Real-time PCR-based identification methods for <i>Megaselia scalaris</i> (Loew) (Diptera: Phoridae) targeting mitochondrial DNA

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species–derived material in concentrate feed mixtures and fish meals.

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Real-Time PCR Detection of Ruminant DNA

Journal of Food Protection ◽

10.4315/0362-028x-67.3.550 ◽

2004 ◽

Vol 67 (3) ◽

pp. 550-554 ◽

Cited By ~ 36

Author(s):

LUIS MENDOZA-ROMERO ◽

EDWARD L. C. VERKAAR ◽

PAUL H. SAVELKOUL ◽

ARNOLD CATSBURG ◽

HENK J. M. AARTS ◽

...

Keyword(s):

Mitochondrial Dna ◽

Bovine Spongiform Encephalopathy ◽

Real Time ◽

Real Time Pcr ◽

Spongiform Encephalopathy ◽

Meat And Bone Meal ◽

Bone Meal ◽

Dna Targeting ◽

Species Origin ◽

Semiquantitative Method

To control the spread of bovine spongiform encephalopathy, several DNA methods have been described for the detection of the species origin of meat and bone meal. Most of these methods are based on the amplification of a mitochondrial DNA segment. We have developed a semiquantitative method based on real-time PCR for detection of ruminant DNA, targeting an 88-bp segment of the ruminant short interspersed nuclear element Bov-A2. This method is specific for ruminants and is able to detect as little as 10 fg of bovine DNA. Autoclaving decreased the amount of detectable DNA, but positive signals were observed in feeding stuff containing 10% bovine material if this had not been rendered in accordance with the regulations, i.e., heated at 134°C for 3 instead of 20 min.

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Common Deletion (CD) in mitochondrial DNA of irradiated rat heart

Anais da Academia Brasileira de Ciências ◽

10.1590/0001-37652014110912 ◽

2014 ◽

Vol 86 (2) ◽

pp. 685-694 ◽

Cited By ~ 1

Author(s):

RAQUEL G. SIQUEIRA ◽

DAYSE A. DA SILVA ◽

LUIZ D.B. DE MELO ◽

ELIZEU F. DE CARVALHO ◽

SAMARA C. FERREIRA-MACHADO ◽

...

Keyword(s):

Mitochondrial Dna ◽

Ionizing Radiation ◽

Real Time ◽

Real Time Pcr ◽

Rat Heart ◽

Wistar Rats ◽

Post Irradiation ◽

Male Wistar Rats ◽

The Common ◽

Common Deletion

The purpose of this study was to map the common deletion (CD) area in mtDNA and investigate the levels of this deletion in irradiated heart. The assays were developed in male Wistar rats that were irradiated with three different single doses (5, 10 or 15 Gy) delivered directly to the heart and the analyses were performed at various times post-irradiation (3, 15 or 120 days). The CDs area were sequenced and the CD quantified by real-time PCR. Our study demonstrated that the CD levels progressively decreased from the 3rduntil the 15th day after irradiation, and then increased thereafter. Additionally, it was observed that the levels of CD are modulated differently according to the different categories of doses (moderate and high). This study demonstrated an immediate response to ionizing radiation, measured by the presence of mutations in the CD area and a decrease in the CD levels.

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Intraradical Dynamics of Two Coexisting Isolates of the Arbuscular Mycorrhizal Fungus Glomus intraradices Sensu Lato as Estimated by Real-Time PCR of Mitochondrial DNA

Applied and Environmental Microbiology ◽

10.1128/aem.00035-12 ◽

2012 ◽

Vol 78 (10) ◽

pp. 3630-3637 ◽

Cited By ~ 22

Author(s):

Karol Krak ◽

Martina Janoušková ◽

Petra Caklová ◽

Miroslav Vosátka ◽

Helena Štorchová

Keyword(s):

Mitochondrial Dna ◽

Real Time ◽

Real Time Pcr ◽

Glomus Intraradices ◽

Large Subunit ◽

Arbuscular Mycorrhizal ◽

Am Fungi ◽

Content Type ◽

Copy Numbers ◽

Pcr Assays

ABSTRACTReal-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this genomic region does not allow the specific amplification of closely related genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution power, but mtDNA-based quantification has not been previously explored in AM fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to monitor the DNA dynamics of two isolates ofGlomus intraradicessensu lato coexisting in the roots of medic (Medicago sativa). The mtDNA-based quantification was compared to quantification in nrDNA. The ratio of copy numbers determined by the nrDNA- and mtDNA-based assays consistently differed between the two isolates. Within an isolate, copy numbers of the nuclear and the mitochondrial genes were closely correlated. The two quantification approaches revealed similar trends in the dynamics of both isolates, depending on whether they were inoculated alone or together. After 12 weeks of cultivation, competition between the two isolates was observed as a decrease in the mtDNA copy numbers of one of them. The coexistence of two closely related isolates, which cannot be discriminated by nrDNA-based assays, was thus identified as a factor influencing the dynamics of AM fungal DNA in roots. Taken together, the results of this study show that real-time PCR assays targeted to the large subunit of mtDNA may become useful tools for the study of coexisting AM fungi.

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Detection and quantification of mitochondrial DNA deletions in individual cells by real-time PCR

Nucleic Acids Research ◽

10.1093/nar/gnf067 ◽

2002 ◽

Vol 30 (14) ◽

pp. 68e-68 ◽

Cited By ~ 211

Author(s):

L. He

Keyword(s):

Mitochondrial Dna ◽

Real Time ◽

Real Time Pcr ◽

Dna Deletions ◽

Detection And Quantification

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Lamivudine/telbivudine-associated neuromyopathy: neurogenic damage, mitochondrial dysfunction and mitochondrial DNA depletion

Journal of Clinical Pathology ◽

10.1136/jclinpath-2013-202069 ◽

2014 ◽

Vol 67 (11) ◽

pp. 999-1005 ◽

Cited By ~ 19

Author(s):

Hongliang Xu ◽

Zhaoxia Wang ◽

Lemin Zheng ◽

Wei Zhang ◽

He Lv ◽

...

Keyword(s):

Mitochondrial Dna ◽

Mitochondrial Dysfunction ◽

Real Time ◽

Real Time Pcr ◽

Axonal Neuropathy ◽

Nerve Biopsy ◽

Quantitative Real Time Pcr ◽

Electronic Microscopy ◽

Nucleotide Analogues ◽

Mitochondrial Dna Depletion

AimsMyopathy or neuropathy has been associated with lamivudine/telbivudine therapy in hepatitis B patients. We aim to describe the pathological changes of lamivudine/telbivudine-associated neuromyopathy.MethodsWe retrospectively recruited six patients who were diagnosed with nucleotide analogues-associated myopathy or neuropathy. Muscle and nerve biopsy were performed, and the specimens were prepared for the light microscopy and electron microscopy. Genomic DNA was extracted from frozen muscle specimens, and the mitochondrial DNA (mtDNA) content was quantified by real-time PCR.ResultsRecovery of the myopathy can be achieved after the discontinuation or changing the drugs to entecavir. Muscle and nerve biopsy revealed similar changes under either the light or electronic microscopy in all the subjects. Quantitative real-time PCR revealed decrease of mtDNA content in the affected muscle.ConclusionsMtDNA depletion results in mitochondrial dysfunction in the lamivudine/telbivudine-associated neuromyopathy. Myopathy was characterised by mitochondrial dysfunction accompanied with neurogenic damage due to axonal neuropathy. Ultrastructure changes of mitochondria included vacuolisation, simplification of the cristae and homogenised matrix.

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