PRRSV sequencing success dependent upon Cycle Threshold values

PRRSV sequencing success dependent upon Cycle Threshold values

Porcine reproductive and respiratory syndrome virus continues to serve as one of the economically most important pathogens in the global swine industry. Diagnostics for this disease has improved over the years and one of the most routine tests used in diagnostic laboratories is the real-time reverse transcription polymerase chain reaction test.

This diagnostic assay detects the RNA of the PRRS virus from clinical samples and reports a Cycle Threshold value when the virus is detected. The CT provides an approximation of the amount of the virus in the sample. The more virus is present in a sample, the lower CT value will be obtained. Samples with low amounts of virus generally have high CT values. At the Iowa State University Veterinary Diagnostic Laboratory, a commercial PRRSV PCR is used as a screening PCR for the detection and quantification of PRRSV. If no virus is detected after 37 cycles on the PRRSV PCR test, then the sample is considered negative for PRRS virus.

Sequencing of the PRRS virus is commonly used to study molecular epidemiology and/or determine genetic relatedness of strains. This information can be used to determine if a PRRS virus strain found is the one already on the farm or a new introduction. It can also be used to determine the spread of a new strain into a geographical area or even for selection of strains for vaccine selection. Samples containing higher amounts of PRRS virus are much easier to sequence than those with less amounts of virus, but any sequencing information is valuable. One of the most common questions that comes in the ISU VDL is whether we can still get a successful sequence from a sample with a relatively high CT value (less virus)?

In order to help provide some guidance for producers and practitioners, we analyzed ISU VDL data on PRRS virus sequencing results performed on samples received in 2015. There were 4,681 cases where PRRSV open reading frame 5 (ORF5) sequencing was attempted using the Sanger method. Those cases were further filtered to only include the cases where PRRSV RT-PCR CT values were available. In addition, only the cases containing a North American PRRSV strain were included and the cases containing a European PRRSV strain (only 50 cases) were excluded for analysis in this report. After this data filtering, a total of 2,977 samples were used to analyze the correlations of PRRSV screening PCR CT values to the ORF5 sequencing success rate for the North American strain.

Table 1 represents the analysis results. This included all specimen types, which primarily included serum, lung and oral fluid samples. It was shown that samples with a CT less than 30 had a very high ORF5 sequencing success rate (99.85%).

Samples with CT from 30-32 still had over 90% sequencing success rate (92.80%). For samples with Ct 32-34, the ORF5 sequencing success rates were 81.79%. However, when the Ct values of the samples increased over 34, the ORF5 sequencing success rate decreased dramatically (58.92% for samples with Ct 34-36 and 40% for samples with Ct 36-37).

There are other factors that can increase the likelihood of obtaining successful sequencing of high CT PRRS virus PCR samples, such as being sure to get your samples cooled as soon as they are collected and getting the samples to the diagnostic lab within 24-28 hours. The data from the ISU VDL in 2015 seems to indicate that the likelihood of getting an ORF5 sequence from a PRRS virus PCR sample should be very high as long as the CT value is less than 34, regardless of sample type (oral fluids, tissues or blood serum).

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