Impact of oral fluid handling on sensitivity of real time PCR to detect PRRSV

Oral fluid is convenient sample for monitoring of PRRSV in pig herds. However, due to its nature, it is recommended to maintain cold chain during transport and storage. The aim of the study was to evaluate the impact of storage conditions of oral fluid on sensitivity of real time PCR to detect PRRSV.

Oral fluid was obtained from a PRRSV-positive farm, transported to the laboratory, and stored at -20°C. The sample was extracted with RNA Mini Kit (Qiagen) and tested by Real-time PCR with EZ-PRRSV MPX 4.0 reagents (Tetracore Inc.). Obtained Ct was 28.00. The sample was diluted ten-fold in PRRSV negative oral fluid, from 10^-1 to 10^-3. The undiluted sample and each dilution was stored at room temperature (20-25°C) without additives, or after adding equal volumes (1:1) of 0.1% chlorhexidine, 0.01% chlorhexidine or PBS, that served as preservatives. At days 0, 2, 4 and 6 one tube from every treatment and dilution series was placed in -20°C. Next, RNA was extracted from all samples, and Real-time PCR was performed as above. Each extracted RNA was run with PCR in five repeats.

As expected the Real Time PCR Ct values increased from day 0 to day 6. After 6 days of undiluted oral fluid storage at room temperature the best results were obtained for samples preserved with 0.1% or 0.01% chlorhexidine. The mean Ct values were 30.25 (SD 0.73) and 30.88 (SD 0.36), respectively, compared to Ct 32.94 (SD 0.3) for samples mixed with PBS, or Ct 34.27 (SD 0.45) for untreated samples. The increase of mean Ct values between day 0 and 6 was 6.27, 0.73, 1.87 and 4,36 for native oral fluid and preserved with 0.1% chlorhexidine, 0.01% chlorhexidine and PBS, respectively. All oral fluid samples diluted 10-1 were positive at day 6 only when preserved with either chlorhexidine or PBS. PRRSV was detected in preserved oral fluid also diluted 10-2, in 3 or 2 out 5 samples, mixed with 0.1 and 0.01% chlorhexidine, respectively. The mean Ct values of positive samples at day 6 were 35.48 (SD 0.58) and 37.82 (SD 0.93).

Oral fluid may contain many factors that compromise PCR sensitivity. Also, in field conditions it can be exposed for increased temperatures that can also decrease PCR sensitivity. In the experimental set up of this study, the sensitivity of Real Time PCR performed in unpreserved oral fluid dropped more than 100 fold between day 0 and 6 (the rise of the mean Ct value by 6,27). On the other hand, preserving oral fluid samples with chlorhexidine fully protected genetic material of PRRSV and the sensitivity of PCR was not decreased even after 6 days of storage of the samples at room temperature.

Disclosure of Interest: None Declared.