Detection Of Immune Function Of Congenital HCMV Latently Infected Balb/c Mice And Research Of Truncated Pp65 Antigen Application In Detection Of Specimen Of Kidney Transplantation

Objective Part 1 Detection of immune function of congenital HCMV latently infected Balb / c mice:On the foundation of the successful established mouse model of innate HCMV latent infection,observe the infection phase of HCMV and the status of immune function by test the peripheral blood of mouse, in order to search for application value of mouse model with correlated clinical disease . Part 2 Research of truncated pp65 antigen application in detection of specimen of kidney transplantation: On HCMV infection in renal transplant patients tested, and test kit of this room made pp65ELISA specificity and sensitivity, evaluate its practical value clinically.Methods Part 1:Three groups mouse with innate HCMV latent infection (A group), HCMV reactivation infection (B group) or HF cell control (C group) were selected randomly, every group includes 6 mouses aged 18~20 months. After mouses are killed by bleeding from mouse eyepit, peripheral blood of mouses were tested by following technical: (1) How HCMV infection estate of these mouse was validated by virus separation in base of cells co-cultivation of HF, gene testing through PCR and RT-PCR. (2) HCMV IgG,IgM of mouse serum were tested by indirect enzyme linked immunosorbent assay; T lymphocyte subgroups including CD3+/CD4+/CD8+ molecule in peripheral blood were tested by flow cell meter. Part 2:From the First Affiliated Hospital of Anhui Medical University and the Armed Police Hospital collected 77 patients after renal transplantation in urine specimens, 78 blood samples, according to experimental design were treated as follows: (1) HCMV virus isolation: The samples were inoculated into human embryonic fibroblasts (HF), the observation of cytopathic effect (Cytopathic effect, CPE); (2) by PCR test specimens HCMV IE, UL54 and UL83 gene; (3) by indirect ELISA checkerboard titration of various purity pp65 antigen screening specificity; (4) were screened using the pp65 protein assembly of the ELISA kit, Zhuhai Haitai ELISA kit, and Italy DIESS kit plasma samples of 77 patients; (5), data processing, assembly, respectively, pp65 ELISA reagents box test results were compared with the genetic test results to analyze whether the difference between the two. And international well-known brand kit Italy DIESS kit as the reference, compare the room pp65 kit and a kit made its specificity, sensitivity, the same rate.Result Part 1:The results of relativity between HCMV innate latent infection and immune foundation of mouse showed following: (1) Infection state of HCMV in mouse was identified. Result showed that mouses of A group were infected latently by HCMV, mouses of B group were infected actively by HCMV. These results were testified by PCR and RT-PCR. PCR of genomic DNA of the mouses could detect HCMV UL83 gene, but RT- PCR can not detect HCMV UL83 mRNA. The results of PCR and RT-PCR were positive. (2) Detection of imunne estate showed following: Value of HCMV IgG in group A/B/C mouse shown that group A/B were positive while group C was negative; Value of HCMV IgM in group A/B/C mouse shown that group B was positive while group B/C were negative; Detection of T limphocyte subgroup find quantity of CD3+ T limphocyte was 33.05%±3.39%,6.58%±3.38%and 4 3.18%±1.67% serveraly in peripheral blood from A/B/C group mouse, and difference between every group was significative (p<0.01). Quantity of CD4+ is 66.08%±1.618%,59.98%±2.29% and 65.78%±2.17%, serveraly in peripheral blood from A/B/C group mouse, difference compared with A group and B group mouse was significative (p<0.01). But different between A group and C group was not significative (p>0.05). Quantity of CD8+ T limphocyte was 23.26%±2.99%,11.50%±2.79%,18.61%±0.94% serveraly in peripheral blood from A/B/C group mouse, and difference between any two groups was significative(P<0.01).Part 2:(1) HCMV virus isolation results were negative. (2) 77 renal transplant patients blood samples HCMV PCR detection of HCMV IE, 67 were positive, the positive rate was 87.0%; HCMV UL54 total of nine cases, the positive rate was 11.7%, HCMV UL83 total of 10 cases, the positive rate 13.0%. 78 HCMV UL83 PCR testing of urine samples, 13 samples were positive, the positive rate was 16.7%. (3) the board after the titration that only pure showed pp65 as an antigen-specific responses, the optimal dilution was 1:40. (4) The results of each group by chi-square test detected no significant differences appear consistent to some extent. And the Italian DIESS kit, as a reference HCMV IgM kit are Haitai sensitivity of 7.7% and the specificity was 98.4%; the room pp65 HCMV IgM sensitivity was 15.4%, specificity of 72.0%.Conclusion (1)Due to the use of high-dose immunosuppression, reactivation of infection in mice showed immunosuppression, and immune suppression in the mice groups, polished truncated antigen pp65 was used as antigen ELISA test results and virus isolation and PCR, RT-PCR were completely consistent. That polished truncated pp65 antigen for HCMV IgG, IgM detection.(2)pure as the post-pp65 antigen ELISA detection of various types of samples, the sensitivity of HCMV IgM was 15.4%, higher than similar domestic double kit; the specificity was 72.0%, compared with 98.4% of similar products low.