Practical strategies for improving swine oral fluid PCR performance

Objective

Early work attempting to improve swine oral fluid PCR performance involved “fine tuning” extraction protocols (Chittick et al., 2011).  Later work focused on improving PCR performance by chemically degrading potential PCR inhibitors (guanidinium chloride, trypsin, dithiothreitol, sodium borohydride, and others) (Weiser et al., 2018).  Improvements were made, but the significant breakthroughs sought were not achieved.  The goal of this current research was to identify practical methods to improve the detection of PRRSV, IAV-S, and Mycoplasma hyopneumoniae (MHP) nucleic acids in swine oral fluids by evaluating sample treatments reported to achieve improved nucleic acid detection by qPCR: (1) Heat.  Ranoa et al. (2020) reported that direct PCR was possible when human oral fluids spiked with SARS-CoV-2 were heated at 95°C for 30 minutes.  (2) Diluent.  Diluents can affect the oral fluid matrix by changing the mucin configuration and reducing aggregation (Hughes et al., 2019; Ridley et al., 2014).  The assumption behind this approach is that mucins are less able to bind PCR targets if they are dispersed.  (3) Targeted concentration.  Tian et al. (2008) reported a 2-log improvement in sensitivity for norovirus detection using “targeted concentration”, i.e., magnetic beads with a norovirus-compatible ligand.

Materials and Methods  

TREATMENT 1.  Oral fluid samples known to contain PRRSV, IAV, PEDV or MHP (n = 8 each) were 2-fold serially diluted (neat, 1:2, 1:4, 1:8) using oral fluid known to be free of PRRSV, IAV, PEDV and MHP (n = 32 diluted samples per pathogen), and split into 4 aliquots, each of which was randomized to one of 4 protocols (see Table 1): (P1) heat (95°C × 30 m) and direct qPCR; (P2) heat, cool (25°C × 20 m) and direct qPCR; (P3) heat, cool, nucleic acid extraction, and direct qPCR; (P4, i.e., control) extraction and qPCR.

TREATMENT 2.  Oral fluid samples containing PRRSV (n = 9), IAV (n = 10), PEDV (n = 10) or MHP (n = 10) were split into 3 aliquots: (D1) diluted 1:2 with TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA); (D2) diluted 1:2 with oral fluid free of PRRSV, IAV, PEDV and MHP; (D3) undiluted (“neat”).  Samples were randomly ordered and then tested.

TREATMENT 3.  A pool of PRRSV-specific biotin-tagged probes targeting sequences of different PRRSV-specific genes were attached to streptavidin-conjugated magnetic beads.  A PRRSV modified live vaccine (MLV) was rehydrated and ten-fold diluted with molecular grade water, and dilutions 10⁴ and 10⁵ were processed in duplicates by a probe-based hybridization capture protocol, and compared to the standard protocol (extraction and amplification). 

Results and Discussion

TREATMENT 1.  P4 (control) produced 32/32 positives for PRRSV, IAV, PEDV, and 31/32 for MHP. Cumulatively, P1, P2, and P3 produced 1/96 positive for PRRSV, 5/96 for IAV, 15/96 for PEDV, and 47/96 for MHP. Among positives, P4 produced the lowest Cqs, i.e., produced the strongest positive results.

TREATMENT 2. Overall, D1 and D2 did not improve detection, with D3 producing the lowest Cqs.

Overall, the results clearly showed that the methods described in the literature for heat and dilution treatments not only did not improve the process but were actually detrimental to the detection of PRRSV, IAV, PEDV, and MHP nucleic acids in oral fluid samples by qPCR.  To understand these unanticipated results, we re-examined the original reports.  Most notably, we found that the work often did not include comparisons with standard methods.  That is, quantitative comparisons, e.g., standard method vs the proposed alternative, was typically lacking.  Thus, quantitative measures of the gains or losses in performance achieved by alternative methods was not provided.

TREATMENT 3.  We were unable to complete the work on targeted concentration within the allotted time.  However, preliminary results showed that capture of PRRSV-specific nucleic acids is achievable with the probe-based hybridization capture protocol we developed.  Further optimization is needed but the probe-based hybridization capture protocol could result in development of specific target capture methodology that would improve qPCR detection.