Development Of Rapid Detection Technique And Expression Of Rpf Gene Of Micrococcus Luteus

Micrococcus luteus (formerly Micrococcus lysodeikticus) is a specialized aerobic gram-positive bacteria. Skin is considered to be a primary habitat of the bacterium, and it has also beenwidely detected in water and soil. Recently, this organism was recognized as an opportunisticpathogen and has been implicated in not only immunosuppressed patients but also immunosuppressed pigs and birds. However, the research of pathogenicity,drug resistance and assays ofdetection is less. In this paper, the four aspects were done: Micrococcus leteus isolated from theYongzhou, Liuyang, Li Ling, Chaling, and other farms were identified; development of a PCRassay to detect M.leteus; development of LAMP assay to detect M.leteus; expression of rpf geneof M.leteus.A routine systematic pathological check of porkers caused by a opportunistic pathogen, were conducted.The main pathological anatomic changes were characterized swell and hemorrhagein the liver, intestines and lung could also been observed. The histological-pathological changeswere degeneration, necrosis in liver cells and lymph node and in mucous membrane cells ofstomach and intestines;hyperemia and hemorrhage in lung; Punctate hemorrhage in parenchumalorgans such as liver, spleen, inflammatory cells in most tissue were usually observed.Based on the 23SrRNA gene sequence of Micrococcus luteus, a pair of specific primers wasdesigned to detect M.luteus.Through optimizing the amplified condition, a fast,sensitive andaccurate PCR assay was developed and its accuracy and sensitivity were confirmed. The 1326 bpspecific fragment could be amplified with M.luteus standard strain by this assay, but could not beamplified with other bacterial species such as Escherichia coli,Salmonella typhisuis,staphylococcus aureus,Streptococcus suis. 1058 CFU/mL was the lowest detecting limit formixed bacterial solution of M.luteus.A highly specific set of four primers F3,B3,FIP,BIP that recognize a total of six distinctsequences, was designed to target 1125-1323bp region of the 23SrRNA gene from Micrococcusluteus, we optimized the conditions needed for the specific amplification of the 23SrRNA geneand evaluated the specificty an sensitivity of the method. These observations indicate that noamplification of the target 23 SrRNA gene was detected in other related bacteria when the LAMPprimers were used, detection of the clinical samples, the LAMP assay is an extremely rapid, highly sensitive and specific method.A gene-specific primers was designed to amplify the DNA fragment with length of 564bp.The aplified DNA were subcloned into the pET32a plasmid vetor and transformed into thebacterial host BL21(DE3). After sequencing the corresponding clone, we found that thisfragment of this clone was the same as a part of the ORF of rpf. The transformant was efficientlyexpressed.