Viral, bacteria strains and clinical samples
The common pathogens causing bovine diarrhea were used in this study. BRVA (BRVA, HB-LF/2021), Bovine viral diarrhea virus (BVDV, C24V), Bovine coronavirus (BCoV, HB-SJZ/2021), Bovine enterovirus (BEV, HB-BD/2021), Bovine kobuvirus (BKV, HB-ZJK/2022), Enterotoxigenic Escherichia coli (ETEC, CICC24190) and Clostridium perfringens (C.perfringens, CICC22949) were reserved in our laboratory.
One hundred and thirty-four fecal samples from the dairy cattle with diarrhea were collected from 23 cattle farms in 7 different regions of Hebei province and Datong of Shanxi province between May 2021 and March 2022. Seventy-eight samples were collected from calves under 3 months and the remaining 56 samples were collected from adult cattle (Table 1).
DNA/RNA extraction of the viruses, bacteria and the cattle fecal samples
BRVA, BVDV, BCoV, BEV and BKV viral RNA were extracted using TIANampViral RNA kit (Tiangen, Beijing, China), and ETEC and C.perfringens bacterial genomic DNA were extracted using the TIANamp bacterial DNA kit (Tiangen, Beijing, China), which were all performed according to the manufacturer’s instructions. The fecal samples were homogenized with phosphate buffered saline (PBS, pH7.4) as a 10% (w/v) suspension and centrifuged for 5 min at 6000 g at 4 °C. Two hundreds microliter of the supernatant was used for viral RNA extraction using the TIANLONG Magnetic Viral DNA/RNA kit (Tianlong, XiAn, China) according to the manufacturer’s instructions. The viral RNA was extracted using the Automatic Nucleic Acid Extraction Instrument (np968-c, Tianlong, XiAn, China). Viral RNA and bacteria DNA were quantified using ND-2000c spectrophotometer (NanoDrop, Wilmington, USA). All RNA and DNA templates were used immediately or stored at −80 °C.
Generation of the BRVA RNA standard
The BRVA RNA standard for the RT-RPA assays was generated as previously described . The BRVA viral RNA was reverse transcribed using the PrimeScriptTM 1st strand cDNA Synthesis Kit (TaKaRa，Dalian, China) and the generated cDNA was used as template in the following PCR assay. The PCR primers (BRVA-VP6-F and BRVA-VP6-R) were designed based on the conserved sequence of VP6 gene (Table 2). The 1177 bp PCR product were purified using the TIANgel Midi Purification Kit (Tiangen, Beijing, China), ligated into a pGEM-T Easy vector (Promega, Madison, USA) and transformed into E. coli DH5α. The in vitro transcribed BRVA standard RNA was produced with RiboMAX Large Scale RNA Production System-T7 (Promega, Madison, USA), quantified using ND-2000c and the copy number of RNA molecules was calculated by the formula: Amount (copies/μL)=[RNA concentration (g/μL)/(transcript length in nucleotides×340)] ×6.02×1023. The in vitro transcribed RNA was diluted in 10 fold serial dilutions to achieve RNA concentrations ranging from 1.4×105 to 1.4×100 copies/μL and stored at −80°C, which were used as the standard RNA in the following study.
Primers and probes of the RT-RPA assays
The VP6 gene is highly conserved among the different BRVA strains. Thus it was set as the target of the RT-RPA assays. The RPA primers, exo probe and nfo probe were designed based on the conserved region of VP6 gene, which were collected from the different BRVA strains deposited in GenBank (Accession numbers: MN047454.1, MT240631.1, MK250428.1, MK638874.1, MT240629.1, X53667.1, LC336590.1, AF411322.2, HM988974.1, K02254.1). The RPA primers and probes were synthesized by Generay (Shanghai, China), and the information of them was presented in Table 2.
Real-time RT-RPA assay for BRVA
Real-time RT-RPA assay for BRVA was performed using a ZC BioScience™ exo kit (ZC BioScience, Hangzhou, China) as previously described with minor modifications . The reaction volume was 50 μL including 25 μL of Buffer A (rehydration buffer), 2.0 μL of each RPA primer (BRVA-exo-F and BRVA-exo-R, 10 μmol/L), 0.6 μL of exo probe (BRVA-exo-P, 10 μmol/L), 2.5 μL of Buffer B (magnesium acetate, 280 mmol/L) and a total of 17.9 μL of RNase-Free ddH2O and RNA template. Additionally, 1 μL of BRVA standard RNAwas used for the specificity and sensitivity analysis, while 3 μL of sample RNA was used for the clinical sample diagnosis. All reagents except for the Buffer B (magnesium acetate) were distributed into each 0.2 ml freeze-dried reaction tube containing a dried enzyme pellet. Subsequently, magnesium acetate was pipetted into the tube lids, then the lids were closed carefully and the magnesium acetate was centrifuged into the rehydrated material using a minispin centrifuge. The reaction tubes were vortexed briefly and spun down once again, and the tubes were immediately placed in the Genie III scanner device (OptiGene Limited, West Sussex, UK) to start the reaction at 42 ℃ for 20 min.
LFS RT-RPA assay for BRVA
LFS RT-RPA assays for BRVA were performed using a GenDx ERA Kit (GenDx Biotech, Suzhou, China) as previously described with minor modifications . The reaction volume was 50 μL including 20 μL of Buffer A (rehydration buffer), 2.1 μL of each RPA primer (BRVA-nfo-F and BRVA-nfo-R, 10 μmol/L), 0.6 μL of nfo probe (BRVA-nfo-P, 10 μmol/L), 2.0 μL of Buffer B (magnesium acetate, 280 mmol/L) and a total of 23.2 μL of RNase-Free ddH2O and RNA template. Additionally, 1 μL of BRVA standard RNA was used for the specificity and sensitivity analysis, while 3 μL of sample RNA was used for the clinical sample diagnosis. The procedure for adding reagents and samples was the same as the procedure for real-time RT-RPA, then the reaction tubes were incubated at optimal temperature for suitable reaction time in a thermostatic metal bath. The subsequent RT-RPA products were analyzed using the lateral flow strips (GenDx Biotech, Suzhou, China) according to the manufacturer’s instructions.
Optimization of incubation temperature and time of LFS RT-RPA
To determine the optimal incubation temperature, the LFS RT-RPA reactions were carried out on a Gradient PCR instrument (Applied Biosystems, Foster City, California) set at 39, 40, 41, 42, 43, 44 and 45 °C for 20 min using 1.4×102 copies of standard RNA as templates. The amplification assays were performed in parallel for each temperature. The subsequent RT-RPA products were analyzed by the lateral flow strip analysis. To define the optimal incubation time, the reactions were performed at the specified optimal temperature for 10, 15, 20 and 30 min using 1.4×102 copies standard RNA as templates followed by the lateral flow strip analysis. The amplification assays with 4 different incubation time were performed independently and repeated 3 times.
Analytical specificity and sensitivity analysis
The real-time RT-RPA and LFS RT-RPA assays were performed with the nucleic acids of a panel of pathogens including BRVA, BVDV, BCoV, BEV, BKV, ETEC and C.perfringens, which are important pathogens causing diarrhea in cattle. In the assay, ddH2O was used as the non template control. Three independent reactions were performed.
The constructed BRVA RNA standards ranging from 1.4 × 105 and 1.4 × 100 copies/μL were used in the analytical sensitivity analysis of the real-time RT-RPA and LFS RT-RPA assays, in which ddH2O was used as the non template control. One microliter of each dilution was used as template in both RT-RPA assays, and the limit of detection (LOD) was determined to be the highest dilution of the viral RNA detected by the assays. Eight independent real-time RT-RPA and 5 independent LFS RT-RPA repeats were performed.
Real-time RT-PCR assay
Real-time RT-PCR assay was used to detect the BRVA in the cattle fecal samples using primers and probe from a previously published protocol . The reaction volume was 25 μL, which included 12.5 μL of 2×PerfectStartTM Probe One-Step qPCR SuperMix (TransGen Biotech, Beijing, China), 0.5 μL of TransScript@ Probe One-Step RT/RI Enzyme Mix, 0.5 μL of each primers and probe (10 μmol/L), 3 μL of RNA template and 7.5 μL of RNase-Free ddH2O. The reaction condition was 94 °C for 5 min, 94 °C for 30 s, 40 cycles of 94 °C for 5 s and 60 °C for 30 s. Real-time RT-PCR was performed using ABI Quant Studio 5 (Applied Biosystems, Foster City, California).
Validation of the developed RT-RPA assays with the cattle fecal samples
The applicability of the BRVA real-time RT-RPA and LFS RT-RPA assays in the clinical diagnosis was validated using the RNA extracted from 134 cattle fecal samples as template. Then, the results were compared with those obtained with a real-time RT-PCR described previously , which was run in parallel for the above clinical samples.