The Methodological Study Of Detection And Removing Of Inhibitors In Polymerase Chain Reaction

Objectives: Malignant lymphoma has been a difficulty in pathological diagnosis. As a general technique in genie diagnosis, polymerase chain reaction (PCR) has been applied in various aspects. In recent years, PCR for rearrangentment of genes has become an essential step in diagnosis of lymphoma. However, false negetive result is a common event in PCR and has been ignored frequently. Especially the presence of inhibitors may be the reasons resulted to false negtive in PCR. At present, the issue of inhibitors has no systematic studies at home. So, how to detect the presence of inhibitors in templates and how to eliminate its function become the aims of this study. In addition, the effects of partial reagents and different fixatives on PCR are the objectives of this study meanwhile.Methods: (1) The effective measure to demonstrate inhibitors in extractives of DNA is to build an internal control indication systeam. Construct a recombined plasmid containing β -globin gene fraction as a template in PCR (internal control ).Screen negative samples from fresh and embedded tissues and detect if it contains inhibitors. (2) With DNA of lymphoma DG75 as template in PCR.Select regents 10%formalin, 95%ethanol,xylene,Tris.HCl, EDTA,phenol, chloroform, 2%SDS and-5-dilute into 1:10, 1:50, 1:100, l:1000,the four different concentration. Place 1 microlitre of every the regents (including the solution not be diluted) into a 20 microlitre systeam of PCR and observe effects resulted from these substances.(3) 16 samples obtained from simple tonsillitis fresh tissue were sliced up in frozen conditions. Each, of them was divided into 25 parts with 2.5cmX 1.5cmX4u m, and fixed for 4h,12h,24h,72h,and 120h with four different fixatives and O.IM PBS as the control at random. Extracted DNA through proteinase-K digestion method, the ratio of OD260nn/OD280nm, the concentration of DNA and the results of PCR was compared.Then select the best fixative and fixation time.(4)In the first part of experiments, after all samples be amplified twice,we select negative extractives as a substrate of some pretreatments such as boiling, dilution, BSA incubation,endonuclease digestion and observe results of these pretreatments on these negative samples.Results:(1) The positive ratio of 27 fresh tissues is 88.9%(24/27).The positive ratio of 65 paraffin-wax embedded tissues is 78.5%(51/65). However, the positive ratio of lymphoma tissues new-prepared is 89.3%(25/28) and is higher than the sections prepared two years ago 40%(6/15). Within 17 false negative samples , 13 inhibited 3 -globin amplification of a recombined plasmid, account for 76.5%; (2) 10% formalin, hematoxylin, eosin, EDTA, phenol, chloroform, 2%SDS have effects of inhibition on PCR, but 95% ethanol, Tris.HCl, xylene have no obvious effects;(3) With the increases of fix time, the ratio of positive results of samples fixed with 10%formalin has turn down to 66.7%,but the ratio of positive results of samples fixed with 95% ethanol is-6-100%;(4)After all means mentioned above, the results of 14 samples turned into positive in 17 negative samples and the whole efficiency is 82.4%.Conclusions: (l)That the presence of inhibitor(s) is the primary reason for a false positive result; (2) Some reagents such as formalin, hematoxylin, eosin ,EDTA, phenol, chloroform, 2%SDS have inhibition function indeed;(3) After fixed with four different fixatives,we found 95 % ethanol was the best fixative in the amplification of an objective fragment in fresh lymphoid tissue. Especially, the extractives of DNA fixed with 10% formalin perhaps have inhibitors in different concentration and the proper fixation time is less than 72h. (4) BSA pretreatment is the best method for eliminating of inhibition in PCR.