Development of a high-throughput real-time PCR system for detection of enzootic pathogens in pigs

Abstract

Respiratory and intestinal diseases in pigs can have significant negative influence on productivity and animal welfare. A wide range of real-time PCR (rtPCR) assays are used in our laboratory (National Veterinary Institute, Technical University of Denmark) for pathogen detection, and PCR analyses are performed on traditional rtPCR platforms in which a limited number of samples can be analyzed per day given limitations in equipment and personnel. To mitigate these restrictions, rtPCR assays have been optimized for the high-throughput rtPCR BioMark platform (Fluidigm). Using this platform, we developed a high-throughput detection system that can be used for simultaneous examination of 48 samples with detection specificity for 18 selected respiratory and enteric viral and bacterial pathogens of high importance to Danish pig production. The rtPCR assays were validated and optimized to run under the same reaction conditions using a BioMark 48.48 dynamic array (DA) integrated fluidic circuit chip, and the sensitivity and specificity were assessed by testing known positive samples. Performance of the 48.48DA was similar to traditional rtPCR analysis, and the specificity of the 48.48DA was high. Application of the high-throughput platform has resulted in a significant reduction in cost and working hours and has provided production herds with a new innovative service with the potential to facilitate the optimal choice of disease control strategies such as vaccination and treatment.

Objective pathogen monitoring in nursery and finisher pigs by monthly laboratory diagnostic testing

“…In the present study, three monthly samplings were conducted in six nursery and four finisher production units to investigate the value of continuous screening for selected respiratory and enteric viral and bacterial pathogens in different age groups. Oral fluid and faecal sock samples were collected and analysed using the high-throughput diagnostic system described elsewhere [ 22 ]. The use of a high-throughput rtPCR platform, in which multiple samples can be analysed in different assays simultaneously, provides new possibilities for conducting extended pathogen detection at a limited cost.…”

“…Extracted oral fluid and faecal sock samples were both reverse transcribed/pre-amplified and pre-amplified as previously described [22]. For high-throughput rtPCR amplification, the BioMark 48.48 dynamic array (DA) Integrated Fluidic Circuit system (Fluidigm, South San Francisco, USA) was used, which combines 48 pre-amplified samples with 48 assays for 2304 individual and simultaneous rtPCR reactions.…”

“…For high-throughput rtPCR amplification, the BioMark 48.48 dynamic array (DA) Integrated Fluidic Circuit system (Fluidigm, South San Francisco, USA) was used, which combines 48 pre-amplified samples with 48 assays for 2304 individual and simultaneous rtPCR reactions. The rtPCR assays and procedure used for the high-throughput rtPCR analysis in the present study have previously been described and validated in the study by Goecke et al, 2020 [22]. The rtPCR assays were performed in duplicates for each sample.…”

“…The major reason for the limited use of laboratory diagnostics is that the traditional methods are expensive and resource demanding. To overcome these limitations, we have developed a high-throughput real-time PCR (rtPCR) detection system using the BioMark HD platform (Fluidigm, South San Francisco, USA), which is capable of detecting 18 significant porcine viruses and bacteria in the same setup [22].…”

The effects of supplementation of yeast (<i>Saccharomyces cerevisiae</i>) and postbiotic from <i>Lactobacillus acidophilus</i> on the health and growth performance of young Jersey heifer calves

“…It was not possible to obtain samples from all calves treated with antibiotics for diarrhoea. The pathogens in the samples were tested according to Goecke et al (2020); the chip used was 192.24 (192 samples and 24 assays).…”