Study On Association Of Dermatomyositis With Four Types Of Human Herpes Virus

This study is to detect the association of dermatomyositis with four types of human herpes viruse ( HHV) : HHV1 ( herpes simplex virus 1, HSV1) , 2 ( herpes simplex virus 2, HSV2) , 4( Eptein ?Barr virus , EBV) and 5 ( human cytornegalovirus, HCMV) by investigating the occurrence of their DNA sequences in the apparently normal skin biopsies, muscle biopsies and peripheral blood from patients with dermatomyositis.Methods1. Specimens examinedMultiple specimens, which driven from different sites (the apparently normal skin, muscle biopsies and peripheral blood) of two groups of individuals in No. 1 hospital of China Medical University and the Seventh People's Hospital of Shenyang City, were examined.1) The first group was dermatomyositis. Thirty - two apparently normal skin biopsies, twenty - nine muscle biopsies and thirty ?one peripheral blood specimens were collected from forty - two patients, whose diagnosis were confirmed by manifestations, histological findings, elevation of the serum muscle enzyme and electromyographicfeatures of myopathy. The specimens were preserved at - 20@ for DNA extraction.2) The second group was referred to healthy control. Forty - six peripheral blood specimens from healthy donors, while thirty - six normal skin and twenty - six normal muscle biopsies were analyzed.2. DNA extractionUsing phenol - chloroform procedure carried out human genomic DNA isolation. Optical density ( OD) values were measured with spectrophotometry at 260 nm and 280 nm respectively to determine the concentration and purity of the DNA extracted.3. Polymerase chain reaction (PCR) amplificationAll specimens were amplified by different PCR steps with two pairs of primers: one was PC03/PC04 specific to the human B - globulin gene, serving as an internal control; the other was P - 1/P - 2 serving as degenerate primers to amplify DNA fragments of the four types of HHV in samples. The 5uL of each PCR product was resolved on agarose gel containing ethidium bromide. The gels were illuminated with ultraviolet light and analyzed. The internal control's PCR product was 110bp and the purpose PCR products were 518bp, 518bp, 524bp and 589bp corresponding to HSV1, HSV2, EBV and HCMV DNA sequences. HCMV can be distinguished from the other three types by its largest size. Every sample was tested at least twice.4. Retrieval of the positive productsThe 5 uL of PCR mixture containing approximate 520bp fragments were separated in gel. The pcoducts were retrieved with a DNA recovery kit.5. Digestion of the retrieved products with specific restriction en-donucleaseThe virus species were identified by the retrieved products digested through restriction endonuclease ( BamH I and Sma I ) after 8% polyacrylamide gel electrophoresis (PAGE). HSV1 DNA (518bp) can be cleaved into two fragments sized 476bp and 42bp by Sma I , rather than BamH I . BamH I , rather than Sma I , can digest HSV2 DNA (518bp) into 255bp and 293bp. EBV can be cleaved by both BamH I and Sma I , with the digestive fragments of 424 bp and 100bp, 247bp and 277bp respectively.6. Control specimensAppropriate positive control specimens were used throughout the study. DNA, extracted from HSV1 infected cell line and co - infected by EBV and HHV8 cell line, served as the positive controls for HSV1 and EBV. Negative control was using the sterilized water instead of DNA as template.7. Statistical analysisThe significance of the differences in positive rates of four types of HHV between the dermatomyositis and normal control were determined by the continuity correction chi - square ( X2 ) test. The analysis was performed with spss 11.0 statistical software.Results1. The amplification of B- globulin gene DNA was positive in all samples.2. DNA, extracted from HSV1 infected cell line and EBV and HHV8 co - infected cell line, were PCR amplified with P - 1/P -2 primers and approximate 520bp products were obtained.3. There were no PCR products in negative control.4. The presence of EBV D...