BLT Stages

Abstract Bachelor Project 1 FBT 2020-2021:Validation of metals and trace elements in food products and animal feeds from vegetable and animal origin using ICP-MS

The increasing amount of metal pollution is problematic and potentially dangerous for humans and animals. Regular analysis of these metals in food products and animal feeds can help prevent intoxication. Inagro wants to validate a couple of analysis methods that are able to measure the amount of trace elements accurately and reliably. Analysis is done by using inductively coupled mass spectrometry.

These methods focus on analyzing the amount of antimony, chromium, stibium, selenium and molybdenum. After weighing, samples are completely dissolved in acid before they are analyzed in a inductively coupled mass spectrometer. The samples themselves are being split into different matrix groups. For each group the method performance is evaluated using the linearity, limit of quantitation, precision, trueness and the uncertainty.

Analyzing all samples for each matrix group showed that the method performance was on point with the expectations. Improvements could be made to lower the uncertainty for the parameter stibium and selenium. Overall the validation was successfully concluded and the analysis methods proved to be accurate and reliable for its purpose.

Abstract Bachelor Project 2 FBT 2020-2021:Identification molds isolated during the retting proces flax and development identification method Erwinia amylovora

This thesis has two subjects, identification of molds isolated during the dew retting process of flax and the development of an identification method for E. amylovora. Molds are known to have an influence on the process of dew retting and E. amylovora is a bacterium that causes fire blight in the family Rosaceae. The aims of this thesis are developing a method specifically to identify fire blight and identifying what molds are present during the dew retting process. This might give an indication as to what species are interesting to further investigate. For identification of molds the methods include DNA-extractions, PCR using ITS5-4 primers, Sanger sequencing and enzyme tests. For the development of an identification method for E. amylovora, gradient PCR and qPCR with amsC primers are used.

The concentrations after DNA-extractions were strongly dependent on the humidity of the molds, affecting the weight of the molds used to perform the DNA-extraction. PCR gave DNA-fragments between 500 and 900 bp, indicating that the ITS-region was amplified. The concentrations after purifications varied but did not affect the results of Sanger sequencing. Alternaria, Fusarium and Epicoccum were the most recurring results. The results of the qPCR optimalisation for E. amylovora indicate that an annealing temperature of 61°C is optimal, combined with the primers concentration of 900 nM and a probe concentration of 200 nM. Other bacteria do not interfere with the Ct-values. It can be concluded that the most frequent colonizers during dew retting are Alternaria, Fusarium and Epicoccum. For the identification of E. amylovora, it can be concluded that the method is applicable for identification, using the determined optimal parameters.

Abstract Bachelor Project FBT 2019-2020: Development of a genetic deteriminationmethod on Trichoderma and Fusarium oxysporum f.sp. lactucae

Some Fungi like Trichoderma aggressivum and Fusarium oxysporum f.sp. lactuae (Fol) can cause severe losses in yield and quality of crops. This is a problem that keeps expanding through the agriculture community. For diminishing this problem there must be a fast and precise determination method of T. aggressivum and Fol.

The goal of this study is to develop a general polymerase chain reaction (PCR) for the identification of Trichoderma species specially to identify T. aggressivum and a PCR to determine the physio type of Fusarium oxysporum f.sp. lactucae.

First, sections of a raw sample are inoculated on an agar plate. The second step is to determine which kind of fungus there is growing by use of microscopy. If it is the pathogenic Fungus, the Fungus can be subcultured to obtain a pure culture of the pathogen. The next step includes the inoculation of the purified Fungus on potato dextrose broth (PDB). After incubation the desoxyribonucleic acid (DNA) will be extracted from the culture. For the fifth step a PCR set-up is created, run and optimized by using different primers and different conditions. The PCR results were checked by capillary electrophoresis. Sequencing is performed by using the Sanger method, where the sequence is determined using a radioactive primer. The obtained sequences were aligned against a database.

The primers ITS4-ITS5 have for nearly every Trichoderma sample a positive result. The positive results are linked with a Trichoderma spp. and are compared with the results of another laboratory. The primers Tef1-Ef1(728) and ITS1-ITS2 have far fewer positive results but every result can be linked with the right Trichoderma spp. The PCR for Fol physio 1 and physio 4 is both negative.

The most results for Trichoderma are booked with the primers ITS4-ITS5. Although there are some faults in the determination of the Trichoderma spp. The faults are the blame of the general specificity. The primers ITS1-ITS2 can determine the different Trichoderma spp. but not on formae level. The primers Tef1-Ef1(728) have for each positive result the correct determination of the Trichoderma spp. Therefore, it is needed to keep researching if these primers are more specific than ITS4-ITS5. The determination of the physio type of Fol must be further investigated with a more sensitive PCR machine.

Abstract Bachelor Project FBT 2019-2020: Determination of metals in surface, ground and drinking water using ICP-MS and ICP-AES

The request for the determination of metals in water is increasing due to the improved knowledge of the consequences of the metals. So the aim of this study is to find and validate a method to measure metals in ground-,drink- and surface water. With a validated method Inagro can obtain an acknowledgment from an accreditation institute.

The methods are based on the methods that are already used in Inagro. A digestion block is used to pretreat the monsters and the Inductive Coupled Plasma – Mass Spectrometry (ICP-MS) and Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) are used to measure the concentrations of the metals.

The results show that for cadmium, lead, molybdenum, nickel, antimony, cobalt, arsenic and selenium (measured on the ICP-MS) the linearity, limit of quantification and trueness are within the standards for ground-, drink- and surface water. Only for arsenic in surface water the trueness is not within the standards. An interference can be the cause of the bad result. In a next step the intra-reproducibility, repeatability and expanded uncertainty can be determined for the elements accept for arsenic in surface water. For aluminum, boron, chromium, iron, copper, manganese, zinc, phosphorus, calcium, sodium, magnesium and potassium (measured on the ICP-AES) a good wavelength has been found to determine the linearity. These elements except boron and aluminum are now measured on the ICP-MS, so there are no further results. The linearity has to be redetermined.

There can be concluded that the validation is going in a good direction, but it’s not finished.

Abstract Bachelor Project FBT 2018-2019: Development of identification techniques for plant pathogenic fungi and bacteria

Phytopathogens can cause enormous losses in yield and quality of crops. Different crops are threatened by various plant diseases. The increasing problem provides the access to novel findings to characterize fungi and bacteria. Taxonomic identification is important for controlling plant pathogens.

Morphological approaches are routinely used in taxonomic studies for classification of fungi and bacteria. Using morphology alone is too limited, that’s why researchers are using a combination of morphology and molecular data. The goal of this study is to develop a general polymerase chain reaction (PCR) for the identification of fungi and bacteria. In parallel, there was also made a flow chart for a general identification of bacteria.

First, desoxyribonucleic acid (DNA) is extracted from a pure culture of fungi or bacteria. By using universal primers, a selected locus is amplified. PCR results were checked by capillary electrophoresis. When successful, the 16S ribosomal DNA (rDNA) genes and the internal transcribed spacer (ITS) region are ready to be sequenced. Sequencing is performed by using the Sanger method, where the sequence is determined using a radioactive primer. The obtained sequences were aligned against a database.

The flow chart is based on four selective biomedical tests: MacConkey agar, oxidase test, King B agar and Yeast dextrose calcium carbonate agar (YDC).

By using five known fungi and bacteria species an optimized PCR protocol was obtained. The ITS region of fungi was amplified and the primer pair ITS5/4 was selected. The primer pair 27F/1492R was chosen for the amplification of the entire 16S rRNA region of bacteria. The effectivity was tested on unknown fungi species. It is necessary to check the GenBank identifications and to isolate the primary infection.

The theoretical flow chart was tested on known bacteria species. Most test results were as expected, but the color on MacConkey is not always pronounced.

An optimized protocol was developed for the identification of fungi and bacteria. When possible, both morphology and molecular data should be used for correct identification. Otherwise, the optimized protocol is used when the morphology is too unclear. The flow chart gives a general idea of the identification on genus level.

Abstract Bachelor Project 2017-2018: Determination of metals in surface-, ground- and drinking water using ICP-MS

The increased knowledge of the effect of metals in water has led to a raising demand for the determination of metals in water. Therefore, Inagro wants to obtain a recognition from an accreditation institution.

Inductive Coupled Plasma – Mass Spectrometry (ICP-MS) is used to measure the concentrations of metals. A development of the calibration curves, control samples and internal standards will lead to a usable method. Interferences must also be investigated in order to be able to assess the influence on other results. Eventually a validation can be performed to quantify the accuracy of the method.

Results show that some elements are difficult to measure. Especially mercury, aluminium, selenium and boron (only for ground and surface water) are the most problematic. Linearity, Limits of detection and trueness are reached for almost all elements with the ground and drinking water method. Some deviant linearity’s or high reproducibility’s lead to problems. However, the method for surface water shows more problems where especially repeatability has to improve. The measurement uncertainty depends on good results for trueness and repeatability. For this reason, some elements have obtained too high measurement uncertainties.

Taking all results in count, the surface and drinking water method comes up as the best method. For the majority of the elements recognition will be possible. The other method for surface water and some elements of drinking and surface water will require improvements before the method can enter routine measuring.