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Sensitive quantification of mitochondrial mutation using new Taqman probes

100%
Truong H., Nguyen V.-A., Nguyen H.-L., Pham V.-A., Phan T.-N.
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vol. 9
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issue 6
839-848
EN
The A3243G mitochondrial mutation is the major cause of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). The severity of the disease is correlated with the heteroplasmy level of the mutation. Here we describe for the first time the validation of a real-time polymerase chain reaction (PCR) assay with Taqman locked nucleic acid (LNA) fluorescent (FAM for mutant, HEX for wild type) probes for quantification of heteroplasmy levels in a total of 18 family members from 5 Vietnamese MELAS patients carrying A3243G. Almost no background of FAM signals was detected in normal samples, indicating that the probes were allele-specific. Standard curves indicate sensitive detection at 0.1% mutants and high reliability with R2 > 0.985. The correlation line between measured % mutant and expected % mutant was highly reliable, with a slope of 0.993 and R2 of 0.998. All positive A3243G mutant samples pre-screened by PCR-restriction fragment length polymorphism (RFLP) were confirmed, and their heteroplasmy levels quantified to be from 3.68 to 80.85%. The heteroplasmy levels in patients were higher than in their family members and generally correlated well with the severity of their clinical symptoms. Overall, this work is the first demonstration of the application of LNA probes for sensitive and highly reliable quantification of heteroplasmy levels in human mitochondria.
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Identifying the etiologic role of Parvovirus B19 in non-immune hydrops fetalis by histopathology, immunohistochemistry and nucleic acid testing: a retrospective study

86%
Ergunay K., Altinok G., Gurel B., Pinar A., Sungur A., Balci S., Ustacelebi S.
Open Medicine
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2007
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vol. 2
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issue 3
271-279
EN
Intrauterine Parvovirus B19 infections may cause fetal anemia, non-immune hydrops fetalis or abortion. This study focuses on the pathogenic role of Parvovirus B19 in non-immune hydrops fetalis at Hacettepe University, a major reference hospital in Turkey. Twenty-two cases of non-immune hydrops fetalis were retrospectively selected out of a total of 431 hydrops fetalis specimens from the Department of Pathology archieves. Paraffine embedded tissue sections from placental and liver tissues from each case were evaluated by histopathology, immunohistochemistry, nested PCR and commercial quantitative Real-time PCR. Viral DNA was detected in placental tissues by Real-time PCR in 2 cases (2/22, 9.1%) where histopathology also revealed changes suggestive of Parvovirus B19 infection. No significant histopathologic changes were observed for the remaining sections. Nested PCR that targets the VP1 region of the viral genome and immunohistochemistry for viral capsid antigens were negative for all cases. As a result, Parvovirus B19 is identified as the etiologic agent for the development of non-immune hydrops fetalis for 9.1% of the cases in Hacettepe University, Turkey. Real-time PCR is observed to be an effective diagnostic tool for nucleic acid detection from paraffine embedded tissues. Part of this study was presented as a poster at XIIIth International Congress of Virology, San Francisco, USA (Abstract V-572).
3
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Cryptosporidiosis outbreak on a dairy farm: Detection of Cryptosporidium parvum as a causative agent in the water source

86%
Karakavuk M., Can H., Döşkaya M., Karakavuk T., Erkunt-Alak S., Köseoğlu A. E., Gül A., Ün C., Gürüz Y., Değirmenci-Döşkaya A., Karakavuk M., Can H., Döşkaya M., Karakavuk T., Erkunt-Alak S., Köseoğlu A. E., Gül A., Ün C., Gürüz Y., Değirmenci-Döşkaya A.
Polish Journal of Veterinary Sciences
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2021
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