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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. |
- 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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| 3. |
- 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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