Bacteria, virus strains, clinical samples and DNA extraction
Genomic DNA of M. Ovipneumoniae (Y98) and genomic DNA or cDNA of a panel of other close related pathogens considered dangerous to sheep and goats were maintained in our laboratory and used in the study, which were the following 4 mycoplasmas, 2 non-mycoplasma bacteria and 1 virus: M. capricolum subsp. capripneumoniae (F38), M. mycoides subsp. capricolum (PG3), M. arginini (G230), M. agalactiae (PG2), Klebsiella pneumoniae (strain F21W3), Pasteurella multocida (strain F91G3) and Peste des petits ruminants virus (Nigeria 75/1 vaccine strain).
A total of 46 sheep clinical samples (30 nasal swabs and 16 fresh lungs) were collected in Baoding City, Hebei Province from October to November 2019. The nasal swabs were collected from the sheep with coughing symptom in Fangzhuang farm in Dingzhou County, Baoding City, and the sheep fresh lungs were obtained from Zhuanluzhen slaughter house in Tang County, Baoding City. The sheep nasal swabs and lung samples were treated and the total DNA was extracted as described previously [24]. All DNA were quantified using a ND-2000c spectrophotometer (NanoDrop, Wilmington, USA) and stored at -80 °C until use.
Generation of standard DNA
To generate a M. Ovipneumoniae standard DNA for the RPA assays, a PCR product containing 361 bp covering the region of interest of 16SrRNA gene was amplified from the M. Ovipneumoniae DNA using LMF1 and LMR1 as primers (Table 1) and cloned into the pMD19-T (Takara, Dalian, China) for standards. The resulting plasmid, pMO-16SrRNA, was transformed into Escherichia coli DH5α cells, purified with the SanPrep Plasmid MiniPrep Kit (Sangon Biotech, Shanghai, China) and quantified using a ND-2000c spectrophotometer. The copy number of DNA molecules was calculated by the following formula: amount (copies/μL) = [DNA concentration (g/μL)/ (plasmid length in base pairs×660)] ×6.02×1023. Ten-fold dilutions of the pMO-16SrRNA, ranging from 1.0×107 to 1.0×100copies/μL, were prepared in nuclease-free water and aliquots of each dilution were stored at −80 ◦C.
RPA primers and probe
Nucleotide sequence data for different M. ovipneumoniae strains available in GenBank were aligned to identify the conserved regions in the 16SrRNA gene, which was determined as the molecular target for RPA. According to the reference sequences of M. ovipneumoniae (Accession numbers: NR_025989.1, LR215028.1, MN028361, MN028184, MN028079, MH133233), the primers, exo and nfo probes were designed following RPA manufacturer guidelines (TwistDx. Cambridge, UK). Primers and probe are listed in Table 1 and were synthesized by Sangon Biotech Co., Shanghai, China.
Real-time RPA and LFS RPA assays
The M. ovipneumoniae real-time RPA assay was performed as described previously [24]. The total reaction volume was 50 μL including 40.9 μL of Buffer A (rehydration buffer), 2.0 μL of each RPA primers (MO-exo-F and MO-exo-R, 10 μmol/L), 0.6 μL of exo probe (MO-exo-P, 10 μmol/L) and 2.5 μL of Buffer B (magnesium acetate, 280 mmol/L). Furthermore, 1 μL of genomic DNA or recombinant plasmid was used for the specificity and sensitivity analysis, or 2 μL of sample DNA was used for the clinical sample diagnosis.
The M. ovipneumoniae LFS RPA assay were performed as described previously [24]. The total reaction volume was 50 μL including 29.5μL of rehydration buffer, 2.1 μL of each RPA primers (MO-nfo-F and MO-nfo-R, 10 μmol/L), 0.6 μL of exo probe (MO-nfo-P, 10 μmol/L ) and 2.5 μL of magnesium acetate (280 mmol/L). In addition, 1 μL of bacterial genomic DNA or recombinant plasmid was used for the specific and sensitive analysis, or 2 μL of sample DNA was used for the clinical sample diagnosis. The assay was performed in a metal bath incubator at 39 °C for 15 min. Furthermore, the lateral flow strips (Milenia Biotec GmbH, Germany) were used to detect the RPA amplicons dual-labeled with FAM and biotin.
Analytical specificity and sensitivity analysis
Both RPA assays were performed to amplify the nucleic acids of a panel of pathogens including M. Ovipneumoniae, M. capricolum subsp. capripneumoniae, M. mycoides subsp. capricolum, M. arginini, M. agalactiae, P. multocida, K. pneumoniae, PPRV, which are considered to be dangerous to the sheep and goat respiratory system. The analytical specificity analysis was repeated five times.
The standard DNA of M. Ovipneumoniae, ranging from 1.0×107 to 1.0×100 copies/μL, was prepared in nuclease-free water and used for the RPA analytical sensitivity analysis. One microliter of each dilution was amplified by both RPA assays to determine the limit of detection (LOD). The analytical sensitivity analysis was repeated five times. Furthermore, the real-time RPA was tested using the standard DNA in 8 replicates, the threshold time was plotted against the molecules detected and a semi-log regression was calculated using Prism software 5.0 (Graphpad Software Inc., SanDiego, California).
Validation with clinical samples
The real-time RPA assay was assessed with 30 sheep nasal swabs and 16 sheep fresh lungs. All samples tested with the two RPA assays were also tested by a real-time PCR in parallel. The real-time PCR for M. oviopneumoniae was performed on a ABI 7500 instrument (Applied Biosystems, Foster City, California) described previously [4].