Establishment And Preliminary Application Of Detection Method For Mycoplasma Hyopenumoniae By In Situ Hybridization

Mycoplasma pneumoniae of Swine (MPS) caused by Mycoplasma hyopneumoniae (Mhp) is a contagious respiratory disease characterized by chronic cough, growth retardation, low mortality and high morbidity. Mhp can combine closely with the ciliated epithelial cells of porcine respiratory tract and make the cilia amotic. Currently, PCR and EILSA method are used to detect antigen and antibodies in general laboratory detection. However, these methods can not locate Mhp in tissue. In situ hybridization histochemistry (ISHH), one of the most effective technologies in molecular pathology and molecular genetics, can detect and locate specific target DNA or RNA, and show its status and morphological characteristics in tissue.In this experiment, a25bp probe labeled by digoxin were designed according to the P36genome of Mhp published in GenBank. The result of dot blot hybridization shown that the probe is sensitive, specific and widely applicable with different Mhp strains. The further research of ISHH method for Mhp detection on porcine respiratory tract and lung tissue was performed. The reaction conditions such as pepsin digestion time, probe concentration and colored time were optimized. The dynamic distribution of the attenuated vaccine strain or field strain of Mhp on different time post-immunization or post-challenge was detected by the developed ISHH method.The developed ISHH method for Mhp detection and localization in paraffin e--mbedded lung tissue from pigs infected or immunized with Mhp strain is aval-uable technique for the pathogenesis study of Mhp infection.