A Study of Standardization of the In situ PCR ( Polymerase Chain Reaction ) on the Tissues of Borderline Leprosy Patients

Choi, Hae Young

Abstract

BACKGROUND: 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 sita hybridization or immunohistoche mical method.
OBJECTIVE
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.
METHODS
The paraffin-embedded tissues of ten borderline leprosy patients were subjected to in situ 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 concentration of proteinase K such as 10, 15 and 100ug/ml and various incubation time of proteinase K from 3.5 minutes to 15 minutes. Fixation was performed before pretreatment. and after PCR for 15 minutes, after pretreatment and after PCR for 15 minutes, only after pretreatment for 15 minutes or after pretretmant for 60 minutes. The total volume of PCR was 40, 50 or 60ul.
RESULTS
1. The amplified DNA was detected using the HCl-pretreated tissues in four of seven BL patients and one of three BT pat ients. 2. The signals were detected in the cytoplasm of most histiocytes and proliferated Schwann cells, some secretory cells of sweat glands and a 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 in only one tissue used in 10ug/ml of proteinase K for 10 minutes. 4. The proper time for fixation of paraformaldehyde was before treatment of proteinase K or HCl and after PCR reaction. 5. The 40ul of total volume for PCR reaction was enough and minimized the loss of tissues during PCR reaction.
CONCLUSION
When in situ PCR was applied on the paraffin-ernbedded tissues of borderline leprosy patients, pretreatment such as concentration and incubation time of proteinase K or HCL was the most critical parameter.