Effects of sample handling on the detection of porcine reproductive and respiratory syndrome virus in oral fluids by reverse-transcription real-time PCR.

We evaluated effects of handling procedures on detection of porcine reproductive and respiratory syndrome virus (PRRSV) in oral fluids (OFs) by reverse-transcription real-time PCR (RT-rtPCR). The experiments were conducted using a composite sample of PRRSV-positive OF collected from 5-wk-old pigs vaccinated 15 d earlier with a modified-live PRRSV vaccine. Five pre-extraction sample-handling steps and all combinations thereof were evaluated: 1) thaw temperature (4°C or 25°C); 2) sample diluent (1:1 dilution with nuclease-free water or guanidinium thiocyanate-phenol); 3a) sonication of the sample (yes or no); 3b) temperature (4°C or 25°C) at which step 3a was conducted; and 4) temperature at which the sample was maintained after step 3b and until RNA extraction was initiated (4°C or 25°C). All combinations of the 5 sample-handling steps (i.e., 32 unique treatments) were tested in a completely randomized factorial design with 4 replicates and 1 negative control for each treatment. The entire experiment was repeated on 5 separate days to produce a total of 800 PRRSV RT-rtPCR results. Binary (positive or negative) data were analyzed by logistic regression and results (Ct) were analyzed using a generalized linear model. Overall, 1 false-positive result was observed among 160 negative controls (99.4% specificity), and 85 false-negative results were observed among the 640 known-positive samples (86.7% sensitivity). The most significant factor affecting test outcome was thaw temperature (4°C or 25°C); samples thawed at 4°C had higher positivity rate (94% vs. 80%, p < 0.0001) and lower Ct (36.2 vs. 37.5, p < 0.0001).

High-throughput whole genome sequencing of Porcine reproductive and respiratory syndrome virus from cell culture materials and clinical specimens using next-generation sequencing technology.

Next-generation sequencing (NGS) technologies have increasingly played crucial roles in biological and medical research, but are not yet in routine use in veterinary diagnostic laboratories. We developed and applied a procedure for high-throughput RNA sequencing of Porcine reproductive and respiratory syndrome virus (PRRSV) from cell culture-derived isolates and clinical specimens. Ten PRRSV isolates with known sequence information, 2 mixtures each with 2 different PRRSV isolates, and 51 clinical specimens (19 sera, 16 lungs, and 16 oral fluids) with various PCR threshold cycle (Ct) values were subjected to nucleic acid extraction, cDNA library preparation (24-plexed), and sequencing. Whole genome sequences were obtained from 10 reference isolates with expected sequences and from sera with a PRRSV real-time reverse transcription PCR Ct ≤ 23.6, lung tissues with Ct ≤ 21, and oral fluids with Ct ≤ 20.6. For mixtures with PRRSV-1 and -2 isolates (57.8% nucleotide identity), NGS was able to distinguish them as well as obtain their respective genome sequences. For mixtures with 2 PRRSV-2 isolates (92.4% nucleotide identity), sequence reads with nucleotide ambiguity at numerous sites were observed, indicating mixed infection; however, individual virus sequences could only be separated when 1 isolate identity and sequence in the mixture is known. The NGS approach described herein offers the prospect of high-throughput sequencing and could be adapted to routine workflows in veterinary diagnostic laboratories, although further improvement of sequencing outcomes from clinical specimens with higher Ct values remains to be investigated.