In this assay, we developed and evaluated a multiplex PCR (mPCR) for its ability in detecting multiple infections of swine simultaneously. Four pairs of primers were used to detect five viruses. Specific primers were designed for classical swine fever virus (CSFV), African swine fever virus (ASFV) and pseudorabies (PRV). A pair of primers was designed prudently for two different types of porcine reproductive and respiratory syndrome virus that respectively were porcine reproductive and respiratory syndrome virus (PRRSV), highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). The detection limits of the mPCR were 1. A total of 49 clinical specimens were tested by the mPCR, and the result showed that co-infection by two or three viruses was 51%. In conclusion, the PCR is a useful tool for clinical diagnosis of not only single infections but also mixed infections in swines.

Aim:  The main objective of our study is to develop a reverse transcriptase loop‐mediated isothermal amplification (RT‐LAMP)‐based system for rapid and specific detection of H3 swine influenza virus (SIV). Methods and Results:  The system, H3 RT‐LAMP, contained a set of six novel primers that targeted eight distinct regions of the viral haemagglutinin (HA) gene that are highly conserved among H3 influenza A viruses but not between H3 and other subtypes. H3 RT‐LAMP accurately and specifically detected H3 SIV of different isolates from culture and from swine lung samples. The system is at least 10‐fold more sensitive than the conventional RT‐PCR assay and even comparable to the real‐time RT‐PCR method, with the detection limit of about one plaque‐forming unit per reaction. Of 27 swine lung samples tested, 11 samples were positive in reactions with the RT‐LAMP and real‐time RT‐PCR methods, while only 7 were positive with the conventional RT‐PCR assay. Importantly, the assay can be completed within 45 min and is faster than the conventional RT‐PCR and real‐time RT‐PCR approaches. Conclusions:  Our results provide the first direct evidence that RT‐LAMP is highly specific and sensitive for detecting H3 SIV. Significance and Impact of the Study:  These results suggest that LAMP offers a promising alternative tool for rapid, inexpensive and specific diagnosis of influenza virus infection of swine and other animals in frontline settings.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important swine pathogen, causing huge economic losses each year worldwide. Immunization with vaccines containing the glycoprotein 5 (GP5) of PRRSV is the main measure to induce neutralizing antibodies and control the disease. Here, we developed a GP5 protein-based ELISA for detecting antibodies against PRRSV. The overall yield of purified GP5 in E. coli flask culture was more than 45 mg/L cell culture. Western blot and IFA indicated that the GP5 protein was highly immunogenic. After optimization and validation with IDEXX PRRS using 566 clinical sera, the DSN, DSP, and accuracy of GP5-ELISA were 81.39%, 75.96%, and 80.39%, respectively. Besides, GP5-ELISA is highly specific, showing no cross-reactions with sera against other important swine pathogens. Hence, GP5 is a good diagnostic antigen and the GP5 protein-based ELISA has the potential to be used in the field.