Plasmids and in vitro transcription

The HA segment of AIV type A/H5N1 (GISAID Accession #EPI1985974) of viral isolate A/turkey/Indiana/22-003707-003/2022 (|HA|4|WSS3056019) was used as a template for the synthetic gene sequence. The H5 subtype HPAIV.HA was synthesized and cloned into the pBluescript II KS (+) plasmid provided by Genescript Biotech under the stream of the T7 promoter sequence. The sequence had been terminated by the T7 terminator. SacI and NotI restriction endonuclease recognition sequences were placed upstream and downstream of the HA gene, respectively. The recombinant plasmid carrying the target HA gene insert was then linearized by the restriction enzyme NotI-HF^® (NEB R3189; Ipswich, MA, USA) using rCutSmart buffer for 15 min. The linearized plasmid DNA template was then subjected to a plasmid purification step using the Wizard Clean-up System (Promega A7280; Madison, WI, USA). The purified linearized DNA, including the T7 promoter, was then used as a template for in vitro transcription using HiScribe^® T7 Quick High Yield RNA Synthesis Kit (NEB E2050; Ipswich, MA, USA) per the supplier’s instructions. The in vitro transcribed RNA was subjected to 2 µl of DNase enzyme treatment for 30 min in DNase buffer to eliminate any remnants of the template DNA. The in vitro transcribed RNA was purified using Monarch RNA Clean-up Kits (NEB T2030; Ipswich, MA, USA). Subsequently, the nanodrop 8000 spectrophotometer (Thermo Scientific, Wilmington, Delaware) was used to measure the RNA concentration.

Primer design and synthesis

LAMP primer sets were designed and prioritized over the conserved portions of the HA sequence of HPAI virus type A/H5N1 (GISAID Accession #EPI1985974) utilizing the LAMP Primer Explorer v5 program (http://primerexplorer.jp/e; Eiken Chemical) or manual methods following the instructions of Primer Explorer v5 program. The sequences were subjected to multiple sequence alignment using Clustal W by default setting, and some primers were modified as degenerate primers manually (Supplementary Table 1). The feasibility of all primer sets was then validated using the BLAST program (BLASTN). All designed primer sets included loop primers (LF/LB) to increase reaction speed. The LAMP primer sets for the HA gene shown in Supplementary Table 1 were synthesized and desalted by Invitrogen (Waltham, MA, USA). The qPCR primers and probes for the HPAIV strains listed in Table 4 were either from the WHO primer–probe set or newly designed using the PrimerQuest tool from Integrated DNA Technologies (IDT). The qPCR primers were obtained from IDT (USA) in desalted form, while the probes were HPLC-purified and double-quenched.

Fluorescent LAMP assays

The fluorescent LAMP assay was carried out with the primers listed in Supplementary Table 1. The master mix utilized for the assay was composed of 2.5 µl of 10 × HA LAMP primer mix, 12.5 µl of WarmStart^® 2X Master Mix E1700 LAMP Kit (NEB, USA), and 5 µl 5 × LAMP fluorescent dye (diluted in nuclease-free water from 50X LAMP fluorescent dye provided with the kit) per reaction. Subsequently, 20 µl of this mixture was dispensed evenly into a 96-well plate (Thermo Scientific, AB-0800/W), followed by adding 5 µl of purified RNA (at varying concentrations) or molecular grade nuclease-free water in the case of NTC reactions. The plate was then capped using Versicap mat cap strips (Fisher Scientific, Catalog No AB1820100). The sealed plate was then placed in a qTower 3G Touch (Analytic Jena, Germany) or qTower 3G (Analytic Jena, Germany) and incubated at 65 °C with a ramp rate of 0.1 °C/sec for 60 min. Real-time fluorescence was detected on the blue channel using settings for the FAM dye. Fluorescence measurements were taken every 60 s during the reactions.

LAMP screening and scoring

Primer sets were screened by performing 4 replicates of positive control reactions containing 5 µl of 2.0 × 10³ copies/reaction of synthetic HA gene IVT RNA and varying the primer set. In the case of NTC reactions, 5 µl of nuclease-free water was used in place of synthetic HA gene IVT RNA. LAMP reactions were carried out as detailed in the entitled “Fluorescent LAMP assays”.

Primer sets were scored according to a weighted schema previously designed by our lab⁶⁹. Briefly, primer sets were scored based upon the average reaction time, standard deviation of reaction time, average maximum fluorescent intensity, and standard deviation of maximum fluorescent intensity across all 4 replicates, as well as the number of false positives with weights of 20%, 15%, 5%, 5%, and 55% of the primer set score, respectively (Table 2). The reaction time for an individual reaction was defined as the time at which the second derivative of the time rate of change of the fluorescent intensity reached a maximum (i.e., roughly when the exponential phase is first beginning).

False positives were determined as any signal in an NTC reaction that displayed non-negligible fluorescent intensity increases over the course of the reaction and that increased above 10% of the maximum intensity of the instrument (approximately 140,000 RFU) in a negative reaction. False positives were penalized with increasing severity as the number of false positives increased. Primer sets were then sorted based on the total score. Primer sets scoring above 95 were selected and one additional primer set was selected in the event that downstream screening failed. Information on accessing the code for primer scoring can be found in the section entitled “Data ”.

Limit of detection experiment for LAMP assay

The LODs for candidate primer sets were established using serial dilutions of HA-IVT in water based on its dilutions of 1980, 500, 250, 125, 50, 25, 5, and 1 copies/reaction in 25 μl reactions. A benchtop (Epson Perfection V800 Photo Color) scanner was used to scan the plate to capture the colors of the reaction mixtures at different time points in the colorimetric liquid LAMP. An assessment of LOD is based on a primer set with the lowest virus concentrations, resulting in a distinct color change across all three replicates in colorimetric liquid/paper-based LAMP or exhibiting a fluorescent amplification curve in the three triplicates.

Cross-reactivity assay of HPAIV.HA.5 against panel of avian and bovine pathogens

A comprehensive assessment of the specificity for the HPAIV.HA.5 LAMP primer set was conducted using genomic extracts from a range of bovine and avian pathogens. The pathogens tested included BRAV (SD-1 strain, ATCC, catalog number: VR-668), BRBV (EC-11, ATCC, catalog number: VR-1806), BCV (Mebus, BEI, catalog number: NR-445), IDV (D/bovine/660, provided by Dr. Benjamin Hause, South Dakota State University, USA), BAV-3 (WBR-1 strain, ATCC, catalog number: VR-639), BHV-1 (Los Angeles strain, ATCC, catalog number: VR-188), BVDV-1 (NADL strain, ATCC, catalog number: VR-534), BAV-7 (Fukuroi VR-768™ strain, ATCC VR-768), BRSV (A 51908 strain, ATCC, catalog number: VR-1339), and BPI3V (SF-4 strain, BEI, catalog number: NR-3234). In addition, six avian pathogens were also evaluated for cross-reactivity tests: NDV (non-virulent), IBV, ILT, MG, ORT, and Av. paragallinarum. Prior to this specificity analysis, all genomic extract materials were confirmed using qPCR, and 10⁶ copies/reaction were used for input as templates.

Colorimetric liquid LOD assay

New England BioLabs^® Inc. has developed a pH-dependent master mix that incorporates the highly modified Bst 2.0 WarmStart^® DNA Polymerase in a special buffer containing essential co-factors MgSO₄ and phenol red as a visible pH-sensitive indicator. This indicator changes from pink to yellow when the pH of the reaction mixture drops. This is due to the large amount of proton produced by Bst 2.0 WarmStart^® DNA polymerase due to extensive nucleic acid amplification activity. LAMP results were more visible and detectable with this type of master mix. DNA polymerase I from Bacillus stearothermophilus has been genetically engineered to be highly active in an isothermal environment without the need for a denaturation step. The WarmStart Master Mix is designated because the enzyme is inactive at lower temperatures but becomes active once the reaction solution reaches a temperature of 40 °C (NEB, USA). Using the enzyme and indicator of this recent invention, a simple device can be used for incubation, and the color of the amplicon could be detectable by the naked eye within a short time. Since the reaction was performed in a water bath at 65 °C, all samples were incubated simultaneously for detection without needing a thermocycler. In addition, color detection of the amplicon simplified the reading of the results, eliminating the need for a post-amplification processing step.

The colorimetric assay was performed according to the instructions in the NEB WarmStart Colorimetric LAMP 2X Master Mix Kit containing UDG (NEB M1804). This mix provides a distinctive red-to-yellow transition in the presence of a pH indicator dye to indicate a positive result. Reactions consisted of 2.5 μL 10 × primer mix (1.6 μM FIP and BIP primers, 0. 4 μM each of LF and LB primers, 0.2 μM each of F3 and B3 primers), 12.5 μL WarmStart Colorimetric LAMP 2X Master Mix, 5 μL nuclease-free water, and 5 μL template RNA or molecular biology water for NTCs in a total volume of 25 μl. All reactions were then incubated in a qPCR cycler at 65 °C for 60 min with the lid heated to 95 °C. During the RT-LAMP procedure, the Bst 2.0 WarmStart^® DNA Polymerase (NEB, USA) was fortified with dUTP to avert carryover contamination. Furthermore, the antarctic thermolabile UDG (NEB, USA) was employed to eliminate DNA contamination and degrade any dU-containing DNA. The WarmStart Colorimetric LAMP 2X Master Mix (DNA and RNA) (NEB, USA) is comprised of a specialized reaction mixture incorporating a modified and WarmStart strand-shifting DNA polymerase (Bst 2.0) and reverse transcriptase (RTx), enables rapid and highly reliable detection of both DNA (LAMP) and RNA (RT-LAMP).

Colorimetric paper-based LAMP assay

We used a formulation previously described by our group for the on-paper colorimetric assay to detect SARS-CoV-2^65,70: Final colorimetric paper-based RT-LAMP master mix included MgSO₄ (8 mM), KCl (50 mM), WarmStart^® RTx reverse transcriptase (0.3 U/μL), Bst 2.0 WarmStart^® DNA polymerase (0.32 U/μL), antarctic thermolabile UDG (0.0004 U/μL), dUTP (0.14 mM), dNTP mixture (1.4 mM each dNTP), phenol red (0.25 mM), tween^® 20 (1% v/v), trehalose (10% w/v), BSA (500 μg/mL), and betaine (20 mM). Upon device assembly, 25 µl of the aforementioned master mix was added to each pad along with the appropriate primer set as specified in the section entitled “Colorimetric liquid LOD assay”. The devices were then left for 1 h to dry, after which 20 µl of either RNA-free water or a solution containing RNA was added to each pad individually for rehydration. The rehydrated devices were then placed in 2 × 2 inch 2 mil polypropylene bags (ULINE S-17954). To maintain a constant temperature of 149°F (or 65 °C), a 12-quart EVERIE Sous Vide Container (Amazon B07GQWP85C) was filled with water and an Anova Culinary AN500-US00 Sous Vide Precision Cooker (Amazon B07WQ4M5TS) was used. Each appliance bag was securely attached to a standard clear film (617993, Office Depot, USA) and then placed inside a larger 1-gallon Savour Sous Vide Cooking Bag (Amazon B07NCSXMNN). After heating the water bath, the sealed and capped cooking bag was inserted. The samples were allowed to remain in the water for 60 min. Then, images for the experiments were captured at 0 and 60 min with an Epson Perfection V800 Photo Scanner (Amazon B11B223201) configured to Pro Mode, 48-bit color image type, with a resolution of 600 dpi.

Fabrication and optimization of devices for paper-based LAMP assay

This device comprises reading and reaction layers and spacers that prevent crosstalk among strips. The 3 mil optically clean MELINEX (Tekra MELINEX[r] 454 polyester [PET]) backing was used to create the reading area. The double-sided adhesive was adhered to the reaction strips, which were 5 mm by 5 mm of chromatography paper (Ahlstrom-Munksjö Grade 222). These strips were then separated by the 2.5 × 6 mm 20-mil polystyrene spacers (Tekra Double White Opaque HIPS Polystyrene Litho Grade). 20 µl of water was added to saturate the strips during rehydration.

Evaluation of qPCR and paper LAMP assays on contrived samples

All animal-based procedures were approved by Purdue University Institutional Animal Care and Use Committee (PACUC # RT00000127). Briefly oropharyngeal swabs (sterile, BBL Culture Swab, Becton Dickinson, Sparks, MD USA) were collected from 60 adult, apparently healthy white laying hens. Hens were all approximately 40 weeks of age collected from enriched colony cages, gently restrained and the oropharyngeal cavity swabbed for approximately 30 s. Swabs were immediately capped, labeled, and stored on wet ice until processed. Each bird was marked after swabs collected to ensure no bird was repeatedly sampled. 1000 copies per reaction (2 × the LOD in water) of HPAIV-IVT were spiked into resuspended 30 oropharyngeal swabs collected from healthy poultry for use in the assay, while the other 30 samples were kept negative without spiking. Two assays were conducted. 7.5 μL of either spiked or non-spiked samples were tested in 3 mm*3 mm paper size, having dried 7.5 μL of master of paper LAMP. The same samples were tested with two qPCR assays for comparison and diagnostic assay evaluation.

One-step RT-qPCR assay

A one-step reverse transcription-quantitative PCR (RT-qPCR) assay was conducted utilizing the NEB Luna Universal One-Step RT-qPCR Kit (NEB #E3006) and the qTOWER^3G real-time system (Analytik Jena AG, Jena, Germany). The reaction mixture was prepared in accordance with the manufacturer’s instructions. Each 20 μL reaction contained the following components: 10 μL of Luna Universal One-Step Reaction Mix (2X), 1 μL of Luna WarmStart RT Enzyme Mix (20X), 0.8 μL of Forward Primer (10 μM), 0.8 μL of Reverse Primer (10 μM), 0.4 μL of Probe (10 μM), 5 μL of RNA template/oropharyngeal swab sample, and 3 μL of nuclease-free water. The used primers and probes are shown in Table 4. The RT-qPCR assay was performed under the following thermal cycling conditions: Reverse transcription at 55 °C for 10 min, followed by initial denaturation at 95 °C for 1 min. This was followed by 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 45 s.