Dierengezondheidszorg Vlaanderen Torhout
Abstract Bachelor Project FBT 2019-2020: The optimization of the real-time PCR protocol for Chlamydia psittaci, Chlamydia suis and Streptococcus suis
The aim of this thesis was to optimize the Real-Time PCR protocol for Chlamydia psittaci, Chlamydia suis and Streptococcus suis. Chlamydia psittaci and Streptococcus suis both cause infectious diseases in humans. Chlamydia suis species only infects pigs. The protocols of Chlamydia psittaci and Chlamydia suis are optimized using positive controls. The streptococcus suis protocol is tested on strains from the bacteriology department. The animal health care lab aims to implement an in-house PCR protocol for these bacteria.
In the first test, two methods to determine Chlamydia psittaci were compared: a Kylt kit and a protocol based on SYBR Green. This comparison was made using two positive controls from the Kylt kit and an Amplirun positive control. The second test was performed using two positive samples for Chlamydia suis and a Taqman probe protocol. In the final test, several primer pairs were applied on Streptococcus suis strains grown in the bacteriology department using a SYBR Green-based protocol. These primer pairs target virulence factors, housekeeping genes and capsular polysaccharides of the Streptococcus suis strains. For Streptococcus suis, two tests were performed. First, an initial set of primer pairs was tested, of which only the primer pairs of suilysine, muramidase released protein, IPC16S and arginine deiminase were tested in the second test.
When comparing the two methods of Chlamydia psittaci, the three control samples included in the kit showes a positive result. In the method based on SYBR Green, only the Amplirun positive control gave a positive result. This was due to the fact that no complete DNA sequence is present in the positive controls from the kit. The added pimers could not bind in the correct place. Therefore, the positive controls of the kit could not be used in the SYBR Green based method. When testing Chlamydia suis strain 4-29 b samples, the result of the HEX channel is doubtful because the obtained Ct values are higher than 35. The values of the FAM-channel of these samples are lower than 35 so the values are positive. Next, both channels at C.suis strains S45/6 are positive because the Ct-value is lower than 35. At least one channel has to be positive to have a positive result. The tested C.suis samples, C.suis strain S45/6 and C.suis strain 4-29 b, are positive but C.suis strain S45/6 is used as positive control because both channels are positive.
In the first test of Streptococcus suis, the primer pairs of the extracellular factor and the capsular proteins are negative. The housekeeping gene 16S should be present in each strain, but is absent in the undiluted strain A. The genes suilysin (sly), muramidase released protein (mrp), 16S and arginin deiminase (arcA) are absent in both strains of A and B undiluted. The mrp gene is present on all strains except the undiluted strain B. The IPC16S gene is present in all samples except on both samples of strain A. In the second test, the best primer pairs from the previous test were used. Sly is not detected in both dilutions of strain 29 and 38 and mrp is absent in both dilutions of strain 38. Finally, arcA and IPC16S were detected in all strains. In both tests, the melting temperatures of the PCR products are the same. For the primer pairs of sly and arcA the annealing temperature is 78,5 °C, mrp has an annealing temperature of 79 °C and for IPC16S it is 86 °C.
For Chlamydia psittaci, the kit method is used because all controls give a positive result. In the case of C.suis the sample C.suis strains S45/6 is used as a positive control, because both the HEX and FAM channels are positive. The melting temperatures in the S.suis are the same in both tests of the corresponding PCR products. Nevertheless, all tests still need to be thoroughly validated before they can be used in the lab.
Abstract Bachelor Project FBT 2018-2019: Determination of iodine, selenium and zinc in serum using ICP-MS
Research has shown that there are deficiencies of minerals such as selenium, zinc and iodine in cattle on many cattle farms. These deficiencies cause a decrease in milk and meat production. Therefore, a reliable analysis method to detect these minerals in serum is important. The purpose of this research was to develop and optimize a method to measure the concentrations of these minerals in serum via inductively coupled plasma mass spectrometry (ICP-MS).
Firstly, a calibration curve was measured and compared to a reference run, showing that zinc contamination had occurred at some point. To detect where the contamination happened, 1- butanol was replaced by ethanol to prepare the diluent solution. The results showed a strong decrease of the zinc level in the calibration standards. Afterwards, the diluent solution was made with demineralized water instead of ultrapure water, resulting in a small decrease of the zinc level. To optimize the detection of selenium, four different ethanol concentrations in the diluent solution were tested. This showed that a higher ethanol concentration led to better results.
However, because a higher ethanol concentration resulted in a higher zinc level, a middle ground had to be chosen. To finish this research, a calibration curve was measured using a few samples with known concentrations of zinc, selenium and iodine. When these results were compared, no substantial difference was observed.
From these results, it can be concluded that the used method is almost ready for measurement of mineral concentrations in cattle serum. In the future, a validation must be performed while keeping the zinc contamination as low as possible.