Multiplex PCR and Microarray for Detection of Swine Respiratory Pathogens

Abstract

Porcine respiratory disease complex (PRDC) is one of the most important health concerns for pig producers and can involve multiple viral and bacterial pathogens. No simple, single-reaction diagnostic test currently exists for the simultaneous detection of major pathogens commonly associated with PRDC. Furthermore, the detection of most of the bacterial pathogens implicated in PRDC currently requires time-consuming culture-based methods that can take several days to obtain results. In this study, a novel prototype automated microarray that integrates and automates all steps of post-PCR microarray processing for the simultaneous detection and typing of eight bacteria and viruses commonly associated with PRDC is described along with associated multiplex reverse transcriptase PCR. The user-friendly assay detected and differentiated between four viruses [porcine reproductive and respiratory syndrome virus (PRRSV), influenza A virus, porcine circovirus type 2, porcine respiratory corona virus], four bacteria (Mycoplasma hyopneumoniae, Pasteurella multocida, Salmonella enterica serovar Choleraesuis, Streptococcus suis), and further differentiated between type 1 and type 2 PRRSV as well as toxigenic and non-toxigenic P. multocida. The assay accurately identified and typed a panel of 34 strains representing the eight targeted pathogens and was negative when tested with 34 relevant and/or closely related non-target bacterial and viral species. All targets were also identified singly or in combination in a panel of clinical lung samples and/or experimentally inoculated biological material.

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assay showed a reduction in sensitivity for some target pathogens both in this and other studies (Lung et al., , and unpublished results) when compared with the RT‐PCR component of the assay.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.…”

“…Although an RT step was not required for DNA viruses, previous studies have shown better amplification of the DNA targets when a RT step was included (Lung et al., ). This could be due to the utilization of messenger RNAs as well as genomic DNA as template (Lung et al., ).…”

“…Although an RT step was not required for DNA viruses, previous studies have shown better amplification of the DNA targets when a RT step was included (Lung et al., ). This could be due to the utilization of messenger RNAs as well as genomic DNA as template (Lung et al., ). Further improvements in assay sensitivity and integration of the PCR with the electronic microarray in the new NanoChip400 XL electronic microarray platform could make this novel automated microarray technology a useful tool for multipathogen detection and subtyping.…”

“…The reverse primers were synthesized with the reverse complementary sequence of the NGEN™ Red Universal Reporter Probe (Nexogen, San Diego, CA) at the 5′ end (Lung et al., ). The primers were also evaluated in silico as previously described (Lung et al., ) against non‐target viruses using NCBI's electronic PCR (e‐PCR, Rotmistrovsky, Jang, & Schuler, ; Schuler, ) to evaluate potential cross‐reactivities. Furthermore, all unique primer pairs used in the multiplex PCR were tested against all non‐redundant nucleotide sequences of non‐FMDV picornaviruses ( n = 61,995 unique sequences).…”