A novel method to quantify deleted mitochondrial DNA in a real time PCR

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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The ZKIR Assay, a novel Real-Time PCR Method for the Detection of Klebsiella pneumoniae and Closely Related Species in Environmental Samples

10.1101/855668 ◽

2019 ◽

Author(s):

Elodie Barbier ◽

Carla Rodrigues ◽

Geraldine Depret ◽

Virginie Passet ◽

Laurent Gal ◽

...

Keyword(s):

Public Health ◽

Klebsiella Pneumoniae ◽

Real Time ◽

Real Time Pcr ◽

Environmental Samples ◽

Health Concern ◽

Complex Matrices ◽

Animal Pathogens ◽

Novel Method ◽

Pcr Method

ABSTRACTKlebsiella pneumoniae (Kp) is of growing public health concern due to the emergence of strains that are multidrug-resistant, virulent, or both. Taxonomically, Kp includes seven phylogroups, with Kp1 (K. pneumoniae sensu stricto) being medically prominent. Kp can be present in environmental sources such as soils and vegetation, which could act as reservoirs of animal and human infections. However, the current lack of screening methods to detect Kp in complex matrices limits research on Kp ecology. Here we analysed 4222 genome sequences and found that existing molecular detection targets lack specificity for Kp. A novel real-time PCR method, the ZKIR assay, was developed and used to detect Kp in 96 environmental samples. Results were compared to a culture-based method using SCAI agar medium coupled to MALDI-TOF mass spectrometry identification. Whole-genome sequencing of environmental Kp was performed. The ZKIR assay was positive for the 48 tested Kp reference strains, whereas 88 non-Kp strains were negative. The limit of detection of Kp in spiked soil microcosms was 1.5 × 10-1 CFU g-1 after enrichment for 24 h in LB supplemented with ampicillin, and 1.5 × 103 to 1.5 × 104 CFU g-1 directly after soil DNA extraction. The ZKIR assay was more sensitive than the culture method. Kp was detected in 43% of environmental samples. Genomic analysis of the isolates revealed a predominance of phylogroups Kp1 (65%) and Kp3 (32%), a high genetic diversity (23 MLST sequence types), a quasi-absence of antibiotic resistance or virulence genes, and a high frequency (50%) of O-antigen type 3. This study shows that the ZKIR assay is an accurate, specific and sensitive novel method to detect the presence of Kp in complex matrices, and indicates that Kp isolates from environmental samples differ from clinical isolates.IMPORTANCEThe Klebsiella pneumoniae species complex (Kp) includes human and animal pathogens, some of which are emerging as hypervirulent and/or antibiotic resistant strains. These pathogens are diverse and classified into seven phylogroups, which may differ in their reservoirs and epidemiology. Proper management of this public health hazard requires a better understanding of Kp ecology and routes of transmission to humans. So far, detection of these microorganisms in complex matrices such as food or the environment has been difficult due to a lack of accurate and sensitive methods. Here, we describe a novel method based on real-time PCR, which enables detection of all Kp phylogroups with high sensitivity and specificity. We used this method to detect Kp isolates from environmental samples, and show based on genomic sequencing that they differ in antimicrobial resistance and virulence gene content, from human clinical Kp isolates. The ZKIR PCR assay will enable rapid screening of multiple samples for Kp presence and will thereby facilitate tracking the dispersal patterns of these pathogenic strains across environmental, food, animal and human sources.

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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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Novel Method for Genotyping Hepatitis B Virus on the Basis of TaqMan Real-Time PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.01442-09 ◽

2010 ◽

Vol 48 (4) ◽

pp. 1105-1111 ◽

Cited By ~ 11

Author(s):

S. Malmstrom ◽

I. Berglin-Enquist ◽

M. Lindh

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Real Time ◽

Real Time Pcr ◽

B Virus ◽

Novel Method

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The ZKIR Assay, a Real-Time PCR Method for the Detection of Klebsiella pneumoniae and Closely Related Species in Environmental Samples

Applied and Environmental Microbiology ◽

10.1128/aem.02711-19 ◽

2020 ◽

Vol 86 (7) ◽

Cited By ~ 1

Author(s):

Elodie Barbier ◽

Carla Rodrigues ◽

Geraldine Depret ◽

Virginie Passet ◽

Laurent Gal ◽

...

Keyword(s):

Public Health ◽

Klebsiella Pneumoniae ◽

Real Time ◽

Real Time Pcr ◽

Environmental Samples ◽

Health Concern ◽

Complex Matrices ◽

Content Type ◽

Novel Method ◽

Pcr Method

ABSTRACT Klebsiella pneumoniae is of growing public health concern due to the emergence of strains that are multidrug resistant, virulent, or both. Taxonomically, the K. pneumoniae complex (“Kp”) includes seven phylogroups, with Kp1 (K. pneumoniae sensu stricto) being medically prominent. Kp can be present in environmental sources such as soils and vegetation, which could act as reservoirs of animal and human infections. However, the current lack of screening methods to detect Kp in complex matrices limits research on Kp ecology. Here, we analyzed 1,001 genome sequences and found that existing molecular detection targets lack specificity for Kp. A novel real-time PCR method, the ZKIR (zur-khe intergenic region) assay, was developed and used to detect Kp in 96 environmental samples. The results were compared to a culture-based method using Simmons citrate agar with 1% inositol medium coupled to matrix-assisted laser desorption ionization–time of flight mass spectrometry identification. Whole-genome sequencing of environmental Kp was performed. The ZKIR assay was positive for the 48 tested Kp reference strains, whereas 88 non-Kp strains were negative. The limit of detection of Kp in spiked soil microcosms was 1.5 × 10−1 CFU g−1 after enrichment for 24 h in lysogeny broth supplemented with ampicillin, and it was 1.5 × 103 to 1.5 × 104 CFU g−1 directly after soil DNA extraction. The ZKIR assay was more sensitive than the culture method. Kp was detected in 43% of environmental samples. Genomic analysis of the isolates revealed a predominance of phylogroups Kp1 (65%) and Kp3 (32%), a high genetic diversity (23 multilocus sequence types), a quasi-absence of antibiotic resistance or virulence genes, and a high frequency (50%) of O-antigen type 3. This study shows that the ZKIR assay is an accurate, specific, and sensitive novel method to detect the presence of Kp in complex matrices and indicates that Kp isolates from environmental samples differ from clinical isolates. IMPORTANCE The Klebsiella pneumoniae species complex Kp includes human and animal pathogens, some of which are emerging as hypervirulent and/or antibiotic-resistant strains. These pathogens are diverse and classified into seven phylogroups, which may differ in their reservoirs and epidemiology. Proper management of this public health hazard requires a better understanding of Kp ecology and routes of transmission to humans. So far, detection of these microorganisms in complex matrices such as food or the environment has been difficult due to a lack of accurate and sensitive methods. Here, we describe a novel method based on real-time PCR which enables detection of all Kp phylogroups with high sensitivity and specificity. We used this method to detect Kp isolates from environmental samples, and we show based on genomic sequencing that they differ in antimicrobial resistance and virulence gene content from human clinical Kp isolates. The ZKIR PCR assay will enable rapid screening of multiple samples for Kp presence and will thereby facilitate tracking the dispersal patterns of these pathogenic strains across environmental, food, animal and human sources.

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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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