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KMID : 0363219960340030415
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Korean Journal of Dermatology
1996 Volume.34 No. 3 p.415 ~ p.421
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A Study of Standardization of the In situ PCR(Polymerase Chain Reaction) on the Tissues of Borderline Leprosy Patients
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Abstract
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Background :
@EN PCR from paraffin-embedded tissues has been recently applied on the diagnosis of leprosy. PCR is a highly sensitive technique and the amplified product is detected by gel electrophoresis and Southern blot hybridization. But conventional PCR
does not
give an information about histopathologic features. On the other hand in situ PCR informs the histopathological location of DNA amplified inside the cells and detected by in situ hybridization or immunohistochemical method.
@ES Objective :
@EN In situ PCR was applied on the paraffin-embedded tissues of borderline leprosy patients. The optimal condition of in situ PCR in leprosy was searched with the parameters of pretreatment of the tissues. Fixation time of paraformaldehyde and
total
volume of PCR.
@ES Methods :
@EN The parffin-embeddded tissues of then borderline leprosy patients were subjected to in sutu PCR. Amplified DNA product within the cells was incorporated with Digoxigenin and was directly detected by immunohistochemical method using
anti-Dig-alkaline
phosphatase conjugated antibody. The tissues were pretreated with 0.2N HCl or various oncentration of proteinase k such as 10. 15 and 100μg/ml and various incubation time of proteinase K from 3.5 minutes to 15 minutes. Fixation was performed
before
pretreatment and after PCR fort 15 minutes, after pretreatment and after PCR for 15 minutes, only after pretreatmetn for 15 minutes or after pretretmant for 60 minutes. The total volume of PCR was 40, 50 or 60μ.
@ES Results :
@EN 1. The amplified DNA was detected using the HCCl-pretreated tissues in four of seven BL patients and one of three BT patients.
2. The signals were detectedin the cytoplasm of most histiocytes and proliferated Schwann cells, some secretory cells of sweat glands and as few endothelial cells in the tissues of the BL patients and the cells composing of the granuloma in
those
of
the BT patients.
3. Proteinase K-treated tissues showed positive reaction only one tissue used in 10μg/ml of proteinase K for 10 minutes.
4. The proper time for fixation of parafoumaldehyde was before treatment of proteinase K or HCl and after PCR reaction.
5. The 40μ of total volume for PCR reaction was enough and minimized the loss of tissues during PCR reaction.
@ES Conclusion :
@EN When in situ PCR was applied on the paraffin-embedded tissues of borderline leprosy patients, pretreatment such as concentration and incubation time of proteinase K or HCL was the most critical parameter. (Kor J Dermatol 1996;34(3) : 415~421)
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KEYWORD
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