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
Chinese Journal of Virology
; (6): 410-414, 2013.
Artigo
em Chinês
| WPRIM (Pacífico Ocidental)
| ID: wpr-339936
Biblioteca responsável:
WPRO
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.
Texto completo:
Disponível
Contexto em Saúde:
ODS3 - Meta 3.4 Reduzir as mortes prematuras devido doenças não transmissíveis
Problema de saúde:
Neoplasias do Rim, Pelve Renal e Ureteral
Base de dados:
WPRIM (Pacífico Ocidental)
Assunto principal:
Especificidade da Espécie
/
Fatores de Tempo
/
Infecções Tumorais por Vírus
/
Virologia
/
Sangue
/
DNA Viral
/
Reprodutibilidade dos Testes
/
Sensibilidade e Especificidade
/
Transplante de Rim
/
Tacrolimo
Tipo de estudo:
Estudo diagnóstico
/
Guia de prática clínica
Limite:
Adolescente
/
Adulto
/
Idoso
/
Feminino
/
Humanos
/
Masculino
Idioma:
Chinês
Revista:
Chinese Journal of Virology
Ano de publicação:
2013
Tipo de documento:
Artigo