| Objectives To compare the sensitivity and specificity of different PCR methods in detecting tubercle bacillus DNA in pathologic tissue embedded in paraffin. Chose the best method for differential diagnosis on atypical tuberculosis and sarcoidosis, then evaluating its clinical application value.Methods 1. Comparison and establishment of the best PCR method: 1). Choose the specimens: tissue embedded in paraffin from 68 typical tuberculosis and sarcoidosis patients whose diagnosis had been confirmed and they have recovered now. Of them, 37 specimens were tuberculosis with caseous necrosis, 31 specimens were from sarcoidosis patients. 2). Use three different PCR methods to amplify tubercle bacillus DNA: (1) indirect nest-PCR in situ: select the confirmed patients'specimen slices,2 pairs of primers (internal & external) were designed, with IS6110 being the target sequence. Amplify in situ 1 cycle with the external primers, and then amplify another cycle with the internal primers. Hybridize in situ with digoxin-marked probe, and examine the results by horseradish peroxidase (HRP) examination system, negative and positive controls were set. (2) Liquid nest-PCR: extract the DNA in specimen mentioned above by hydroxybenzene-chloroform extraction method, then amplify 2 cycles with the above primers in the eppendorf tubes, examine by gel-electrophoresis. Negative and positive controls were set. (3) Usual liquid PCR: Amplify the extracted template 1 cycle with the internal primers mentioned above, examined by gel-electrophoresis. Negative and positive controls were set. 3). Compare the sensitivity and specificity of three different PCR methods, considering the clinical facts, choose the best method. 2. to detect the atypical tuberculosis and sarcoidosis pathologic specimens by nest-PCR in situ, which have been selected for its performance superiority: 1). Choose the specimens: tissue embedded in paraffin. 30 from tuberculosis patients with atypical pathology whose... |
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