Computational protocol: Prevalence and molecular characterization of canine and feline hemotropic mycoplasmas (hemoplasmas) in northern Italy

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Protocol publication

[…] Since the amplicon produced by the screening rPCR was too short (127 bp) for good species identification [], all hemoplasma-positive samples were amplified using a newly designed SYBR green rPCR based on conserved regions of the 16S rRNA gene (MycE929f: 5′-ACG GGG ACC TGA ACA AGT GGT G-3′ and MycE1182r: 5′-AGG CAT AAG GGG CAT GAT GAC TTG-3′). This PCR was designed to amplify a 259 bp PCR product, to allow species identification following sequencing.The reactions were carried out in a total volume of 20 μl, containing 10 μl of QuantiFast SYBR Green PCR Master mix 2× (Qiagen GmbH, Germany), 0.1 μM of sense and reverse primer (MycE929f - MycE1182r) and 3 μl of extracted DNA. Amplifications were performed in a StepOnePlus™ instrument (Applied Biosystems, Foster City, CA, USA). The thermal profile consisted of 5 min at 95 °C, followed by 40 cycles at 95 °C for 15 s, 60 °C for 30 s and 60 °C for 30 s. Following amplification, melting curve analysis was performed by slowly raising the temperature of the thermal chamber from 60 to 95 °C to distinguish between hemoplasma amplicons (Tm range 76.2–77.3 °C) and non-specific amplification products. Negative (sterile water) and positive controls (DNA of Mhc) were included in each run.The sensitivity of this rPCR was determined using synthetic DNA of Mhc. After spectrophotometrically determining the concentration, the plasmid DNA copy numbers were calculated with the formula: Y = X/(a × 660) × 6.022 × 1023, where: Y = molecules/μl; X = g/μl dsDNA; a = plasmid plus insert length in nucleotides; 660 is the average molecular weight per nucleotide of dsDNA. The detection limit, evaluated using 10-fold serial dilutions of synthetic DNA, tested in triplicate, was 101 DNA copies/rPCR. The inclusivity of the assay was confirmed by analyzing canine and feline hemoplasma reference strains of Mhc, “CMhp”, Mhf, “CMhm” and “CMt”.In addition, since the 16S rRNA gene sequence is identical for Mhf and Mhc, the positive samples of these two species were amplified, using the RNase P gene primers RNasePFor1 and RNasePrev1, which more reliably differentiate between the two species, and sequenced []. The reactions were carried out in a total volume of 20 μl, containing 10 μl of QuantiFast SYBR Green PCR Master mix 2× (Qiagen GmbH, Germany), 0.1 μM of sense and reverse primer and 3 μl of extracted DNA. Amplifications were performed in a StepOnePlus™ instrument (Applied Biosystems, Foster City, CA, USA). The thermal profile consisted of 5 min at 95 °C, followed by 40 cycles at 95 °C for 15 s, 58 °C for 30 s and 60 °C for 30 s. Following amplification, dissociation was performed by slowly raising the temperature of the thermal chamber from 60 to 95 °C. Negative (sterile water) and positive controls (DNA of Mhc and Mhf) were included in each run.The PCR products obtained with 16S rRNA and the RNase P gene were directly sequenced. Sequencing was performed with 16S rRNA gene primers MycE929f and MycE1182r and with RNase P gene primers RNasePFor1 and RNasePrev1, using the Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). The products were purified using the PERFORMA DTR Ultra 96-Well kit (Edge BioSystems, Gaithersburg, MD, USA) and sequenced in a 16-capillary ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequence data were assembled and edited with SeqScape software v2.5 (Applied Biosystems, Foster City, CA, USA). The sequence data were compared with representative sequences available in GenBank, using the Basic Local Alignment Search Tool (BLAST) [] to identify hemoplasma species. […]

Pipeline specifications

Software tools SeqScape, BLASTN
Application Sanger sequencing
Organisms Canis lupus familiaris, Felis catus, Mycoplasma haemocanis, Candidatus Mycoplasma haemominutum, Feline immunodeficiency virus
Diseases Anemia, Infection, Mycoplasma Infections, Coinfection