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

Abstract

Microarray technology can be useful for pathogen detection as it allows simultaneous interrogation of the presence or absence of a large number of genetic signatures. However, most microarray assays are labour-intensive and time-consuming to perform. This study describes the development and initial evaluation of a multiplex reverse transcription (RT)-PCR and novel accompanying automated electronic microarray assay for simultaneous detection and differentiation of seven important viruses that affect swine (foot-and-mouth disease virus [FMDV], swine vesicular disease virus [SVDV], vesicular exanthema of swine virus [VESV], African swine fever virus [ASFV], classical swine fever virus [CSFV], porcine respiratory and reproductive syndrome virus [PRRSV] and porcine circovirus type 2 [PCV2]). The novel electronic microarray assay utilizes a single, user-friendly instrument that integrates and automates capture probe printing, hybridization, washing and reporting on a disposable electronic microarray cartridge with 400 features. This assay accurately detected and identified a total of 68 isolates of the seven targeted virus species including 23 samples of FMDV, representing all seven serotypes, and 10 CSFV strains, representing all three genotypes. The assay successfully detected viruses in clinical samples from the field, experimentally infected animals (as early as 1 day post-infection (dpi) for FMDV and SVDV, 4 dpi for ASFV, 5 dpi for CSFV), as well as in biological material that were spiked with target viruses. The limit of detection was 10 copies/μl for ASFV, PCV2 and PRRSV, 100 copies/μl for SVDV, CSFV, VESV and 1,000 copies/μl for FMDV. The electronic microarray component had reduced analytical sensitivity for several of the target viruses when compared with the multiplex RT-PCR. The integration of capture probe printing allows custom onsite array printing as needed, while electrophoretically driven hybridization generates results faster than conventional microarrays that rely on passive hybridization. With further refinement, this novel, rapid, highly automated microarray technology has potential applications in multipathogen surveillance of livestock diseases.

Keywords: African swine fever; classical swine fever; foot-and-mouth disease; microarray; multiplex PCR; swine vesicular disease.

Figure 1

Amplification of target and non‐targets using the seven‐plex RT‐PCR. (a) QIAxcel gel image of representative amplified products after RT‐PCR amplification of representative strains of the seven targeted swine viruses and oral swab from healthy pigs. Asterisk denotes in vitro transcribed RNA was used as a template. (b) QIAxcel gel image of amplified products from amplification of 11 non‐target virus and bacteria that affect livestock: swine influenza virus (SIV), porcine respiratory coronavirus (PRCV) KIVA, transmissible gastroenteritis virus (TGEV) TC1998, porcine circovirus 1 (PCV1), vesicular stomatitis virus (VSV) 89 GAS, Streptococcus suis, Pasteurella multocida, Salmonella enterica choleraesuis, Mycoplasma hyopneumoniae, bovine viral diarrhoea virus (BVDV) type 1 Singer, and Border disease virus (BDV) CoosBay. (c) Amplification of nucleic acid from non‐target bacteria and viruses spiked with PRRSV YNL RNA as an exogenous control. NTC: no template control

Figure 2

Multiplex RT‐PCR amplicons visualized using agarose gel and QIAxcel System (a) and heat map depicting the reactivity of samples against 27 virus‐specific capture probes and one non‐specific binding probe (NSBP) (b). The panel of samples includes 58 strains of the seven targeted swine viruses, 11 oral swab samples: 267, 270, 271, 272, 273, 274, 277, 278, 279, 281, pooled 2‐day piglet, taken from healthy pigs and 11 non‐target virus and bacteria that are associated with livestock: swine influenza virus (SIV), porcine respiratory coronavirus (PRCV) KIVA, transmissible gastroenteritis virus (TGEV) TC1998, porcine circovirus 1 (PCV1), vesicular stomatitis virus (VSV) 89 GAS, Streptococcus suis, Pasteurella multocida, Salmonella enterica choleraesuis, Mycoplasma hyopneumoniae, bovine viral diarrhoea virus (BVDV) type 1 Singer, and Border disease virus (BDV) CoosBay. The viral strains tested include representatives of each of the seven FMDV serotypes, three CSFV genotypes, two ASFV genotypes and genotypes NA and EU of PRRSV. A positive signal in red represents a positive‐to‐negative ratio (P/N) of >2, while negative results in black represent any P/N ≤ 2

Figure 3

Multiplex RT‐PCR amplicons visualized using QIAxcel System (a) and heat map depicting the reactivity of 10 genetically diverse Canadian PRRSV‐positive serum samples from diagnostic submissions against 27 virus‐specific capture probes and one non‐specific binding probe (NSBP) (b). The panel of samples includes the following: #2 (17‐001377‐0001_1‐57‐1), #7 (17‐007044‐2208_1‐3‐2), #11 (17‐010599‐0006_1‐30‐2), #20 (17‐019861‐0006_1‐1‐1), #21 (17‐020077‐0002_1‐8‐4), #22 (17‐020084‐0001_1‐3‐2),‐#23 (17‐020347‐0011_1‐8‐2), #33 (17‐035‐986_1‐4‐1), #36 (17‐039858‐0022_2‐5‐2), #43 (17‐052259‐0023_1‐57‐1). A positive signal in red represents a positive‐to‐negative ratio (P/N) of >2, while negative results in black represent any P/N ≤ 2