Application of Fluorescence Quantitative RT-PCR for Live-attenuated Mumps Vaccine Manufacturing / 中国药房

OBJECTIVE:To provide reference for improving the practical application of fluorescence quantitative reverse tran-scription-polymerase chain reaction (RT-PCR) for the live-attenuated mumps vaccine manufacturing. METHODS:A regression equation of standard titer-ct value was established;micro cytopathic effect(micro-CPE)and fluorescence quantitative RT-PCR were respectively adopted for the titer of 10 batches of single harvest liquid of live-attenuated mumps vaccine and end products,and the results were compared. RESULTS:According to the t-test analysis,the P were respectively 0.743 and 0.868,and the average val-ues were in the range of (-0.174,0.094) and (-0.113,0.153),with no significant differences. They were similar. CONCLU-SIONS:Fluorescence quantitative RT-PCR can be used for the titer detection of single harvest liquid and end products in the pro-cess of live-attenuated mumps vaccine manufacturing. And it is expected to further upgrade to Chinese Pharmacopoeia method.

The Analysis of HPV Genotypes and Related High Risk Factors in 459 Women of Annual Health Examination / 天津医药

Objective To investigate the cervical human papillomavirus (HPV) infection, genotypes and related high risk factors in 459 women of annual health examination. Methods A total of 459 cervical cytology specimens were de-tected with nest PCR and pyrosequencing methods. Questionnaires were collected simultaneously and the risk factors of HPV infection were analyzed. Results The detection rate of HPV-positive samples was 17.9%. Seven kinds of high-risk genotypes of HPV were found. The detection of HPV 16 was the most common (9.8%), followed by HPV58 (7.0%) and HPV18 (5.2%). The detection rates of simplex infection, dual infections and multiple infections were 9.6%, 4.8%and 1.5%respectively. The risk factors for HPV infection included alcohol consumption,income 1, frequency of sexual activity>4 times/month and cervical erosion (P<0.05). Conclusion HPV DNA genotyping can provide important reference for HPV screening at early time and the application of HPV vaccines, which also provide sig-nificant evidence for the prevention, diagnosis and treatment of cervical cancer at early stage for the high risk population.

Quantitative Analysis of SMN1 Gene and Estimation of SMN1 Deletion Carrier Frequency in Korean Population based on Real-Time PCR

Spinal muscular atrophy (SMA) is an autosomal recessive disorder, caused by homozygous absence of the survival motor neuron gene (SMN1) in approximately 94% of patients. Since most carriers have only one SMN1 gene copy, several SMN1 quantitative analyses have been used for the SMA carrier detection. We developed a reliable quantitative real-time PCR with SYBR Green I dye and studied 13 patients with SMA and their 24 parents, as well as 326 healthy normal individuals. The copy number of the SMN1 gene was determined by the comparative threshold cycle (Ct) method and albumin was used as a reference gene. The homozygous SMN1 deletion ratio of patients was 0.00 and the hemizygous SMN1 deletion ratio of parents ranged from 0.39 to 0.59. The delta delta Ct ratios of 7 persons among 326 normal individuals were within the carrier range, 0.41-0.57. According to these data, we estimated the carrier and disease prevalence of SMA at 1/47 and 1/8,496 in Korean population, respectively. These data indicated that there would be no much difference in disease prevalence of SMA compared with western countries. Since the prevalence of SMA is higher than other autosomal recessive disorders, the carrier detection method using real-time PCR could be a useful tool for genetic counseling.

THE METHODS DETECTING VASCULAR ENDOTHELIAL GROWTH FACTOR IN HUMAN BREAST CARCINOMAS / 中国现代普通外科进展

Objective: To establish analysis method of vascular endothelial growth Factor(VEGF)for clinic practice.Methods: the expression of VEGF in breast carcinoma tissue is examined with immunohistochemistry, RT-PCR, Western blot and dot blot. Results: the VEGF is revealed mainly in tumor cells and extracellular matrix, molecular weight of VEGF is 46 KD, including three isoforms. In 53% of the cases the tumor tissue reveals stronger expression of VEGF than paratumor tissue. Conclusion :Dot blot may be used as a preliminary method for detecting VEGF in breast carcinoma tissue.

Study of Cytomegalovirus Infection in Renal Transplant Recipients Who had IgG CMV Antibody in the Pretransplantation Period / 대한신장학회잡지

Cytomegalovirus(CMV) infection occurs more frequently in renal transplant recipients than in the normal population. But the incidence and severity of CMV infection and antibody positivity are different between countries. We studied the incidence of CMV infection with a long term follow up in renal transplant recipients who were IgG CMV positive and whose donors were also IgG CMV positive preoperatively. We studied the difference and usefulness of various detection methods including IgM CMV antibody by ELISA, shell vial culture, and quantitative dual polymerase chain reaction(PCR). We also studied possible factors that may affect CMV infection and the function of the grafted kidney in CMV infected patients. This study included 36 patients, 20 males and 16 females, who received renal transplantation at Hanyang University Hospital between July, 1994 and March, 1995. IgG and IgM CMV antibodies were detected and shell vial cultures were performed in recipient candidates and donor candidates preoperatively. Postoperatively, we checked IgM CMV and performed shell vial cultures in renal transplant recipients every month after the operation and when CMV infections were suspected. We also performed dual PCR with endpoint titration to quantify the amount of CMV DNA. The total incidence of CMV infection was 30.6% (11 patients among 36 patients). Three patients had CMV disease, and only one patient was severe enough to need gancyclovir therapy. The average onset of infection was 12.9 weeks after the operation(earliest 5weeks-latest 33weeks). In all patients with CMV disease, CMV positivity appeared by all three detection methods. But detection time and duration of positivity were different. The amount of CMV DNA in patients with active CMV disease was higher than those of asymptomatic patients and one patient following antiviral therapy. Age, sex, donor type, HLA matching and rejection did not affect CMV infection. Incidence of CMV infection was higher in patients who were transfused within 3 weeks before the operation(6/8 vs 5/28, p=0.048). Changes of creatinine level from initial outpatient department(OPD) visit to last OPD visit which were corrected by OPD follow up time showed no significant difference between CMV infected and non-infected patients In conclusion, the incidence of CMV infection and disease was relatively low, and the degree of disease severity was mild. In our renal transplant recipients, CMV infection may not be a serious problem. Quantitative dual PCR using end-point titration is a good method to detect CMV infections and monitor the clinical course. Because it is easy to use, detect disease earlier and can quantify the amount of CMV DNA. Among various factors, transfusion affected CMV infection significantly in our patients. In an average of 231 days of OPD follow up time, CMV infected patients showed no impairment of grafted kidney function, but a more long term follow up is needed.