Detection of Multiple Human Herpes Viruses by DNA Microarray Technology

Földes-Papp, Zeno; Egerer, Renate; Birch-Hirschfeld, Eckhard; Striebel, Hans‐Martin; Demel, Ulrike; Tilz, Gernot P.; Wutzler, Peter

doi:10.1007/bf03260041

Cited by 26 publications

(9 citation statements)

References 30 publications

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“…For this reason, multiplex PCR has been used for monitoring of viral pathogens, and several groups have developed multiplex PCR-based methods for detection of human herpesviruses using nested PCR [Pozo and Tenorio, 1999;Hudnall et al, 2004], restriction enzyme digestion of PCR products [Rozenberg and Lebon, 1991;Johnson et al, 2000], stair primers [Robert et al, 2002], or hybridization with a DNA array system [Wang et al, 2002;Foldes-Papp et al, 2004]. Especially, Hudnall et al [2004] developed the nested PCR assay for detection of all eight herpesviruses and their approach yielded high sensitivities of 10-100 virus copies even using a crude DNA sample containing 0.5 mg of human genomic DNA.…”

Section: Discussionmentioning

confidence: 99%

“…Especially, Hudnall et al [2004] developed the nested PCR assay for detection of all eight herpesviruses and their approach yielded high sensitivities of 10-100 virus copies even using a crude DNA sample containing 0.5 mg of human genomic DNA. The microarray-based diagnosis procedure established by Foldes-Papp et al [2004] is also a sensitive method with a lower limit of 100 viral copies per ml sample. Our multiplex PCR method is thought to have similar sensitivity to the above methods because our assay can detect 1-10 copies of purified viral DNA cloned into the plasmid.…”

Section: Discussionmentioning

confidence: 99%

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Epidemiology of Epstein–Barr virus, cytomegalovirus, and kaposi's sarcoma-associated herpesvirus infections in peripheral blood leukocytes revealed by a multiplex PCR assay

et al. 2006

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A multiplex polymerase chain reaction (PCR) has been developed for the simultaneous detection of Epstein-Barr virus (EBV), cytomegalovirus (CMV), and Kaposi's sarcoma-associated herpesvirus (KSHV) in a clinical sample. Primers of multiplex PCR were designed to amplify specific regions of the EBV EBNA1, CMV IE2, and KSHV LANA genes. This multiplex PCR assay was found to have detection sensitivities of 1-10 copies of purified viral DNA cloned into the plasmid. To assess diagnostic and pre-clinical applications with this method, we utilized KSHV-positive primary effusion lymphoma (PEL) cells, EBV-positive Burkitt's lymphoma cells, CMV-infected fibroblast cells, and clinically prepared peripheral blood leukocytes (PBLs) that had been infected with viruses. We found that this multiplex PCR assay has high sensitivity and specificity for simultaneous detection of EBV, CMV, and KSHV genomes in a single amplification from a clinical material. Using this multiplex PCR assay, we investigated the prevalence of EBV, CMV, and KSHV in PBL samples from normal Japanese randomly selected. KSHV, EBV, and CMV genomes were detected in samples from 2 (0.2%), 377 (39.5%), and 27 (2.8%) of the 953 blood donors, respectively. Interestingly, both EBV and CMV genomes were detected in samples from all KSHV-positive donors.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Epidemiology of Epstein–Barr virus, cytomegalovirus, and kaposi's sarcoma-associated herpesvirus infections in peripheral blood leukocytes revealed by a multiplex PCR assay

et al. 2006

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“…Major drawbacks in using DNA microarrays as a standard technique for pathogen detection are linked to the low representation of pathogen DNA in the analytes, but also to the relatively low sensitivity of fluorescence-based microarrays. The amount of specific pathogen DNA present in clinical, environmental, and food samples is sometimes as low as few femtograms [8-14], while the detection limit for genomic DNA in fluorescence-based microarrays, without any pre-amplification, is in the range of micrograms to nanograms [1,3,4,7,15]. …”

Section: Introductionmentioning

confidence: 99%

Large scale multiplex PCR improves pathogen detection by DNA microarrays

et al. 2009

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BackgroundMedium density DNA microchips that carry a collection of probes for a broad spectrum of pathogens, have the potential to be powerful tools for simultaneous species identification, detection of virulence factors and antimicrobial resistance determinants. However, their widespread use in microbiological diagnostics is limited by the problem of low pathogen numbers in clinical specimens revealing relatively low amounts of pathogen DNA.ResultsTo increase the detection power of a fluorescence-based prototype-microarray designed to identify pathogenic microorganisms involved in sepsis, we propose a large scale multiplex PCR (LSplex PCR) for amplification of several dozens of gene-segments of 9 pathogenic species. This protocol employs a large set of primer pairs, potentially able to amplify 800 different gene segments that correspond to the capture probes spotted on the microarray. The LSplex protocol is shown to selectively amplify only the gene segments corresponding to the specific pathogen present in the analyte. Application of LSplex increases the microarray detection of target templates by a factor of 100 to 1000.ConclusionOur data provide a proof of principle for the improvement of detection of pathogen DNA by microarray hybridization by using LSplex PCR.

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“…The cost of these methods in this diagnostic strategy can be undervalued when compared with the benefit of the most correct diagnosis of the infection. New assays based on molecular techniques have become available for routine CMV diagnosis (HalwachsBaumann et al 2001) and a lot of another based on different strategies have been developed recently (Hong et al 2004, Foldes-Papp et al 2004. The critical point is the determination of clinically relevant CMV activation from latency and the earliest time for accurate detection of this activation.…”

Section: Discussionmentioning

confidence: 99%

A follow up study of cytomegalovirus infection in a group of Turkish renal transplant recipients using molecular assays

Çırak

Rota

Maral

et al. 2005

Mem. Inst. Oswaldo Cruz

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The clinical value of an in-house cytomegalovirus nested polymerase chain reaction (CMV-PCR) and a commercial molecular assay hybrid capture CMV DNA assay (HCA) was evaluated in monitoring a group of renal transplant patients for six months follow up. In this study, the sensitivity, specificity, positive predictive value, and negative predictive value of nested CMV DNA PCR assay and HCA at the beginning of the study were 70, 42.9, 46.7, 66.7, and 60, 78.6, 66.7, and 73.3% respectively. After six months, they were 80, 66.7, 80, 66.7 for CMV PCR and 73.3, 88.9, 91.7, 66.7% Human cytomegalovirus (CMV) infection is responsible for significant mortality and morbidity and graft failure in renal transplant recipients (Tong et al. 1998). The early identification of CMV is important as the majority of patients develop infection and the best result is obtained when treatment is started earlier (Rubin & Tolkoff-Rubin 1993). CMV must be detected before clinical symptom appearance, but virus detection is not always followed by CMV disease (Eckart et al. 1997). Laboratory diagnosis of CMV infection is based primarily on the detection of CMV viremia and active CMV infection after the onset of symptoms. By means of advanced laboratory assays, it is possible to have positive laboratory assay results before the onset of symptoms. However, laboratory diagnosis of active CMV infection is not always associated with symptomatic disease (Tong et al. 1998) and the identification of these patients for clinically relevant disease is difficult.Various qualitative and quantitative molecular assays are used to monitor renal transplant recipients and they appear to be more convenient and sensitive than the traditional methods such as serology, virus cultures and shell vial assays. The detection CMV DNA in leukocytes or plasma by polymerase chain reaction (PCR) is the most sensitive method of identifying CMV disease. However a highly sensitive CMV-PCR may detect latent virus that is not involved in disease (Rollag et al. 1998). The commonly used DNA hybridization assays offer the advantage of objective CMV DNA detection and quantification without the hazards of PCR such as contamination or inhibition (Aitken et al. 1999). The aim of this study was to monitor a group of adult Turkish renal transplant patients for six months follow up by using PCR and hybrid capture CMV DNA assay (HCA) in detecting and predicting CMV disease and to evaluate the relationship between the CMV-DNA presence and the symptomatic human CMV infection. As far as we are concerned this is the first follow up study in renal transplant patients using molecular assays in Turkey. MATERIALS AND METHODSPatients and specimens -Blood samples were obtained from 24 kidney transplant recipients who were CMV IgG positive. EDTA-anticoagulated blood samples were collected at monthly intervals during six months. All of the blood samples were evaluated with both PCR and HCA at the beginning of this follow up study and after six months. In the same way, the clinical symptoms of the pat...

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Detection of Multiple Human Herpes Viruses by DNA Microarray Technology

Cited by 26 publications

References 30 publications

Epidemiology of Epstein–Barr virus, cytomegalovirus, and kaposi's sarcoma-associated herpesvirus infections in peripheral blood leukocytes revealed by a multiplex PCR assay

Epidemiology of Epstein–Barr virus, cytomegalovirus, and kaposi's sarcoma-associated herpesvirus infections in peripheral blood leukocytes revealed by a multiplex PCR assay

Large scale multiplex PCR improves pathogen detection by DNA microarrays

A follow up study of cytomegalovirus infection in a group of Turkish renal transplant recipients using molecular assays

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