Positive Control Preparation

Positive controls — represented by target-sequence-containing plasmids — were used to optimize and validate each pathogen testing assay. All plasmids contained the target sequence for the pathogen of interest with the primer and probe-binding sites. These plasmids containing the artificial DNA fragments for each pathogen were synthesized by Twist Bioscience (San Francisco, CA), except for the Mycoplasma species, which were all cloned in the pBluescript plasmid. All the synthesized plasmids were sequenced to confirm the presence of the specific target sequence. DNA concentration was quantified using Qubit® 3.0 Fluorometer (Invitrogen) and the Qubit® dsDNA HS Assay Kit (Life Technologies), and the DNA copy number was calculated using the following formula:

$$\textrm\ \textrm\ \textrm=\frac\ \textrm\ \textrm\ \textrm\ \textrm\times 6.022\times ^\kern0.5em }\ \textrm\ \textrm\ \left(\textrm\right)\times 1\ \textrm\ ^9\times \kern0.5em 660}$$

Sample Collection and Direct Pathogen Testing of the MSC Batches

MSCs were extracted from visceral adipose tissue acquired as surgical waste, donated by the dogs’ owner via informed consent, following standard ovariectomy of clinically healthy female mixed-breed dogs. The isolated cells were cultured and harvested according to the methodology previously described by Kriston-Pál et al. [31]. To assess the efficacy of our nucleic acid-based pathogen testing methods in detecting the specified pathogens, we separately obtained samples from dog owners and rescue centres that may contain extraneous agents. For this purpose, we processed tissues that did not necessarily meet the originally established isolation criteria, and — monitoring the isolated cells in culture — searched for MSC batches that displayed any abnormal phenotypes such as reduced cell division or altered cell morphology and viability. We collected both supernatants and cell pellets from these samples for further examination. A total of nine MSC batches of uncertain purity were chosen. Furthermore, biosafety investigation was carried out on MSC batches intended for therapeutic utilization. Our biosafety study enrolled 12 batches of MSC batches previously selected for pilot production with the view to use in subsequent treatment.

Exogenous, natural spike-in controls were used as indicators of optimal nucleic acid extraction. In this context, cells from each sample were previously spiked with viable T7 (single-stranded DNA control) and MS2 (RNA control) bacteriophage and E. coli DH5-alpha (double-stranded DNA control). Following spike-in experiments, nucleic acid extraction was carried out for protozoan and bacterial DNA and, separately, total nucleic acid extraction was performed for viruses. Conventional and real-time PCR assays were conducted using the isolated DNA samples, whereas viral RNA was subjected to reverse transcription to produce complementary DNA prior to its inclusion in the PCR reactions. The PCR reactions were performed first to verify the presence and yield from the spike-in controls and then to test for the specific pathogens. The generalized workflow of the pathogen testing procedure is illustrated schematically in Fig. 6.

Fig. 6

Schematic representation of the pathogen testing workflow. Image created with BioRender.com

Spike-in Controls

As indicators of optimal nucleic acid extraction, exogenous IC were used. These IC provide confirmation of the presence of amplifiable DNA and the absence of inhibitors in the sample. Exogenous heterologous IC were utilized with primers and target sequences that were different from those used for pathogens (Supplemental Tables 1, 2 and 3), ensuring non-competitiveness and ease of implementation. We used natural DNA and RNA as external spike-in controls to control nucleic acid extraction: viable E. coli DH5-alpha, MS2 phage, and T7 phage were used as spike-in controls for bacterial/protozoan DNA, viral RNA, and viral DNA extraction, respectively. MS2 bacteriophage (15597-B1TM), T7 bacteriophage (BAA-1025-B2), the appropriate hosts, and E. coli DH5-alpha were obtained from the American Type Culture Collection (ATCC). The standardized amount of 5 × 105 MSC samples were spiked with serial dilutions of each spike-in control. These spike-in controls were diluted in parallel to quantify colony forming units (CFUs) for bacteria and plaque forming units (PFUs) for viruses using traditional plating and double-agar overlay methods, respectively. Following nucleic acid extraction from the spiked samples, real-time PCR assays were performed to validate the presence and efficacy of spike-in controls.

Viral Nucleic Acid Extraction, Quantification, and cDNA Synthesis

Total viral nucleic acid from stem cell samples was extracted using 5 × 105 cells/MSC batch and additionally 100 μl of the corresponding cell supernatant in the case of the nine batches of uncertain purity. Isolation was performed using the MagCore® Viral Nucleic Acid Extraction Kit (RBC Bioscience, Taiwan) following the manufacturer’s recommendation. The final sample was eluted in 100 μl elution buffer. RNA concentration was measured using the Qubit™ RNA High Sensitivity Assay Kit (Invitrogen by Thermo Fisher Scientific), while the concentration of the DNA was measured using the Qubit™ dsDNA High Sensitivity Assay Kit (Invitrogen by Thermo Fisher Scientific) with a Qubit™ 3.0 fluorometer. Following total nucleic acid extraction, High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was utilized to generate cDNA for the detection of RNA viruses. Eluted nucleic acids and synthesized cDNA were stored at − 20 °C.

Bacterial and Protozoan DNA Extraction and Quantification

For bacterial and protozoan DNA extraction, we used the QIAamp® PowerFecal® Pro DNA Kit (QIAGEN®) according to the manufacturer’s protocol. DNA was isolated using 5 × 105 cells/MSC batch and additionally 100 μl of the corresponding cell supernatant in the case of the nine batches of uncertain purity and for Mycoplasma detection in all cases. The final sample was eluted in 100 μl elution buffer. Following DNA extraction, a Qubit™ 3.0 fluorometer was used to measure DNA concentration utilizing the associated Qubit™ dsDNA High Sensitivity assay kit (Invitrogen by Thermo Fisher Scientific). Eluted DNA was stored at − 20 °C.

Primers, Probes and Target Sequences

All primers, probes, and target sequences used in this study were retrieved from previous studies (Table 5). NCBI-BLAST (www.ncbi.nlm.nih.gov) was used to compare and retrieve the nucleotide sequences of each target pathogen. Primer sequences and specific PCR product sizes are described in Table 5. All primer sequences for real-time PCR-based assays were utilized in our study as previously described [36,37,38,39,40], with slight modifications in the application of the primers. Primer sequences for conventional PCRs were used as described previously [32,33,34,35, 41], except for CCoV, where the forward primer was combined with the nested reverse primer in a single PCR reaction [32]. A single PCR reaction was also performed for L. interrogans [20], B. canis, and N. caninum [42], employing the previously described internal primers that were initially utilized in the nested PCR, resulting in high specificity, while minimizing the laboriousness of the second round PCR.

Table 5 Primers and probes used for extraneous agent detectionReal-Time PCR-Based Assays

Real-time PCR-based assays were carried out using the Roche LightCycler® 480 system. The primer concentration was optimized for each real-time PCR-based assay (Supplemental Table 4). Single-tube real-time quantitative PCR (qPCR) was used for Mycoplasma assays. An artificial oligonucleotide was employed as IC, which was simultaneously amplified in a single tube and detected by a specific VIC-labelled TaqMan probe. Eight different forward primers, a single FAM-labelled TaqMan probe, and a single reverse primer were used to detect the strains of interest. A primer mixture of the eight forward primers (4 pmol/μl each) and the FAM-labelled TaqMan probe (1.6 pmol/μl) was used. Real-time PCR reaction was performed in 20 μl reaction volume, containing 10 μl Brilliant III Ultra-Fast qPCR Master Mix (Agilent Technologies), the forward primer mixture, 3.5 pmol/μl reverse primer, and 8 pmol/μl VIC-labelled TaqMan probe. For optimization and validation, 1 μl of the M. arginini positive control serial dilution was used as template DNA. In the case of pathogen testing, 1 μl of the DNA template was used for the PCR detection, which was run together with the positive control serial dilution. Nuclease-free water was used as negative control for each PCR run.

Real-time quantitative PCR (qPCR) followed by High Resolution Melting Analysis (HRM) was used for Bartonella spp., Neorickettsia spp., Rickettsia spp., Anaplasma spp., Ehrlichia spp., Borrelia spp., and Leishmania spp. assays. Real-time qPCR amplification reactions were performed in 20 μl reaction volumes containing 10 μl 2x Luna® Universal qPCR Master Mix (NEB), 0.8 μl MgCl2 (25 mM, Promega), and 0.4–2.5 μl primers (Supplemental Table 4). For optimization and validation, 1 μl of the positive control serial dilution was used as template DNA. In the case of pathogen testing, 1 μl of the DNA template for the PCR detection, which was run together with the positive control serial dilution. Nuclease-free water was used as negative control for each PCR run. Primers and target sequences for real-time PCR-based assays are available in supplemental Tables 5 and 6.

Conventional PCR-Based Assays

Conventional PCR-based assays were carried out using a Biometra TAdvanced thermocycler (Analytic Jena). Conventional PCR amplification reactions were performed in 25 μl reaction volumes containing 5 μl 5x colourless GoTaq® Flexi buffer (Promega), 2–3 μl MgCl2 (25 mM, Promega), 2 μl of 2.5 mM dNTPs (100 mM, Thermo Fisher Scientific), and 0.125 μl of GoTaq® G2 Hot Start Polymerase (Promega, 5 U/μl). The primer concentration was optimized for each assay (Supplemental Table 7). For optimization and validation, 1 μl of the positive control serial dilution was used as template DNA. In the case of direct pathogen testing, 1 μl of the DNA or 1 μl synthesized cDNA was used as template for the PCR detection, which was run together with the positive control serial dilution. Nuclease-free water was used as negative control for each PCR run. Primers and target sequences for conventional PCR-based assays are described separately in Supplemental Tables 8, 9, 10, 11, 12, 13 and 14.

Analytical Sensitivity

The analytical sensitivity of each assay was evaluated through experimental determination of the LOD, representing the lowest copy number of the target at which at least 95% of the samples give positive signal. LOD was determined by performing a dilution series of the positive control within the anticipated range of detection limits, which was determined during the optimization phase. Three concentrations (1 μl/reaction) of the positive controls were used as follows: expected LOD, 10-fold dilution above the expected LOD, and 10-fold dilution below the expected LOD. Ten replicates were subjected to PCR analysis in two test runs for the three concentrations of the positive control for each pathogen of interest, resulting in 20 independent replicates. A known and standardized concentration of canine DNA (1 ng/μl) isolated from three independent MSC batches, which may potentially influence the detection limit of the assays, was added to each PCR reaction during the sensitivity assessments.

Specificity

Specificity was evaluated for each pathogen assay by analyzing three different and independent canine DNA isolates within a range of 1–30 ng/μl concentrations. Nuclease-free water was included as negative control in all specificity assays.

Amplification Efficiency and Linearity

To assess the efficiency of the real-time PCR assays, serial dilutions of the positive control DNA of the relevant pathogen (104–100 copies/μl) were prepared. Each concentration was analysed in triplicate. A linear regression analysis was performed by plotting the average of cycle threshold (Ct) values against the average of the log10 of the corresponding DNA copy number. For each real-time PCR assay, the slope of the regression curve indicates PCR efficiency, which should be between − 3.9 and − 2.9, corresponding to PCR efficiencies ranging from 80% up to 120%. The amplification efficiency was calculated using the following formula: E = [10(−1/slope) − 1] × 100%. Linear regression analysis also allows for the determination of the linearity of our real-time PCR-based assays by the calculation of the coefficient of determination (R2).