Abstract Current detection technologies for diagnosis of animal diseases is mostly targeted at single pathogen, but the prevalence of animal diseases is characterized by mixed infection with more than one pathogen. In order to resolve the trouble, here we describe the new multiparameter assay, which is based on multiplex ligation-dependent probe amplification(MLPA) technology with the advantages of specificity, sensitivity and high-throughput. Five pairs of specific probe targeted at Swine influenza virus(SIV), Pseudorabies virus(PRV), Foot and mouth disease virus (FMDV), Transmissible gastroenteritis virus (TGEV) and Porcine reproductive and respiratory syndrome virus (PRRSV), were designed, respectiviely. The mixture of five standard RNA/DNA was used as template, together with the mixture of these probes as probe and the PCR universal primer, one MLPA method for simultaneous detection of the five porcine viruses was developed. The result of specificity test showed that the designed probes had good specificity without mismatch between each virus-specific probe pair and other six viruses, and that the mixture of the five pairs of probe only amplified the corresponding one specific fragment from the eight virus templates, respectively, no amplification signal was produced among Porcine parvovirus(PPV), Classical swine fever virus(CSFV) and Porcine epidemic diarrhea virus(PEDV) with the same probe mixture. The result of sensitivity test showed that the concentration of nucleic acid of single virus in one MLPA reaction was up to 3 000~6 000 copies. All the results showed that the developed MLPA method in this article accomplished the simultaneous detection of five viruses in one reaction, which indicates MLPA technology may be an alternative to simultaneous detection of many pathogens in the future in the field of veterinary medicine.
