| 1. |
|
|
| 2. |
- El Bagoury, Gabr F., et al.
(författare)
-
Development and evaluation of one-step real-time RT-PCR assay for improved detection of foot-and-mouth disease virus serotypes circulating in Egypt
- 2022
-
Ingår i: Journal of Virological Methods. - : Elsevier. - 0166-0934 .- 1879-0984. ; 306
-
Tidskriftsartikel (refereegranskat)abstract
- Foot-and-mouth disease (FMD) is an extremely contagious and economically important viral disease affecting livestock. Rapid and precise diagnosis of FMD is of critical importance for efficient control and surveillance strategies of the disease. In this study, one-step real-time reverse transcription-polymerase chain reaction (RTqPCR) assays were developed using newly designed primers/probe sets in the conserved regions within the VP1 coding sequence for specific detection of FMDV serotypes SAT 2 and O with their different lineage circulating in Egypt. The assays were validated for efficacy to detect different lineages of these endemic FMDV serotypes in Egypt; the detection limit was 10 genomic copies for serotype SAT 2 and one genomic copy for serotype O, with no cross-reactivity observed. These findings were confirmed by the specific and sensitive detection of FMDV in clinical samples obtained from different regions in Egypt and representing a range of subtypes within the SAT 2 and O serotypes. The results illustrated the potential of tailored RT-qPCR methods for the rapid detection and serotyping of FMDV belonging to different lineages of serotypes SAT 2 and O circulating in Egypt with high sensitivity and specificity. The developed assays could be easily deployed for routine surveillance and hence improving the disease control measures.
|
|
| 3. |
|
|
| 4. |
- Kaliff, Malin, 1985-, et al.
(författare)
-
Optimization of droplet digital PCR assays for the type specific detection and quantification of five HPV genotypes, also including additional data on viral load of nine different HPV genotypes in cervical carcinomas
- 2021
-
Ingår i: Journal of Virological Methods. - : Elsevier. - 0166-0934 .- 1879-0984. ; 294
-
Tidskriftsartikel (refereegranskat)abstract
- The droplet digital PCR (ddPCR) system enables high-sensitivity detection of nucleic acids and direct absolute quantification of the targets. The aim of this research was to evaluate this system for viral load (VL) analysis of the human papillomavirus (HPV) genotypes HPV31, 35, 39, 51 and 56 measured in number of viral particles per cell. The sample types used for the optimization of the ddPCR assay were formalin-fixed paraffin-embedded (FFPE) tissues and cervical liquid cytology samples. The presently optimized ddPCR assays, together with assays optimized previously for HPV16, 18, 33 and 45, with the same ddPCR method, were used for the VL analysis of cervical tumor samples. Results published previously on the present study cohort showed that women with a cervical tumor containing multiple high-risk HPV genotypes had a worse prognosis compared to women with single-genotype-infected tumors. The VL was therefore analyzed in this study for the same cohort, as a possible explanatory factor to the prognostic differences. The results of the optimization part of the study, with analysis of VL using ddPCR in DNA from varying sample types (FFPE and liquid cytology samples), showed that each of the five assays demonstrated good inter- and intra-assay means with a coefficient of variation (CV) under 8% and 6% respectably. The cohort results showed no difference in VL between tumors with multiple and single HPV infections, and therefore did most likely not constitute a contributing factor for prognostic differences observed previously. However, tumors from women aged 60 years or older or containing certain HPV genotypes and genotype genera were associated with a higher VL.
|
|
| 5. |
- Mujtaba, Tahir
(författare)
-
Isolation of biotic stress resistance genes from cotton (Gossypium arboreum) and their analysis in model plant tobacco (Nicotiana tabacum) for resistance against cotton leaf curl disease complex
- 2020
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 276
-
Tidskriftsartikel (refereegranskat)abstract
- Cotton production is widely effected by Cotton Leaf Curl Virus (CLCuV) in world posing serious losses to cotton yield. The CRT genes from CLCuV resistant G. arboreum and CLCuV susceptible G. hirsutum were cloned and sequenced to know the differences of protein composition in both species. Molecular techniques were used to isolate full length putative biotic stress resistance genes from G. arboreum besides the analysis of identified novel genes in model plant tobacco (Nicotiana tabacum) for resistance to cotton leaf curl disease complex. It was found that transgenic plants over expressing Hydroperoxidelyase (HPL) genes exhibited higher enzyme activity than wild type. In addition the genome sequence information was used for the purpose of gene isolation. Even for the enhanced expression of Calreticulin (CRT), AOS and HPL in G. hirsutum, it still showed susceptibility against CLCuV suggesting alternative genes and pathways involved for the expression of resistance.
|
|
| 6. |
- Persson, Sofia, et al.
(författare)
-
A new assay for quantitative detection of hepatitis A virus
- 2021
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 288
-
Tidskriftsartikel (refereegranskat)abstract
- Hepatitis A virus (HAV) is mainly transmitted via contaminated food or water or through person-to-person contact. Here, we describe development and evaluation of a reverse transcription droplet digital PCR (RTddPCR) and reverse transcription real-time PCR (RT-qPCR) assay for detection of HAV in food and clinical specimens. The assay was evaluated by assessing limit of detection, precision, matrix effects, sensitivity and quantitative agreement. The 95 % limit of detection (LOD95 %) was 10 % higher for RT-ddPCR than for RTqPCR. A Bayesian model was used to estimate precision on different target concentrations. From this, we found that RT-ddPCR had somewhat greater precision than RT-qPCR within runs and markedly greater precision between runs. By analysing serum from naturally infected persons and a naturally contaminated food sample, we found that the two methods agreed well in quantification and had comparable sensitivities. Tests with artificially contaminated food samples revealed that neither RT-ddPCR nor RT-qPCR was severely inhibited by presence of oysters, raspberries, blueberries or leafy-green vegetables. For this assay, we conclude that RT-qPCR should be considered if rapid, qualitative detection is the main interest and that RT-ddPCR should be considered if precise quantification is the main interest. The high precision of RT-ddPCR allows for detection of small changes in viral concentration over time, which has direct implications for both food control and clinical studies.
|
|
| 7. |
- Achá Alarcón, L., et al.
(författare)
-
Development and performance evaluation of an In-House ELISA for the detection of group A rotavirus in diarrheal stool samples from children and domestic South American camelids
- 2022
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934. ; 301
-
Tidskriftsartikel (refereegranskat)abstract
- Group A rotavirus (RVA) is a prevalent pathogen causing acute gastroenteritis (AGE) in young children and animals. We developed an in-house ELISA (ROTA-GeFeK) for RVA detection, based on the expression of native recombinant VP6 protein in E. coli. To detect the RVA antigen, rabbit polyclonal IgG antibodies, produced against rVP6,were used as capture and detector antibodies in a sandwich ELISA. To validate the ROTA-GeFeK, 252 stool samples from children with AGE, were evaluated by conventional RT-PCR and commercial ELISA. Compared to RT-PCR, the ROTAGeFeK had a sensitivity of 88.2 % and a specificity of 94.4 %. Total detection rates with the ROTA-GeFeK, commercial ELISA and RT-PCR were 58 %, 58 % and 64 % respectively. The limit of detection was equal to 2.1 × 10 4 CCID 50 of the RVA strain RIX4414. No cross-reactivity with other enteric pathogens was observed. The RVApositive samples detected by the assay belonged to a diversity of G and [P] genotypes.This assay displayed reactivity and was proved to be useful for the detection of RVA in diarrheal samples of domestic South American Camelids. We suggest that the ROTAGeFeK can be used as an epidemiologic tool for rotavirus surveillance and for RVA detection in other animal species. © 2021
|
|
| 8. |
- Persson Berg, Linn, 1984, et al.
(författare)
-
Recombinant Epstein-Barr virus glycoprotein 350 as a serological antigen
- 2020
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934. ; 284
-
Tidskriftsartikel (refereegranskat)abstract
- Epstein-Barr virus (EBV) glycoprotein 350 (gp350) is the most abundant glycoprotein expressed on the EBV envelope, the major target for neutralizing antibodies and also essential for virion attachment to B lymphocytes. Several studies have addressed EBV gp350 as a vaccine candidate, but less commonly as a potential antigen for serological assays. The aim of the current study was to develop a diagnostic tool to quantify EBV gp350-specific IgG in previously EBV-infected individuals. A construct encoding the extracellular domain of EBV gp350 (amino acid (aa) 1-860) was developed for expression in Chinese hamster ovary cells. Serum samples (n = 360) with known IgG serostatus against viral capsid antigen (VCA) and Epstein-Barr nuclear antigen 1 (EBNA1) were divided into three groups based on the differences in their serostatus: VCA + EBNA1+ (n = 120), VCA + EBNA1-(n = 120) and VCA-EBNA1-(n = 120). The samples were analyzed by indirect ELISA using recombinant EBV gp350 aa 1-860 as antigen. A clear majority, 108 of the 120 VCA + EBNA1+ samples, had detectable EBV gp350-specific IgG. Of the 120 VCA + EBNA1-samples, 79 had detectable EBV gp350-specific IgG. Only 2 of the 120 VCA-EBNA1-samples had detectable EBV gp350-specific IgG. The results reported here show that use of the EBV gp350 aa 1-860 ELISA can serve as a sensitive method for EBV-specific IgG detection in serum samples.
|
|
| 9. |
- Ringlander, Johan, et al.
(författare)
-
Deep sequencing of hepatitis B virus using Ion Torrent fusion primer method
- 2022
-
Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934. ; 299
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Hepatitis B virus (HBV) infection is worldwide a major cause of liver cirrhosis and hepatocellular carcinoma. Thousands of years ago, several HBV genotypes (A-I) evolved and have, as a result of human migration, become globally disseminated. Sequencing of HBV is used for genotyping, and investigation of out -breaks or of antiviral resistance. The present study describes a simplified deep sequencing of the whole HBV genome. Methods: Sequencing by Ion Torrent was evaluated and its performance compared with Sanger sequencing on clinical samples. Results: Amplification of overlapping segments spanning the entire HBV genome was successful at HBV DNA levels in serum as low as 100 IU/mL. The use of primers carrying adapter tags generated libraries without the need for fragmentation and ligation steps, and inclusion of barcode sequences allowed parallel analysis of multiple samples. A streamlined bioinformatic platform generated consensus sequences and superior mutation assessment as compared with Sanger sequencing, with which there was a 99.8 % average agreement. Conclusion: Deep sequencing of the whole HBV genome by using PCR primers tagged with adapters that prepare overlapping amplicons for Ion Torrent analysis was efficient and accurate.
|
|
