ABSTRACT
To establish a fluorescent quantitative PCR method (FQ-PCR) with TaqMan probe for simultaneous detection of polyomavirus (BKV) and cytomegalovirus (CMV) and to evaluate its clinical application in the renal transplantation recipients. The conservative sequences of BKV and CMV were targeted and amplified by nested PCR technique. The PCR products were cloned into the plasmids pcDNA3. 1(+). The recombinant plasmid containing target sequences of BKV and CMV were constructed as external standards. The TaqMan-based assay was optimized. For evaluating the assay, the sensitivity was determinated by diluted standard (5 X 103-10icopies/mL), and the specificity was verified by negative control and positive control, and the precision was assessed by intra-assay coefficient of variation (ICV) through detecting standard repeatedly (20 times). A total of 480 blood samples of renal transplantation recipients were used to detect BKV and CMV DNA simultaneously with FQ-PCR, and the concentrations of FK506 were measured by ELISA. The association of DNA copy and concentrations of FK506 was analyzed. The cloned target BKV and CMV DNA was confirmed by sequencing and analysis. The sensitivity of the FQ-PCR assay reached 5 X 103 copies/ml in detecting BKV or CMV DNA. Control DNA verified the assay specifically detecting target DNA. The precision of the assay to quantif target DNA copies was acceptable (Intra-assay CV was 3.44% for BKV and 2.23% for CMV; Inter-assay CV was 4. 98% for BKV and 3.76% for CMV;). Of 480 samples, 130 samples (27. 08%) were CMV DNA positive, significantly higher than the BKV DNA positive (13.33%, 64/480, P<0.05). The positive BKV or CMV DNA was found to be associated with high concentrations of FK506 (P<0. 05). In conclusion, the developed real-time PCR assay for detecting both CMV and BKV DNA simultaneously was s high sensitive, precise and time-effectiveand could be applied in the monitoring of the CMV and BKV infection in the renal transplantation recipients.
Subject(s)
Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Conserved Sequence , Cytomegalovirus , Genetics , Cytomegalovirus Infections , Diagnosis , Virology , DNA, Viral , Blood , Immunosuppressive Agents , Blood , Kidney Transplantation , Polyomavirus , Genetics , Polyomavirus Infections , Diagnosis , Virology , Real-Time Polymerase Chain Reaction , Methods , Reproducibility of Results , Sensitivity and Specificity , Species Specificity , Tacrolimus , Blood , Time Factors , Tumor Virus Infections , Diagnosis , Virology , Viral LoadABSTRACT
<p><b>OBJECTIVE</b>To investigate the application of ABO blood group genotyping in complicated ABO blood group type.</p><p><b>METHODS</b>Ten specimens of complicated ABO blood group were genotyped by sequence specific primer PCR (PCR -SSP), and confirmed by DNA sequencing and alignment. Six hundred and ten blood samples typed by ABO immunoassay were as control of genotyping.</p><p><b>RESULTS</b>Ten cases of complicated blood type were identified by high resolution PCR- SSP as rare ABO blood groups: cis-AB01 (3 cases), B(A)04 (2 cases), cisAB02, B(A)02, Bel03, Bw12 and Ael05, confirmed by DNA sequencing. Genotyping and serotype detected 610 cases ABO blood group were coincident, and the frequency of A, B, AB and O were as 28.69%, 27.54%, 8.2% and 35.57% respectively. According to the genotypes, the highest frequency subgroup was O1 (32.87%), the lowest was A2 (0.66%).</p><p><b>CONCLUSION</b>PCR -SSP could type the ABO blood group accurately, but also the sub-group of blood type. However, special designed high resolution PCR -SSP or DNA sequencing is needed to identify the complicated blood groups.</p>
Subject(s)
Humans , ABO Blood-Group System , Genetics , Blood Grouping and Crossmatching , Methods , Genotype , Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
<p><b>OBJECTIVE</b>To detect the velocity and viscosity of blood flow of the vertebral arteries, the apoptotic cell and apoptotic related protein in the brain in order to offer theoretical foundation for the treatment of cervical spondylopathy of the vertebral artery type with the eliminat dampness resolv phlegm method.</p><p><b>METHODS</b>Sixty male Japanese big ear rabbits were divided randomly into normal sodium group (A), Flunarizine group (B), low dosage Wendantang group (C), large dosage Wendantang group (D), Flunarizine group combined with large dosage Wendantang group (E), normal group (F). Each group had 10 rabbits. Xiaozhiling injection was injected around the vertebral arteries of rabbits in group A, B C, D, E to make the model of the cervical spondylopathy of the vertebral artery type. Normal sodium (20 ml x kg(-1)d(-1)) was apply through intragastric administration in group A, F; Flunarizine (0.8 mg x kg(-1)d(-1)), low dosage Wendantang (1 g x ml(-1)d(-1)), large dosage Wendantang (2 g x ml(-1)d(-1)), Flunarizine combined with large dosage Wendantang were respectively apply through intragastric administration in group B, C, D, E. The velocity and viscosity of blood flow of the vertebral arteries, the apoptotic cell and apoptotic protein in the brain were detected before and after the treatment.</p><p><b>RESULTS</b>Satisfactory animal model were obtained in group A, B, C, D, E. The rabbits of group E had the most improvement of the velocity and viscosity of blood flow of the vertebral arteries while at meantime, which had the lowest apoptotic index and apoptotic related protein expression in the brain.</p><p><b>CONCLUSION</b>The routine treatment for the cervical spondylopathy of the vertebral artery combined with eliminat dampness resolv phlegm method could improve velocity and viscosity of blood flow of the vertebral arteries, which maybe relate with reduction of apoptosis in the brain.</p>