Establishment and Application of a Dual PCR for Mannheimia haemolytica and Pasteurella multocida in Sheep
Mannheimia haemolytica(Mh)and Pasteurella multocida(Pm)are conditional pathogenic bacteria that could lead to a variety of clinical symptoms,resulting in certain economic loss to sheep industry.In order to rapidly and accurately identify and diagnose the diseases caused by the two bacteria in clinical practice,primers were designed according to Lkt gene of Mh and kmt gene of Pm,respectively,and a PCR assay was established to simultaneously identify and detect the two bacteria within the same reaction system.The results showed that the established method could detect Mh and Pm genomic DNA at the limits of 7.23×10-6 and 6.99×10-5 ng/μL,respectively,was negative to Streptococcus suis,Staphylococcus aureus,Streptococcus agalactiae and Mycoplasma ovis,and could detect positive samples in multiple repeat tests,and by its reaction reagents,fragments with expected stripe sizes could be amplified.95 suspected clinical samples of sheep origin were detected,the detection rates were 38.95%(37/95),53.68%(51/95)and 12.63%(12/95)for Mh,Pm and mixed infection,respectively,which was generally consistent with that by the single PCR assay.In conclusion,the method,with good specificity,sensitivity,reproducibility and stability,could be used for clinical detection as a convenient,rapid and accurate tool for identification and diagnosis of sheep respiratory diseases,indicating a good prospect in clinical practice.
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