Establishment Of A Multiplex Nest-PCR Method For Detection Of Three Infectious Pathogens In Abortion Dairy Cattle

In recent years,dairy farming abortion staying at a high level,some regions parts of the cow abortion rate and even can reach above 20%,brings to the farmers is difficult to ignore the economic loss.Therefore,rapidly and accurately detect the cause of cows abortion and pathogen,abortion cows on the prevention and control has very important significance.Through random survey,it was found that Zhoukou(District)various parts cow farms(breeding area)abortion rate is as high as 21%.The cow in the 3-4 fetal abortion rate is significantly higher than other times,abortion rate can reach 16.95 %~25.53 %;Cow miscarriage happens in Spring&Autumn,abortion number accounts for 48.76 % of the total number of abortions(236/484).Neospora caninum,Brucella abortus and Toxoplasma gondii are cow three common causes of miscarriage.In order to detect N.caninum,B.Abortus and T.gondii simultaneously,known as three infectious pathogens in abortion dairy cattle.A multiplex nest-PCR method based on N.caninum ITS1 gene(Nc-ITS1),B.Abortus 16 S rRNA gene(Ba-16 S rRNA)and T.gondii 529 bp repetitive sequence gene(Tg-529 bp)were developed and six specific primers pairs were designed respectively.The result revealed that 601 bp,380 bp,245 bp DNA fragments of Nc-ITS1,Ba-16 S r RNA,Tg-529 bp were amplified by this nest-PCR,respectively.The annealing temperatures were 57 °C for external amplification and 53 °C for internal amplification.The concentration of Taq enzyme was 1.0 U,dNTP was 2.0 mmol/L and Mg+ was 1.0 mmol/L respectively.The primers concentrations of Nc-ITS1,Ba-16 S rRNA and Tg-529 bp gene were 0.4 ?mol/L respectively.The sensitivities of the multiplex nest-PCR were 3×102 copies per reaction for Nc-ITS1 and Ba-16 S r RNA genes,3×101 copies per reaction for Tg-529 bp gene,respectively.Target product was not amplified from Sarcocystis,Escherichia coli,Salmonella,Listeria,Staphylococcus aureus and Streptococcus.The method had good sensitivity and specificity.At the same time using this method with single nested PCR of 30 clinical samples in the diagnosis of miscarriage,the same diagnosis showed that the method has the good application effect in clinical samples.The method was used to detect 172 clinical samples in Zhoukou area,Results showed that the 52 samples were positive for N.caninum,4 samples for B.abortus,2 samples for T.gondii,and 2 sample for both N.caninum and B.Abortus.The results show that the Neosporosis is one of the main causes of cows abortion in zhoukou region,This will provide a valuable reference for further preventing and controling cows abortion in the region.