Materials

Escherichia coli strains expressing the LbCas12a protein, plasmids containing the AIV M and NP genes, and H1–H16 subtypes of AIV, newcastle disease virus (NDV), infectious bursal disease virus (IBDV), and infectious bronchitis virus (IBV) were conserved in the State Key Laboratory of Harbin Veterinary Research Institute (HVRI), Chinese Academy of Agricultural Sciences (CAAS). A total of 81 samples were obtained from the National Reference Laboratory for Avian Influenza.

Reagents and instruments

RNase inhibitors were obtained from Thermo Fisher Scientific. The crRNA targeting the RT-RPA of the M and NP genes was synthesized by RuiBiotech. RNA was extracted using a TIANamp Virus RNA Kit. A HiScribe T7 Quick High-Yield RNA Synthesis Kit was purchased from New England Biolabs. The single-stranded DNA reporter was synthesized by Sangon Biotech. The HybriDetect test dipstick was purchased from Warbio. The RNA Rapid Concentration Purification Kit was purchased from Sangon Biotech. Fluorescence signals were acquired using QuantStudio 5 (Applied Biosystems) and the degraded fluorescent reporter group (ssDNA-FQ) was visualized under UV light.

Template RNA preparation

Plasmids containing the M and NP genes from AIV were PCR-amplified using primers containing T7 promoter sequences and then purified using a PCR Product Purification Kit. The purified DNA products were used as templates for in vitro transcription reactions using the HiScribe T7 Quick High Yield RNA Synthesis Kit, and RNA was purified using the RNA Rapid Concentration Purification Kit.

Protein expression and purification

LbCas12a protein used for AIV detection was expressed in E. coli. The gene encoding LbCas12a was cloned into a pET-based expression vector containing a C-terminal 6 × His-tag and a TEV protease cleavage site. The soluble protein was purified as previously described with the following modifications: the bacterial expression plasmid was transformed into RosettaTM2(DE3) cells, and a 10 mL bacterial culture was grown in 1 L Luria–Bertani growth media which was inoculated for growth at 37 °C and 200 RPM until an OD600 of 0.6. Meanwhile, LbCas12a protein expression was induced by supplementation with isopropyl β-d-1-thiogalactopyranoside to a final concentration of 0.5 mM, and the cell was cooled to 16 °C for 18 h. Bacterial cells were harvested, resuspended in lysis buffer (50 mM HEPES pH 7.2, 2 M NaCl, 20 mM imidazole, 2 mL PMSF, and 0.25 mg/mL lysozyme), disrupted by sonication, and purified using an Ni–NTA column. The purified protein was added to TEV enzyme to remove the His-tag and concentrated through an ultrafiltration tube to obtain the purified LbCas12a protein, which was stored at -80 °C or used directly in the assay.

Optimization of RT-RPA and Cas12a detection

RNase H is an endonuclease that specifically hydrolyzes RNA strands in RNA-cDNA hybrids. In RT-RPA, viral RNA is reverse transcribed into cDNA or cDNA-RNA hybrids using reverse transcriptase, which helps to improve amplification efficiency [25]. To obtain the best amplification, different concentrations of RNase H were added to the RT-RPA reaction, and the amplification products were detected for Cas12a by reading the fluorescence signal to determine the optimal concentration.

To establish the optimal reaction conditions for Cas12a detection, the influence of different factors, including pH, ssDNA reporter, buffer composition, Mg2+, and crRNA concentration, on Cas12a cleavage efficiency was investigated.

Design and screening for crRNA and primers of RT-RPA

Based on the conserved sequences of the M and NP genes of AIV, four M-crRNAs and four NP-crRNAs were designed and screened for the best crRNA. Based on the selected crRNA, RT-RPA primers targeting the M and NP genes of AIV were designed as described for Twist-Dx (Maidenhead, United Kingdom). To screen the best pairs, a random combination of primers was used to screen the primer pairs with the most efficient amplification effects, as shown in Table 1. Primer pairs with the most efficient performance were used in subsequent experiments.

Table 1 Oligonucleotides used in this experimentRT-RPA reaction

RT-RPA reactions were performed using a RT-Basic RNA isothermal rapid amplification kit (Genenode, Wuhan, China) according to the manufacturer’s protocol. In brief, the reactions were performed at a total volume of 50 μL comprising RT-RPA enzymes, 2 μL RNA input, 29.4 μL A Buffer, 2 μL Forward primer (10 μM), 2 μL Reverse primer (10 μM), and 2.5 μL B Buffer. All reactions were incubated at 42 °C for 30 min in a PCR instrument or metal bath.

Cas12a detection reactions

Detection assays were performed with 10 μL RT-RPA products or 1 μL dsDNA plasmid, 1.25 μL purified Cas12a protein, 12.5 μL reaction Buffer (20 mM Tris–HCl pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol), 1 μL crRNA (200 nM), 0.25 μL RNase inhibitor (RI), 1 μL ssDNA reporter, and water to make up a total volume of 25 μL. Reactions were incubated in a Real-Time PCR Detection System (ABI QuantStudio 5) or a fluorescence plate reader (Enspire, USA) for 1–2 h at 37 °C with fluorescence signals measured every 5 min.

Lateral flow detection

After amplification was completed, 2 μL of amplification product was mixed with 25 μL reaction buffer, 2.5 μL LbCas12a, 2 μL crRNA, 2 μL probe (FAM-TTATT-Biotin), 0.5 μL RI, and 16 μL ddH2O, and given at 37 °C for 2 h. A side-flow test strip was then added to the reaction tube and the results were observed after 2 min. A single band near the sample pad indicated a negative result, whereas a single band or two bands near the top of the test strip indicated a positive result.

rRT-PCR assay for AIV testing

rRT-PCR detection of the AIV-M gene was performed using the QuantStudio 5 system according to the manufacturer’s instructions (Guanmu Biotechnology, Hunan, China). In brief, single-tube PCRs were prepared including 19 μL reaction buffer, 1 μL enzyme mixture, and 5 μL RNA template. The amplification program was reverse transcription at 50 °C for 2 min, pre-denaturation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing and extension at 60 °C for 30 s. Fluorescence signals were collected during the annealing and extension steps per cycle.

Statistical analysis

The data were analyzed using GraphPad Prism 8.0 (GraphPad Software, Inc.) for analysis of variance. Data are presented as the mean ± standard deviation of three independent experiments.