A Novel GeXP Multiplex PCR Method For The Simultaneous Detection Of Nine Swine Pathogens

Porcine circovirus type II, classical swine fever virus, Porcine reproductive and respiratory syndrome virus, vesicular stomatitis virus, swine vesicular disease virus,porcine transmissible gastroenteritis virus, Mycoplasma hyopneumoniae, Haemophilus parasuis and Actinobacillus pleuropneumoniae bacilli are more common In clinic. They are contagious, widespread, very especially harmful, and not only greatly affect and restrict the development of China’s pig industry, but also cause a huge impact on public health and safety. The nine pathogens often occur simultaneously, and these clinical symptom are too complex to distinguish with the current detection methods. The GenomeLab GeXP Genetic Analysis System provides an alternative technology for multiplex quamtitative gene expression. In the method the combination of the GeXP multiplex PCR and the capillary electrophoresis technology simplifies the reaction steps and improve sensitivity, specificity and throughput by the use of a pair of universal primers fluorescently labeledand and nine pairs of specific chimeric primers.In this study, according to the target gene sequence of PCV-2, CSFV, PRRSV, VSV,SVDV, TGE, M.hyo, HPS and APP in NCBI, we design nine pairs of specific primers after analysis the conserved sequence of these genes. Then verify the specificity of these primers by the conventional PCR in the single primer single template system and single primer mix templates system.. While we successfully construct recombinant plasmids of the PCR products and sequence these plasmids by GeXP. The results showed that these primers had high specificity and there were more than 99.5 % identy between the target genes and the NCBI published sequences. Subsequently we design two two sets of GeXP primer pairs including one pair of cy5 fluorescent-labeled universal primer and nine pairs of specific chimeric primers, and verify their specificity and sensitivity in the GeXP single and multiplex PCR system. The results show that the detection limit of nine kinds of pathogens are 102 copies / μL, at the considerable level of the Lamp method. By optimizing the GeXP system, we find the optimal Tm value is 58.5 ℃ and the optimum dosage of the specific primers is 0.5μL. After the optimization, the stability of the amplification system is greatlyincreased. Finally, we detect other 12 disease pathogens using the method, not found non-specific amplification, further proof that the system has a high specificity。The positive rate of GeXP multiplex PCR assay of the 27 samples was 6.4%,which is consistent with the actual results. So the GeXP multiplex PCR method has a high accuracy and a strong clinical value.