Browsing by Subject "PCR"

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Diagnostics and genome analysis of phloem-limited phytopathogenic bacteria
(2022) Zübert, Christina; Hölzle, Ludwig
This thesis contributes to improving the epidemiological understanding of ‘Candidatus Arsenophonus phytopathogenicus’ and bacteria of the provisional taxon ‘Candidatus Phytoplasma’ by applying genetic markers for identification, differentiation, and phylogenetic reconstruction of this economically relevant plant pathogens.
Entwicklung von spezifischen PCR-ELISA Nachweissystemen für Coxiella burnetii, Francisella tularensis und Orthopockenviren
(2001) Kohlhaußen, Simone; Böhm, Reinhard
In the present work specific systems on the basis of the PCR were developed for the detection of Coxiella burnetii, Francisella tularensis and Orthopoxviruses. The detection of the pathogens is possible without additional cultivation. In order to exclude false-negative PCR results the amplification reaction was established as a competitive PCR in which an internal control DNA is included as an amplification check. With regard to a possible automation the detection-systems were constituted in form of a 'PCR ELISA'. The colorimetric detection of the amplicons takes place thereby in streptavidin coated microtiter plates. For each system biotinylated capture probes for both the internal control DNA (ST) and the pathogen specific DNA (WT) have been developed. For the use of these proof systems in the clinical laboratory, the routine application of the tested decontamination procedure by means of UNG during the execution of the PCR ELISA is recommended. For all three systems a detection of the pathogens is possible within one working day. For the species specific detection of F. tularensis and the genus specific detection of Orthopoxviruses the PCR ELISA systems established here represent the first developments of this type with integrated amplification control and potential for quantification.
Entwicklung, Charakterisierung und Kartierung von Mikrosatellitenmarkern bei der Zuckerrübe (Beta vulgaris L.)
(2001) Dörnte, Jost; Geiger, Hartwig H.
Simple sequence repeats (SSRs) or microsatellites were isolated from a sugarbeet (Beta vulgaris L.) genomic phage library. The size-fractionated library was screened for the occurrence of the motifes (GA)n, (GT)n, (TGA)n, (AGA)n and (CCG)n. The motifes (GA)n and (GT)n were found to occur most frequently in the sugarbeet genome (every 225 kb). In contrast, the trimer motifes were half as frequent (every 527 kb). A total of 217 microsatellite sequences were found in the sequenced clones. Most of the repeats were imperfect and/or compound. Sequence comparison revealed that 23% of the clones wich containing the (GT)n motif are variants of a previously described satellite DNA (SCHMIDT et al. 1991). Of 102 primer pairs tested on sugarbeet DNA, 71 gave a single product in the expected size. On 23 sugarbeet samples 64 of the 71 SSR-markers reveald length polymorphisms. The number of detected alleles per marker ranged from 2 to 13 (average 4,9) and the PIC-values ranged from 0,17 to 0,86 (average 0,58). A cluster analysis of the 23 samples confirms the pedigree data. The developed SSR markers were compared with RFLP and AFLP markers. Therefore nine sugarbeet lines, each with five single plants per line, were analysed. The SSR analyse shows the lowest similarity between the nine lines. The similarity inside the lines revealed no differences between the marker assays. Thirtythree SSR markers were genetically mapped into the RFLP framework maps of 2 F2-populations. The markers are randomly distributed over eight linkage groups of sugar beet.
Epidemiological and clinical description of Candidatus Mycoplasma haemosuis, an emerging pathogen in pigs
(2023) Ade, Julia; Hölzle, Ludwig
Candidatus Mycoplasma haemosuis is an emerging pathogen infecting pigs. It belongs to the group of uncultivable hemotrophic mycoplasmas. This group includes other long-known porcine representatives, i. e. Mycoplasma parvum und Mycoplasma suis. M. suis is the causative agent of infectious porcine anemia (IAP), a disease of great economic importance to the pig industry. Previously, Ca. M. haemosuis was only described in China, South Korea and Thailand, with no knowledge of its occurrence outside Asia or of its general clinical and economic importance in general. The present work investigates the occurrence of the novel hemotrophic bacterium and its clinical importance in Germany for the first time. For this, a quantitative real-time PCR was first successfully developed for the detection of Ca. M. haemosuis in pigs. The SYBR® Green-based PCR amplifies a 177-bp fragment of the Ca. M. haemosuis gap, which encodes the NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Using this PCR, Ca. M. haemosuis was detected in a total of seven pigs during an acute clinical disease in May 2017. This represents the first detailed description of a disease induced by Ca. M. haemosuis and the first detection of this novel HM species outside of Asia. In a further study, the newly established PCR was used to comprehensively investigate the occurrence of Ca. M. haemosuis in clinically healthy animals of different age groups in Southern Germany. Ca. M. haemosuis was prevalent in 6.25% of the sows (n=208), in 4.50% of the piglets (n=622), in 17.50% of the pigs (n=200), and in 0.00% of the breeding boars (n=183). By sampling the piglets immediately after birth and prior to the first colostrum uptake, the possibility of a vertical transmission of Ca. M. haemosuis was also determined within this thesis. Since 76.92% of the Ca. M. haemosuis positive sows gave birth to at least one Ca. M. haemosuis positive piglet, a vertical transmission is regarded as very likely. HMs are known to be transmitted blood-dependent and thus, transmitted iatrogenic or via wounds from animal to animal. The detection of M. suis in blood-free excretions such as saliva, urine, nasal, and vaginal secretions from experimentally infected animals has initiated the discussion of additional, blood-independent transmission routes. Saliva (n=148) and urine samples (n=47) were also collected from the sows examined by blood sampling, semen samples (n=183) were also obtained from the examined boars and applied to Ca. M. haemosuis qPCR. The pathogen was not detected in any of the saliva, urine, or semen samples. On the one hand, this demonstrates the lack of suitability of blood-free sample materials for diagnostics; on the other hand, it highlights the blood-dependent transmission pathways known to date and thus strengthens the potential to limit infections through strict hygiene measures during veterinary procedures and through the control of bloodsucking arthropods. In conclusion, based on the newly established qPCR assay for the sensitive and specific detection of Ca. M. haemosuis, the present work provides the first clinical and epidemiological description of the emerging hemotrophic pathogen in pigs. Further, the qPCR assay will be the basis for future studies regarding the epidemiology as well as the clinical relevance and pathogenesis of Ca. M. haemosuis -infections in pigs.