Establishment of a real-time PCR assay for simultaneously detecting human BKV and CMV DNA and its application in renal transplantation recipients / 病毒学报

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.

Study on KIR gene polymorphisms in 416 renal transplantation recipients from southern Zhejiang / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate polymorphisms of killer cell immunoglobulin-like receptor gene (KIR) in renal transplant recipients from southern Zhejiang.</p><p><b>METHODS</b>KIR genotypes were analyzed by PCR-SSP in 416 renal transplant recipients, and the genotype frequencies were compared with populations from Eastern China and worldwide.</p><p><b>RESULTS</b>All 16 known KIR genes were detected in the renal transplant recipients, and KIR2DL4, 3DL2-3, 3PD1 were found in all. As a pseudogene, 2DP1 has a high genotype frequency (99%). The frequencies of KIR2DL1, 2DL3, 3DL1, 2DS4 have ranged from 92.1% to 98.8%. Compared with 11 groups in Eastern China and other countries, the KIR2DL2 phenotype frequency was higher (34.6%) than those of Shanghai, Zhejiang and Jiangsu populations (P<0.05). Among 41 genotypes, three have not been reported previously. The most common genotype was AA1, with a frequency of 43.51%, which was significantly lower than those of Jiangsu and Northern Zhejiang.</p><p><b>CONCLUSION</b>Renal transplant recipients from southern Zhejiang share similar features with Eastern China Han population with regard to KIR polymorphisms, but also have unique frequencies for KIR genotypes.</p>

Formulation and application of diagnostic models based on clinical biochemical assays in diagnosis of chronic hepatitis and liver cirrhosis associated with viral hepatitis / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To improve the diagnostic ability of routine laboratory items in liver diseases associated with viral hepatitis through constructing assessment models consisting of these items.</p><p><b>METHODS</b>(1) Assessment of routine items and formulation of models. Data of 447 patients seen between May 1997 and August 2003 were collected as the training set and serum specimens of 213 patients taken between June 2004 and March 2005 were examined and used as the validation set. Eleven items (TP, ALB, TBIL, DBIL, ALT, AST, ALP, GGT, TBA, LDH, CHE) were examined with an automated biochemical analyzer. Logistic regression was applied to construct the model for discriminating between chronic hepatitis and liver cirrhosis. The diagnostic value of items and models was assessed by the area under the receiver-operating characteristic (ROC) curve.</p><p><b>RESULTS</b>The model to discrimination between chronic hepatitis and liver cirrhosis consists of five items (CHE, DBIL, ALB, ALT, GLO). The AUCs of model were 0.87 in the training set and 0.83 in validation set, respectively.</p><p><b>CONCLUSION</b>(1) The model consisting of CHE, DBIL, ALB, ALT, GLO improves the diagnostic value of routine laboratory items in discriminating chronic hepatitis from liver cirrhosis.</p>