambodia/E0826360/2020 (H3N2) viral RNA were tested in technical triplicates in mastermix containing no IPC and no IPC detection primers and probe (IAV target, black symbols), and mastermix containing both a constant quantity of IPC RNA (1 × 10³ copies) and IPC detection primers and probe (IAV target, red symbols). Detection of IPC in the VIC channel in duplex mastermix is shown with the green symbols (IPC target). Mean Cq values and standard deviations from three technical replicates are shown. (B) Cq values obtained with IAV-2 singleplex RT-qPCR for samples artificially contaminated with four ten-fold dilutions of IAV and extracted with methods M1 to M4 (same as for Figure 2A). Mean Cq values and standard deviations from three technical replicates are shown. (C) Bland–Altman plot of viral load (in log₁₀ gc/µL) measured with IAV-2 RT-qPCR in singleplex or duplex. Average mean of all differences and 95% limits of agreement are shown with dotted lines. (D) Cq values of detection of commercial IPC with IAV-2 duplex RT-qPCR. The results of three technical replicates of samples contaminated with four IAV dilutions are shown. Lower Cq values are systematically obtained for commercial IPC with IAV-2 duplex compared to duplex detection with HCoV-229E (Figure 2C), due to higher sensitivity of IAV-2 RT-qPCR chemistry for amplification of the commercial IPC.

Figure 4

IAV and IPC detection in bulk milk and individual milk samples from cows with mastitis. A single IAV concentration was seeded into VTM (technical replicates, n = 3), bulk milk (technical replicates, n = 3) and mastitis milk samples (biological replicates, n = 6), while a constant concentration of commercial IPC was added at the lysis step. IAV detection was performed with IAV-2 duplex RT-qPCR. (A) Recovery rates, calculated as the ratio of viral loads in a sample extracted with methods M1–M4 to the average viral load in VTM with method M1. Mean values and standard deviations of the replicates are shown. (B) Cq values (and mean and standard deviations) for commercial IPC detection with IAV-2 duplex RT-qPCR are shown. (C) Viral loads measured in individual mastitis samples extracted with methods M1–M4 are displayed, with samples with curdling highlighted in red.

Figure 5

IAV and IPC detection in a second set of individual milk samples from cows with mastitis. A single IAV concentration was seeded into VTM, bulk milk and individual mastitis milk samples (n = 12) while constant concentration of commercial IPC was added at the lysis step. IAV detection was performed with IAV-2 duplex RT-qPCR. (A) Recovery rates in bulk milk (three technical replicates) and mastitis milk samples (12 biological replicates) obtained with methods M1, M3 and M5 are shown. Mean values and standard deviations of the replicates are shown. (B) Cq values (and mean and standard deviations) for commercial IPC detection with IAV-2 duplex RT-qPCR are shown (C). Viral loads measured in individual mastitis samples extracted with methods M1, M3 and M5 are displayed, with samples with curdling highlighted in red.