Search
Search Results
-
Qualitative detection of genetically modified organisms in plant samples
309-313Views:93We analysed the GMO content of corn samples by polymerase chain reaction following the appropriate optimization of the reaction. The analysis included two main steps: extraction of DNA from the sample, and detection of the GMO content by polymerase chain reaction. The polymerase chain reaction is an in vitro method to multiply chromosomatic or cloned DNA (cDNA) sequences through the enzymatic pathway. The reaction is sensitive enough to produce DNA in sufficient amount for the analysis from a single DNA. We identified the PCR products by agarose gel electrophoresis. When optimizing the reaction, the MgCl2 concentration, reaction time and temperature have to be taken into consideration. The temperature of the anellation has to be increased until the highest specificity and yield is reached. If the temperature of the anellation is too high, the primer is linked to non-specific sites as well; in the gel visualization, more lines can be seen at one sample. If the temperature of the anellation is too high, the primer is insufficiently linked or is not linked at all (too few lines in the gel visualization). After optimization, the GMO content in the unknown sample can be determined along with the appropriate positive and negative controls.
-
Identification and biological examination of some inland Monilinia species
27-29Views:113The aim of this study was to identify and biologically analyse some Monilinia species from Hungary. 146 M. fructigena and 28 M. laxa species out 174 infectious fruit from all over the country were used for the study. For further study 10 isolates were used and apple fruit was inoculated with them according to Koch postulate. 1–2 mm ochre exogen stromas were observbed on infectious plant parts and growing signs on culture of all isolates were identical to M. fructigena. To affirm classical identification, isolates with molecular biological method were also prepared using PCR reaction. Control isolates of M. laxa, M. fructigena and M. fructicola were used. The size of PCR product showed that all isolates had a 415 bp band which was typical of M. fructigena. Results support the previous observation that M. fructigena and M. laxa species occure all over Hungary.
-
Detection of DNA mutations by PCR-TTGE method
21-25Views:163In our study PCR-temporal temperature gelelectrophoresis (TTGE) and MeltINGENY bioinformatic program were used to analyse the mutations in the genes of melanocortin-1 receptor (MC1R) and pituitary adenylate-cyclase activating polypeptide (PACAP) in cattle. Amplification of target DNA by PCR was performed with GC-clamp primers and non-GC-clamp primers in simplex PCR reactions. The fragments were separated by denaturing polyacrylamide gelelectrophoresis (denaturing agents: high temperature, urea) after PCR reactions.MC1R homozygous individuals were used for the reaction.
We concluded that MeltINGENY program makes the decision and detection system easier, and more simple as the melting profile of target sequence is determined by the software. In case of MC1R gene, PCR-TTGE method is appropriate for SNP detection, however PACAP gene polymorphism can not be identified by the method, because PACAP mutations are not included in melting domains, therefore PCR-TTGE cannot detect them.
-
Fruit melanotic ringspot (FMRS) – a disease of resistant Capsicum genotypes infected with Tomato spotted wilt virus (TSWV) on the fruits
64-69Views:64Etiology of pepper fruit melanotic ringspot (FMRS) disease (Salamon, 2009) was studied on fruit samples collected in forced pepper populations. It was noticed that in spite of heavy thrips (Frankliniella occidentalis) infestations and of TSWV epidemy detected in the forcing houses, FMRS occurred only in plants having healthy foliage. Symptomatological surveys strongly suggested that FMRS appeared exclusively in specific pepper genotypes. The size of melanotic ringspots has been observed to grow at room temperature during postripening of diseased fruits. A mechanically transmitted plant virus was isolated from symptomatic parts of 9 white pepper fruits affected by FMRS. On test plants each of the virus isolates caused systemic symptoms characteristic to TSWV. Using cDNA/PCR technique and TSWV N-gene specific primers a ca. 300 bp long DNA fragment has been amplified from total nucleic acid extracted from symptomatic tissues but never from asymptomatic parts of the fruits showing FMRS. Plant progenies grown from seeds of FMRS diseased fruits segregated in respect of resistance and/or susceptibility to TSWV infection. TSWV was also detected in and isolated from three fruits showed non-melanotic yellow rings (one of them was infected with a tobamovirus, too). Seedlings derived from these fruits proved to be susceptible to TSWV. Based on the above results we could conclude that the FMRS disease developed on fruits of “cecei” type white peppers that carry a TSWV resistance gene, most likely the Tsw gene in heterozygous form. These fruits were infected with thrips transmitted TSWV and FRMS appeared as a hypersentive reaction (HR) manifested in fruits.
-
The effect of β-glucan, carotenoids, oligosaccharides and anthocyanins on bacteria groups of excreta in broiler chickens
15-20Views:175This study was conducted to examine the effect of natural compounds, such as β-glucan, carotenoids, oligosaccharides, and anthocyanins in the diet on bacteria gropus of excreta in Ross 308 broiler chickens. Chickens were fed 5 diets: control (basal) diet, a diet supplemented by β-glucan at 0.05%, and diets supplemented by carotenoids, oligosaccharides, or anthocyanins at 0.5% of each compound. On experimental day 19, excreta were collected to determine the proportion of Lactobacillus, Bifidobacterium, Campylobacter, Clostridium, and Escherichia coli. Samples were collected aseptically and snap-frozen in liquid nitrogen. Bacterial DNA was isolated from samples, then polymerase chain reaction using primer pairs designed to the 16S rDNA of bacterial groups were applied to define the proportion of the mentioned bacteria. Another universal primer pair was used to amplify a region of 16S rDNA of all the examined bacteria. Proportion of each bacterial groups was determined relatively to the intensity of universal PCR product band by gel documenting system and ImageLab software. Based on the results, carotenoids and anthocyanins increased the proportion of Bifidobacterium, which might imply the beneficial effects of the mentioned compounds on the bacteria composition of excreta.