PCR ELISA for the quantitative detection of Epstein–Barr virus genome

Bazzichi, Agostino; Guidi, Francesco; Rindi, Laura; Incaprera, Marina; Garzelli, Carlo

doi:10.1016/s0166-0934(98)00060-3

Cited by 12 publications

(10 citation statements)

References 27 publications

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“…The usefulness of the PCR-ELISA was initially evaluated by testing 79 cell culture-adapted human and animal rotavirus reference strains with known G and P types. The G and P types of rotavirus strains for which genotypespecific probes were available (i.e., strains bearing G1, G2, G3, G4, G5, G6, G8, G9, G10, G12, P [4], P [6], P [8], P [9], or P[14] specificity) were correctly identified by the PCR-ELISA, whereas strains for which genotype-specific probes were unavailable (i.e., strains bearing G11, G14, P [3], P [10], or P[11] specificity) were not genotyped (data not shown), demonstrating the genotype specificity of G and P probes used in the assay.…”

Section: Resultsmentioning

confidence: 99%

“…The overall concordance between typing PCR and PCR-ELISA in P-genotyping results was 89.1% (147/165), which was much higher than that for G-genotyping results (Table 6). This may be due to the finding that, unlike a high diversity of G genotypes detected, a vast majority (149/165 [90.3%]) of the tested samples belonged to genotype P [8], the most common human P genotype (Table 5). P genotypes determined by the PCR-ELISA were confirmed by microarray hybridization (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…The PCR products were analyzed by agarose gel electrophoresis and visualized by staining with ethidium bromide. Rotavirus strains Wa (G1P [8]), K8 (G1P [9]), DS-1 (G2P [4]), P (G3P [8]), RRV (G3P [3]), ST-3 (G4P [6]), OSU (G5P [7]), UK (G6P [5]), 69 M (G8P [10]), WI61 (G9P [8]), B223 (G10P [11]), and YM (G11P [7]) were used as control viruses.…”

Section: Pcr-elisamentioning

confidence: 99%

“…Fifteen G genotypes (19,34,70) and at least 27 different P genotypes (10,53,54,55,64,70,73) have been established thus far. The G-P combinations G1P [8], G2P [4], G3P [8], G4P [8], G9P [6], and G9P [8] are most commonly detected in humans (for a review, see reference 69). However, rotavirus strains bearing rare or unusual G and/or P genotypes (e.g., G5, G8, G10, G11, G12, P [11], P [14], and P [25]) associated with human infections have also been reported around the world with an increasing frequency (6,7,18,32,43,56,64,68,78,82).…”

mentioning

confidence: 99%

“…Sensitive and reliable diagnostic techniques that do not bear such disadvantages (11,17,36,49) are needed not only for accurate rotavirus strain surveillance but also for effective rotavirus vaccine development and evaluation (for example, analyses of homotypic versus heterotypic protection). In the present study, we developed a microtiter plate hybridization-based ELISA coupled with PCR (PCR-ELISA) and evaluated its usefulness for the identification of clinically relevant G (G1 to G6, G8 to G10, G12) and P (P [4], P [6], P [8], P [9], and P [14]) genotypes of group A rotavirus.…”

mentioning

confidence: 99%

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Development of a Microtiter Plate Hybridization-Based PCR-Enzyme-Linked Immunosorbent Assay for Identification of Clinically Relevant Human Group A Rotavirus G and P Genotypes

et al. 2008

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A microtiter plate hybridization-based PCR-enzyme-linked immunosorbent assay (PCR-ELISA) has been used for the detection and identification of a variety of microorganisms. Here, we report the development of a PCR-ELISA for the identification of clinically relevant human rotavirus VP7 (G1 to G6, G8 to G10, and G12) and VP4 (P[4], P[6], P[8], P[9], and P[14]) genotypes. The G and P types of reference human and animal rotavirus strains for which specific probes were available were correctly identified by the PCR-ELISA. In addition, reference strains bearing G or P genotypes for which specific probes were unavailable, such as G11, G14, P[3], P[10], and P[11], did not display any cross-reactivity to the probes. The usefulness of the assay was further evaluated by analyzing a total of 396 rotavirus-positive stool samples collected in four countries: Brazil, Ghana, Japan, and the United States. The results of this study showed that the PCR-ELISA was sensitive and easy to perform without the use of any expensive and sophisticated equipment, the reagents used are easy to obtain commercially and advantageous over multiplex PCR since more than one type-specific probe is used and the selection of probes is more flexible.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Pcr-elisamentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Development of a Microtiter Plate Hybridization-Based PCR-Enzyme-Linked Immunosorbent Assay for Identification of Clinically Relevant Human Group A Rotavirus G and P Genotypes

et al. 2008

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Semiquantitative PCR analysis of Epstein‐Barr virus DNA in clinical samples of patients with EBV‐associated diseases

Meerbach¹,

Gruhn

Egerer³

et al. 2001

Journal of Medical Virology

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The laboratory diagnosis of primary and reactivated Epstein-Barr virus (EBV) infection is based on serologic methods in immunocompetent patients. However, in immunocompromised patients, serologic data are difficult to interpret and do not often correlate with clinical data. In order to find a useful and practical marker for diagnosis of EBV-related diseases, a polymerase chain reaction (PCR) assay was established for semiquantitative detection of EBV sequences. The method was based on a nested PCR, using primers of the virus capsid antigen p23 region and an endpoint dilution. This method was carried out on 68 plasma samples, 68 samples of peripheral blood mononuclear cells and 5 cerebrospinal fluid samples of 39 patients with various diseases to evaluate the EBV-genome copy number. Samples from patients suffering from infectious mononucleosis served as positive controls for active EBV infection. In 5 patients with infectious mononucleosis, high copy numbers of EBV genomes in peripheral blood mononuclear cells were detected within a range of 1,000-40,000 copies in 10(5) peripheral blood mononuclear cells. In contrast, samples from 19 latently infected persons either showed low copy numbers (10-100 in 10(5) peripheral blood mononuclear cells) or were EBV PCR negative. Comparable results were observed in seven renal transplant patients without any symptoms. The practical value of the semiquantitative detection of EBV DNA was demonstrated in three bone marrow transplant recipients. Two developed a lymphoproliferative disease associated with extremely high amounts of EBV DNA in plasma (16,000 and 50,000 copies/ml, respectively) and peripheral blood mononuclear cells (100,000 and 6.5 million copies in 10(5) peripheral blood mononuclear cells, respectively). The high EBV load in plasma and peripheral blood mononuclear cells was reduced dramatically after successful antiviral therapy in one case. The third bone marrow transplant recipient developed an EBV-induced transverse myelitis with an increased number of EBV-genome copies in peripheral blood mononuclear cells and EBV-positive cerebrospinal fluid samples. After combined antiviral and immune therapy, the EBV-genome copy numbers decreased and the patient recovered completely. These data demonstrate a good correlation between semiquantitative detection of EBV genomes and clinical findings. The method is recommended for the diagnosis of EBV-associated diseases in patients after transplantation, as well as for monitoring the response to therapy.

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Quantitative PCR-ELAHA for the Determination of Retroviral Vector Transduction Efficiency

et al. 2001

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Current methods to detect transduction efficiency during the routine use of integrating retroviral vectors in gene therapy applications may require the use of radioactivity and usually rely upon subjective determination of the results. We have developed two competitive quantitative assays that use an enzyme-linked, amplicon hybridization assay (ELAHA) to detect the products of PCR-amplified regions of transgene from cells transduced with Moloney murine leukemia virus vectors. The quantitative assays (PCR-ELAHA) proved to be simple, rapid, and sensitive, avoiding the need for Southern hybridization, complex histochemical stains, or often subjective and time-consuming tissue culture and immunofluorescence assays. The PCR-ELAHA systems can rapidly detect proviral DNA from any retroviral vector carrying the common selective and marker genes neomycin phosphotransferase and green fluorescent protein, and the methods described are equally applicable to other sequences of interest, providing a cheaper alternative to the evolving real-time PCR methods. The results revealed the number of copies of retrovector provirus present per stably transduced cell using vectors containing either one or both qPCR targets.

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PCR ELISA for the quantitative detection of Epstein–Barr virus genome

Cited by 12 publications

References 27 publications

Development of a Microtiter Plate Hybridization-Based PCR-Enzyme-Linked Immunosorbent Assay for Identification of Clinically Relevant Human Group A Rotavirus G and P Genotypes

Development of a Microtiter Plate Hybridization-Based PCR-Enzyme-Linked Immunosorbent Assay for Identification of Clinically Relevant Human Group A Rotavirus G and P Genotypes

Semiquantitative PCR analysis of Epstein‐Barr virus DNA in clinical samples of patients with EBV‐associated diseases

Quantitative PCR-ELAHA for the Determination of Retroviral Vector Transduction Efficiency

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