A multiplex real-time RT-PCR system to simultaneously diagnose 16 pathogens associated with swine respiratory disease

Abstract

Aims: Swine respiratory disease (SRD) is a major disease complex in pigs that causes severe economic losses. SRD is associated with several intrinsic and extrinsic factors such as host health status, viruses, bacteria, and environmental factors. Particularly, it is known that many pathogens are associated with SRD to date, but most of the test to detect those pathogens can be normally investigated only one pathogen while taking time and labor. Therefore, it is desirable to develop rapidly and efficiently detectable methods those pathogens to minimize the damage caused by SRD.

Methods and results: We designed a multiplex real-time RT-PCR (RT-qPCR) system to diagnose simultaneously 16 pathogens, including nine viruses and seven bacteria associated with SRD, on the basis of single qPCR and RT-qPCR assays reported in previous studies. Multiplex RT-qPCR system we designed had the same ability to single RT-qPCR without significant differences in detection sensitivity for all target pathogens at minimum to maximum genomic levels. Moreover, the primers and probes used in this system had highly specificity because the sets had not been detected pathogens other than the target and its taxonomically related pathogens. Furthermore, our data demonstrated that this system would be useful to detect a causative pathogen in the diagnosis using oral fluid from healthy pigs and lung tissue from pigs with respiratory disorders collected in the field.

Conclusions: The rapid detection of infected animals from the herd using our system will contribute to infection control and prompt treatment in the field.

Keywords: Actinobacillus pleuropneumoniae; Bordetella bronchiseptica; Haemophilus parasuis; Mycoplasma hyopneumoniae; Pasteurella multocida; Salmonella spp; Streptococcus suis; Aujeszky’s disease virus; classical swine fever virus; diagnosis; multiplex real-time RT-PCR; porcine circovirus 2; porcine circovirus 3; porcine cytomegalovirus; porcine reproductive and respiratory syndrome virus European genotype; porcine reproductive and respiratory syndrome virus North American genotype; porcine respiratory coronavirus; swine influenza virus A; swine respiratory disease.