Fully automated and integrated multiplex detection of high consequence livestock viral genomes on a microfluidic platform

Abstract

Differential diagnosis of diseases that share common clinical signs typically requires the performance of multiple independent diagnostic tests to confirm diagnosis. Diagnostic tests that can detect and discriminate between multiple differential pathogens in a single reaction may expedite, reduce costs, and streamline the diagnostic testing workflow. Livestock haemorrhagic diseases like classical swine fever (CSF), African swine fever (ASF), and vesicular diseases, such as foot-and-mouth disease (FMD), vesicular stomatitis (VS), and swine vesicular disease (SVD) can have an enormous impact on the livestock industry and economy of countries that were previously free of the diseases. Thus, rapid diagnosis of these diseases is critical for disease control. Here, we describe the development and initial laboratory validation of a novel fully automated user-developed assay for simultaneous detection and differentiation of multiple viruses of veterinary importance in a single reaction with minimal user-intervention. The user only performs sample loading, placement of consumables and reagents, selection and initiation of assay while all other processes (i.e., nucleic acid extraction, multiplex RT-PCR, reverse dot blot detection and result reporting) are performed fully automated. The current assay has a turn-around time of approximately 6 hr and can simultaneously process up to 24 samples. The automated assay accurately and specifically detected 37 laboratory amplified strains of the five target viruses, including all seven serotypes of FMD virus, three genotypes of CSF virus, and two serotypes of VS virus. The assay also detected targeted viruses in a variety of clinical samples collected from infected animals, such as oral fluid, oral swab, nasal swab, whole blood, serum, as well as tonsil, spleen, kidney, and ileum. No cross-reactivity was observed with 15 nontarget viruses that affect livestock and samples from clinically healthy animals. To our knowledge, this is the first fully automated and integrated assay for simultaneous detection of multiple high consequence veterinary pathogens.

African Swine Fever Diagnosis in Africa: Challenges and Opportunities

“…A gap that was identified was the inability to diagnose coinfections with other pathogens, in particular pathogens causing diseases that are differential diagnoses for ASF, at the same time as testing for ASF. Several assays to simultaneously detect ASF and classical swine fever have been described [ 60 , 61 , 62 , 63 ] as well as multiplex assays for simultaneous testing for more than two porcine pathogens [ 42 , 64 , 65 , 66 , 67 , 68 , 69 ] but not all are likely to be widely available commercially. Using the methods described in the literature, well-equipped laboratories might be able to develop and validate their tests for simultaneous detection of porcine viruses other than ASF present in their region.…”

Point of care diagnostics and non-invasive sampling strategy: a review on major advances in veterinary diagnostics

“…However, there has been a very limited application of this technology in the veterinary field to date (Busin et al 2016). Such fully automated microfluidic platform has been developed only recently, for multiplex detection of high threat livestock diseases such as FMD, classical swine fever (CSF) and vesicular stomatitis (VS) (Lung et al 2019). Another feature of microfluidic technology includes microfluidic paper-based analytical devices (Busin et al 2016).…”

Recent Advances in the Diagnosis of Classical Swine Fever and Future Perspectives

“…The improved real-time RT-PCRs and advanced alternatives have been designed to help resolve these issues. One-step and automated RT-PCRs can reduce the risk of contamination [ 27 , 28 , 29 ]. The primer-probe energy transfer RT-PCR assay provides a higher specificity by analyzing the melting curve following PCR amplification [ 30 , 31 ].…”