Establishment Of Blood Direct PCR For M.Suis Assay And Screening Natural Compounds With Anti-M.Suis Activity In Vitro

Objective:Establish a method used to detect Mycomplasma suis (M.suis) and screen natural compounds which are able to kill Mycomplasma suis.Method:Blood direct PCR method was established to detect M.suis. The best dissolvent was screened by using water, DMSO or Anhydrous ethanol to dilute natural compounds. Visualization of cytopathologic effect assay and MTT method were used to determine the maximum no-cytotoxic concentration of natural compounds in PK-15 cell culture system. Natural compounds were added to positive blood infected M.suis and blood direct PCR method was applied to amplify the specific gene segments of M.suis at different times. The gray values of electrophoretic bands were used to preliminarily assess the anti.-M.suis activity of tested natural compounds. The qPCR method was used to detect the M.suis copy numbers of positive blood that interacted with valid compounds at different times and the killing ratio was calculated to evaluate the anti-M.suis activity of candidates.Results:(1) A 396 bp DNA fragment of the 16S rRNA gene from M.suis had been amplified by blood direct PCR. No PCR products were amplified from the samples of PRRSV, PCV2, PPV, PRV, E.coli and S.aureus. The sensitivity of blood direct PCR was equivalent to that of conventional PCR. The difference in detecting blood sample from swine by blood direct PCR and conventional PCR was not significant(p> 0.05).(2) The best dissolvents of natural compounds had been screened and the maximum no-cytotoxic concentration had been determined in PK-15 cell culture system.(3) Five anti-M.suis natural compounds in vitro had been preliminarily screened by using blood direct PCR. They were Dipotassium Glycyrrhizinate, Sodium Tanshinone ⅡA sulfonate, Aesculin, Baicalin and Cinnamic Acid.(4) With the increased of reaction time, the killing ratio of five valid compounds was rising significantly. After reacted 18 hours, the killing ratio of Sodium Tanshinone IIA sulfonate, Dipotassium Glycyrrhizinate and Aesculin had reached at 76.9%,90.6% and 79.2%, respectively.Conclution:(1) The blood direct PCR method had been developed for the detection of M.suis infection.(2) There were five natural compounds possessed anti-M.suis activity had been screened in vitro. Sodium Tanshinone ⅡA sulfonate, Dipotassium Glycyrrhizinate and Aesculin may be the ideal anti-M.suis drugs because the killing ratio had reached above 70% after reacted 18 hours.