Multiplex RT-PCR and Automated Microarray for Detection of Eight Bovine Viruses

Abstract

Microarrays can be a useful tool for pathogen detection as it allow for simultaneous interrogation of the presence of a large number of genetic sequences in a sample. However, conventional microarrays require extensive manual handling and multiple pieces of equipment for printing probes, hybridization, washing and signal detection. In this study, a reverse transcription (RT)-PCR with an accompanying novel automated microarray for simultaneous detection of eight viruses that affect cattle [vesicular stomatitis virus (VSV), bovine viral diarrhoea virus type 1 and type 2, bovine herpesvirus 1, bluetongue virus, malignant catarrhal fever virus, rinderpest virus (RPV) and parapox viruses] is described. The assay accurately identified a panel of 37 strains of the target viruses and identified a mixed infection. No non-specific reactions were observed with a panel of 23 non-target viruses associated with livestock. Vesicular stomatitis virus was detected as early as 2 days post-inoculation in oral swabs from experimentally infected animals. The limit of detection of the microarray assay was as low as 1 TCID /ml for RPV. The novel microarray platform automates the entire post-PCR steps of the assay and integrates electrophoretic-driven capture probe printing in a single user-friendly instrument that allows array layout and assay configuration to be user-customized on-site.

A multiplex reverse transcription PCR and automated electronic microarray assay for detection and differentiation of seven viruses affecting swine

“…When the PCR primers for PRRSV and PCV2 were removed and placed in a separate RT‐PCR, an increase in sensitivity was observed for FMDV, SVDV, VESV and CSFV. The electronic microarray component of the assay showed a reduction in sensitivity for some target pathogens both in this and other studies (Lung et al., , and unpublished results) when compared with the RT‐PCR component of the assay. Possible explanations for this reduction in sensitivity at the microarray stage could be the formation of secondary structure of certain capture probes, the amplicon or the annealing of the two strands of the amplicons which could prevent efficient hybridization between the capture probes and the amplicon under the experimental conditions.…”

“…The use of asymmetric PCR to generate single‐stranded products, increased amount of capture probe and/or amplicon may also further increase the sensitivity of the assay. Although not tested in this study due to resource constraints, previously published electronic microarray assays for detection of avian, swine and bovine viruses (Lung et al., , , ), were able to detect the presence of more than one target. Due to potentially high loads of viruses such as PCV2 in many countries, the splitting of the seven‐plex RT‐PCR into a duplex RT‐PCR for PRRSV and PCV2 and a five‐plex RT‐PCR for the other viruses, as described in this study, will improve the sensitivity of the assay for the detection of reportable diseases.…”

“…Microarrays can accommodate multiple probes for each target that can provide redundancy and broader coverage of viral variants when compared with single probe assays. The development and evaluation of traditional microarrays for subtyping and multiplex detection of viruses that affect livestock have been previously described (Banér et al., ; Hindson et al., ; Jack et al., ; Lenhoff et al., ; Lung et al., , ). The assay described by Banér et al.…”

A novel multiplex PCR-electronic microarray assay for rapid and simultaneous detection of bovine respiratory and enteric pathogens

“…Furthermore, depending on the number of samples to be tested, not all probe binding sites on an electronic microarray cartridge need to be used on the initial run, and unused sites can be used in subsequent sessions (up to 10 times per cartridge). Electronic microarray based assays for detection of several avian, bovine, and swine viruses and bacteria have been described previously (Lung et al, 2012;Lung et al, 2015;Lung et al, 2016).…”

Co-Infection of Bovine Papular Stomatitis Virus, Rotavirus and Cryptosporidium Spp. in a Calf

“…cDNA and DNA were used as templates in the PCR stage. For the diagnosis of panpestivirus (BVDV) (p324-326) [3], vesicular stomatitis (VSV) [4], bovine papilloma virus type-2 (BPV-2) (P1-2-3) [5], bovine papular stomatitis virus (BPSV) (PPP1-3-4) [6], foot and mouth disease virus (FMDV) (2BF-2BR) [7], rotavirus (BRV) [8] and coronavirus (BCV) [9] reference primers and PCR protocols were used. The obtained amplicons were analyzed by electrophoresis (110V / 40 min) in 1.5% (w/v) agarose gel stained with ethidium bromide (EtBr).…”