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- Gharizadeh, B., et al.
(författare)
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Type-specific multiple sequencing primers - A novel strategy for reliable and rapid genotyping of human papilloma viruses by pyrosequencing technology
- 2005
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Ingår i: Journal of Molecular Diagnostics. - 1525-1578 .- 1943-7811. ; 7:2, s. 198-205
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Tidskriftsartikel (refereegranskat)abstract
- DNA sequencing is the gold standard method for accurate microbial and viral typing. However, DNA sequencing techniques have been facing limitations in typing of human papillomaviruses when the specimen harbors multiple genotypes and yields nonspecific amplification products, resulting in nonspecific and noninterpretable sequence data. To address these limitations we have developed a type-specific multiple sequencing primer DNA-sequencing method. This new strategy is suitable for sequencing and typing of samples harboring different genotypes (co-infections with multiple genotypes) and yielding nonspecific amplifications, thus eliminating the need for nested polymerase chain reaction (PCR), stringent PCR conditions, and cloning. The new approach has also proved useful for amplicons containing low PCR yield or subdominant types, avoiding reperforming of amplifications. We have applied the multiple sequencing primer method for genotyping of clinically relevant human papillomaviruses in a clinical test panel by using a combined pool of seven type-specific sequencing primers for HPV-6, -11, -16, -18, -31, -33, and -45. Furthermore, we introduced a sequence pattern recognition approach when there was a plurality of genotypes in the sample to facilitate typing of more than one target DNA in the sample. The multiple sequencing primer method has proved to be a multifaceted approach for typing of human papillomaviruses by DNA sequencing technologies.
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| 2. |
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| 4. |
- Ciftci, Sibel, et al.
(författare)
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Digital Rolling Circle Amplification-Based Detection of Ebola and Other Tropical Viruses
- 2020
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Ingår i: Journal of Molecular Diagnostics. - : Elsevier BV. - 1525-1578 .- 1943-7811. ; 22:2, s. 272-283
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Tidskriftsartikel (refereegranskat)abstract
- Emerging tropical viruses have caused serious outbreaks during the recent years, such as Ebola virus (EBOV) in 2014 and the most recent in 2018 to 2019 in Congo. Thus, immediate diagnostic attention is demanded at the point of care in resource-limited settings, because the performance and the operational parameters of conventional EBOV testing are Limited. Especially, their sensitivity, specificity, and coverage of other tropical disease viruses make them unsuitable for diagnostic at the point of care. Here, a padlock probe (PLP)-based rolling circle amplification (RCA) method for the detection of EBOV is presented. For this, a set of PLPs, separately targeting the viral RNA and complementary RNA of all seven EBOV genes, was used in the RCA assay and validated on virus isolates from cell culture. The assay was then translated for testing clinical samples, and simultaneous detection of both EBOV RNA types was demonstrated. For increased sensitivity, the RCA products were enriched on a simple and pump-free microfluidic chip. Because PLPs and RCA are inherently multiplexable, we demonstrate the extension of the probe panel for the simultaneous detection of the tropical viruses Ebola, Zika, and Dengue. The demonstrated high specificity, sensitivity, and multiplexing capability in combination with the digital quantification rendered the assay a promising diagnostic tool toward tropical virus detection at the point of care.
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| 5. |
- Edin, Alicia, et al.
(författare)
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Development and Laboratory Evaluation of a Real-Time PCR Assay for Detecting Viruses and Bacteria of Relevance for Community-Acquired Pneumonia
- 2015
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Ingår i: Journal of Molecular Diagnostics. - : Elsevier BV. - 1525-1578 .- 1943-7811. ; 17:3, s. 315-324
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Tidskriftsartikel (refereegranskat)abstract
- Community-acquired pneumonia may present with similar clinical symptoms, regardless of viral or bacterial cause. Diagnostic assays are needed to rapidly discriminate between causes, because this will guide decisions on appropriate treatment. Therefore, a quantitative real-time PCR (qPCR) assay with duplex reactions targeting eight bacteria and six viruses was developed. Technical performance was examined with linear plasmids. Upper and Lower respiratory tract specimens were used to compare the qPCR assay with standard microbiological methods. The limit of detection was 5 to 20 DNA template copies with approximately 1000-fold differences in concentrations of the two competing templates. SDs for positive controls were <5%. The use of the qPCR assay resulted in 113 positive identifications in 94 respiratory specimens compared with 38 by using standard diagnostics. Diagnostic accuracy of the qPCR assay varied between 60% positive agreement with standard tests for Streptococcus pneumoniae and 100% for Mycoplasma pneumoniae, Moraxella catarrhalis, and Staphylococcus aureus. Negative percentage of agreement was >95% for M. pneumoniae, Streptococcus pyogenes, respiratory syncytial virus, and influenza A virus; whereas it was only 56% for Haemophilus influenzae. Multiple microbial agents were identified in 19 of 44 sputum and 19 of 50 nasopharynx specimens. We conclude that in parallel qPCR detection of the targeted respiratory bacteria and viruses is feasible. The results indicate good technical performance of the assay in clinical specimens.
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| 6. |
- Gréen, Anna, et al.
(författare)
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Assessment of HaloPlex Amplification for Sequence Capture and Massively Parallel Sequencing of Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Genes
- 2015
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Ingår i: Journal of Molecular Diagnostics. - : Elsevier BV. - 1525-1578 .- 1943-7811. ; 17:1, s. 31-42
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Tidskriftsartikel (refereegranskat)abstract
- The genetic basis of arrhythmogenic right ventricular cardiomyopathy (ARVC) is complex. Mutations in genes encoding components of the cardiac desmosomes have been implicated as being causally related to ARVC. Next-generation sequencing allows parallel sequencing and duplication/deletion analysis of many genes simultaneously, which is appropriate for screening of mutations in disorders with heterogeneous genetic backgrounds. We designed and validated a next-generation sequencing test panel for ARVC using HaloPlex. We used SureDesign to prepare a HaloPlex enrichment system for sequencing of DES, DSC2, DSG2, DSP, JUP, PKP2, RYR2, TGFB3, TMEM43, and TIN from patients with ARVC using a MiSeq instrument. Performance characteristics were determined by comparison with Sanger, as the gold standard, and TruSeq Custom Amplicon sequencing of DSC2, DSG2, DSP, JUP, and PKP2. All the samples were successfully sequenced after HaloPlex capture, with >99% of targeted nucleotides covered by >20x. The sequences were of high quality, although one problematic area due to a presumptive context-specific sequencing error causing motif Located in exon 1 of the DSP gene was detected. The mutations found by Sanger sequencing were also found using the HaloPlex technique. Depending on the bioinformatics pipeline, sensitivity varied from 99.3% to 100%, and specificity varied from 99.90/0 to 100%. Three variant positions found by Sanger and HaloPlex sequencing were missed by TruSeq Custom Amplicon owing to Loss of coverage.
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| 7. |
- Hjelmevoll, Stig Ove, et al.
(författare)
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A fast real-time polymerase chain reaction method for sensitive and specific detection of the Neisseria gonorrhoeae porA pseudogene
- 2006
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Ingår i: Journal of Molecular Diagnostics. - : Elsevier BV. - 1525-1578 .- 1943-7811. ; 8:5, s. 574-581
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Tidskriftsartikel (refereegranskat)abstract
- Ever since the advent of molecular methods, the diagnostics of Neisseria gonorrhoeae has been troubled by false negative and false positive results compared with culture. Commensal Neisseria species and Neisseria meningitidis are closely related to N. gonorrhoeae and may cross-react when using molecular tests comprising too-low specificity. We have devised a real-time polymerase chain reaction (PCR), including an internal amplification control, that targets the N. gonorrhoeae porA pseudogene. DNA was automatically isolated on a BioRobot M48. Our subsequent PCR method amplified all of the different N. gonorrhoeae international reference strains (n = 34) and N. gonorrhoeae clinical isolates (n = 176) but not isolates of the 13 different nongonococcal Neisseria species (n = 68) that we tested. Furthermore, a panel of gram-negative bacterial (n = 18), gram-positive bacterial (n = 23), fungal (n = 1), and viral (n = 4) as well as human DNA did not amplify. The limit of detection was determined to be less than 7.5 genome equivalents/PCR reaction. In conclusion, the N. gonorrhoeae porA pseudogene real-time PCR developed in the present study is highly sensitive, specific, robust, rapid and reproducible, making it suitable for diagnosis of N. gonorrhoeae infection.
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| 8. |
- Howell, Viive M, et al.
(författare)
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Rapid mutation screening for HRPT2 and MEN1 mutations associated with familial and sporadic primary hyperparathyroidism.
- 2006
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Ingår i: Journal of Molecular Diagnostics. - : Elsevier BV. - 1525-1578 .- 1943-7811. ; 8:5, s. 559-66
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Tidskriftsartikel (refereegranskat)abstract
- Familial hyperparathyroidism, a disease of the parathyroid glands, may occur in conjunction with pituitary and pancreatic tumors (multiple endocrine neoplasia type I), kidney and bone tumors (hyperparathyroidism jaw tumor syndrome), or alone (familial isolated hyperparathyroidism). This study describes the development and validation of rapid scanning for mutations in two tumor suppressor genes linked to familial hyperparathyroidism-MEN1 and HRPT2. Denaturing high-performance liquid chromatography mutation scanning for MEN1 was performed using a set of 10 amplicons covering the nine coding exons and flanking intronic regions and for HRPT2 using a set of three amplicons for exons 1, 2, and 7 and flanking intronic regions, in which 80% of the mutations identified to date are located. All 52 MEN1 mutations or polymorphisms, 46 known and six unknown, were successfully detected. Mutation detection in exon 9 was not confounded by the presence of the common polymorphism D418D. In addition, all 10 HRPT2 mutations were successfully detected, and a two-step approach was able to distinguish IVS2 common polymorphisms from exon 2 mutations. The development of rapid denaturing high performance liquid chromatography mutation scanning of MEN1 and HRPT2 facilitates a molecular diagnosis of the associated familial syndromes for both clinically affected and at-risk family members.
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