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Huang J.,Chinese National Institute for the Control of Pharmaceutical and Biological Products | Gao C.,Soochow University of China | Ding X.,Peking Union Medical College | Qu S.,Chinese National Institute for the Control of Pharmaceutical and Biological Products | And 6 more authors.
Cancer Biomarkers | Year: 2012

Aim: For full-scale analysis of Human Papillomavirus (HPV) status in humans, two minor groove binder (MGB)-based one-step multiplex real-time PCR systems were developed: one to screen 16 high-risk HPV (HR-HPV) types, and one to screen a broader spectrum of HPV types (common HPV or C-HPV). Methods: Sensitivity and specificity were evaluated using diluted reference plasmids and 20 control human DNA samples. For clinical evaluation, 510 cervical scrape samples were evaluated. Results: The sensitivity assays revealed that the C-HPV detection system could detect 10 ∼ 100 plasmid copies/reaction, while the HR-HPV detection system detected 10 ∼ 500 copies. The specificity test revealed that the systems did not yield positive signals from the 20 human genomic DNA samples. Performance was tested on 510 usable clinical samples. The HR-HPV Results were compared to those from the Hybrid Capture 2 (HC2) test, which assesses 13 HR-HPV types; the concordance level between the two Methods was 90.8% with a kappa value of 0.813. Conclusions: These Results showed that our novel MGB-based one-step multiplex real-time PCR method may be used for the diagnosis and mass screening of HPV in clinical and large-scale epidemiological studies. © 2013-IOS Press and the authors. All rights reserved.

Chen Y.,CAS Institute of Microbiology | Chen Y.,CAS Beijing Institute of Genomics | Zhang H.,Capital Medical University | Xiao X.,Capital Medical University | And 8 more authors.
International Journal of Cardiology | Year: 2013

Background Peripheral blood-based gene expression patterns have been investigated as biomarkers to monitor the immune system and rule out rejection after heart transplantation. Recent advances in the high-throughput deep sequencing (HTS) technologies provide new leads in transcriptome analysis. Methods By performing Solexa/Illumina's digital gene expression (DGE) profiling, we analyzed gene expression profiles of PBMCs from 6 quiescent (grade 0) and 6 rejection (grade 2R&3R) heart transplant recipients at more than 6 months after transplantation. Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR) was carried out in an independent validation cohort of 47 individuals from three rejection groups (ISHLT, grade 0,1R, 2R&3R). Results Through DGE sequencing and qPCR validation, 10 genes were identified as informative genes for detection of cardiac transplant rejection. A further clustering analysis showed that the 10 genes were not only effective for distinguishing patients with acute cardiac allograft rejection, but also informative for discriminating patients with renal allograft rejection based on both blood and biopsy samples. Moreover, PPI network analysis revealed that the 10 genes were connected to each other within a short interaction distance. Conclusions We proposed a 10-gene signature for heart transplant patients at high-risk of developing severe rejection, which was found to be effective as well in other organ transplant. Moreover, we supposed that these genes function systematically as biomarkers in long-time allograft rejection. Further validation in broad transplant population would be required before the non-invasive biomarkers can be generally utilized to predict the risk of transplant rejection. © 2013 Elsevier Ireland Ltd. All rights reserved.

Liu L.,Chinese Institute of Microbiology and Epidemiology | Sun Y.,China Mobile | Kargbo B.,Ministry of Health and Sanitation | Zhang C.,National Institutes for Food and Drug Control | And 16 more authors.
Journal of Virological Methods | Year: 2015

During the 2014 Ebola virus disease (EVD) outbreak, a real-time quantitative polymerase chain reaction was established to detect and identify the Zaire Ebola virus. We describe the use of this assay to screen 315 clinical samples from EVD suspected person in Sierra Leone. The detection rate in blood samples was 77.81% (207/266), and there were relatively higher detection rate (79.32% and 81.42%, respectively) during the first two weeks after onset of symptoms. In the two weeks that followed, the detection rate declined to 66.67% and 25.00%, respectively. There was the highest virus load at the first week and then decreased. The detection rate in swab samples was 89.79% (44/49). This may be benefit from the included patients. 46 of 49 swab samples were collected from died patients. Taken together, the results presented here indicate that the assay specifically and sensitively detects Zaire Ebola virus. © 2015 Elsevier B.V.

Fan X.,PLA Fourth Military Medical University | Jiang J.,CAS Beijing Institute of Genomics | Liu Y.,Changchun University of Chinese Medicine | Huang X.,U.S. Center for Disease Control and Prevention | And 7 more authors.
Virus Genes | Year: 2013

During 2009, an outbreak of hand, foot, and mouth disease (HFMD) enrolled 490 people in Henan Province, causing the death of two children. In order to investigate the pathogens responsible for this outbreak and characterize their genetic characteristics, a total of 508 clinical specimens (stool, throat swab, and vesicle fluid) were collected from the Center for Disease Control and Prevention of Henan Province. Virological investigations (virus isolation, conventional reverse transcription PCR, and real-time reverse transcription PCR) and phylogenetic analysis were performed. It was found that human enterovirus 71 (EV71) was the main pathogen causing this outbreak, while Coxsackievirus A16 (CoxA16) played only a subsidiary role. Phylogenetic analysis of 24 EV71 isolates collected during the period from March 11 to July 24, 2009 showed that they belonged to subgenotypes C4 and C5. Our study for the first time characterizes the epidemiology of HFMD and EV71 infection in Henan Province in 2009 and provides the first direct evidence of the genotype of EV71 circulating in Henan Province at that time. Our study should facilitate the development of public health measures for the control and prevention of HFMD and EV71 infection in at-risk individuals in China. © 2012 Springer Science+Business Media, LLC.

Zhao J.,CAS Beijing Institute of Genomics | Zhao J.,Peking Union Medical College | Huang J.,Chinese National Institute for the Control of Pharmaceutical and Biological Products | Chen Y.,CAS Beijing Institute of Genomics | And 10 more authors.
Lung Cancer | Year: 2011

Background: For the rapid and sensitive screening of epidermal growth factor receptor (EGFR) hot-spot mutations, we developed a novel method combining mutant-enriched PCR with amplification refractory mutation system (ARMS) TaqMan real-time PCR in a one-step reaction tube. Methods and results: We designed two pairs of primers to enrich and genotyping each mutation (E746_A750del and L858R): nest primers and ARMS primers. Before the PCR assays were carried out, the restriction enzymes were used to cut wild alleles. The results showed that this method could detect mutant alleles mixed samples containing 0.1% with a cutoff ΔCt value of 12. We used this method in a survey of 73 non-small cell lung cancer (NSCLC) samples, detecting 14 mutant samples of E746_A750del and 12 mutant samples of L858R. The results well agreed with the results of DxS. All unmatched samples were identified by sequencing and the results showed that our method has high specificty. Conclusion: The mutant-enriched ARMS TaqMan PCR could be useful in the detection of mutation in clinical samples containing only a small number of mutant alleles. © 2011 Elsevier Ireland Ltd.

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