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The association of rpoβ gene polymorphism and multi-drug resistance pattern of clinical isolates of Mycobacterium tuberculosis
Abstracts / New Biotechnology 33S (2016) S1–S213
S175
Fusion DNA polymerase as a useful tool in molecular diagnostic and genetic engineering
(polymerase chain reaction) in 69.6% of non-converged MDR-TB patients indicated that most of drug resistance development is due to mutation at this position and the high prevalence of mutant rpoB allele.
∗ , Beata Krawczyk, Marcin Olszewski ´ Marta Spibida
http://dx.doi.org/10.1016/j.nbt.2016.06.1327
P23-7
Gda´ nsk University of Technology, Poland DNA polymerase is an enzyme which plays crucial role in replication and DNA repair. One of the most important steps in polymerisation activity of this enzyme, which is responsible for their final efficiency, is initiation step connected with binding to matrix DNA. Therefore, it is reasonable to modify well known DNA polymerases in order to facilitate binding to polymerised DNA strand. Example of such modification may be creation of fusion DNA polymerases with proteins which naturally binds to single and double stranded DNA. However there is only few examples described in scientific literature. There is lack of information about fusion of the most frequently used in molecular diagnostic Taq polymerase with single/double stranded binding proteins. The aim of our study was determined the influence on particular properties of Taq DNA polymerase essential in molecular diagnostic and genetic engineering. The polymerase was connected with with NeqSSB – small proteins with ability to bind ds and ssDNA from bacteria Nanoarcheum equitans by 6-amino acides linker. Taq DNA polymerase was produced in E. coli cell, purified with using metal affinity chromatography and concentrated to a dozen units per microliter. Properties of obtained protein was determined using different PCR sets and compared to wild enzyme. Obtained fusion polymerase showed increasing of processivity and resistant to inhibitors form blood sample in comparison to unmodified Taq polymerases. The fusion with DNA binding protein proved to favorably influence on properties useful in molecular diagnostic and genetic engineering.
P23-9 HOOF-print technology assay for rapid differential detection of strains of Brucella melitensis Hui Zhang Shihezi University, China Identifying and discriminating the source of Brucella melitensis from vaccination or natural infection using the HOOF-Prints technology. 8 pairs of primers were respectively designed based on the biological analysis of 8 flanking sequences of Brucella melitensis by the software in present work. Phylogenetic analysis and the determination of vaccine mutation for M5 using HOOF-Prints technology also have been carried out in present work. Take alterable octameric nucleotides as the primers, analyzing and comparing the amplified short polymorphic fragments. On the phylogenetic analysis of short polymorphic fragments, there are significant differences between the vaccine strain M5 and wild type M0217. The result of experiments provides an important insight to identify and discriminate the infection source between vaccine strain and wild type of Brucella. Thus, some losses due to the seropositive elimination of livestock can be avoided. In parallel, it solved the quarantine of importing and exporting which is hard to distinguish the infection between vaccine and wild during the trade circulation of animals. http://dx.doi.org/10.1016/j.nbt.2016.06.1328
http://dx.doi.org/10.1016/j.nbt.2016.06.1326
Genomics P23-8
P24-1
The association of rpo gene polymorphism and multi-drug resistance pattern of clinical isolates of Mycobacterium tuberculosis
Analysis of bacteria isolated from honey and honeybee stomach
Nega Berhane Tessemma University of Gondar, Ethiopia Tuberculosis (TB) is the leading cause of death in the world due to bacterial infection. Despite the use of effective chemotherapy in the past years, drug-resistant TB especially multidrug resistance (MDR). Rifampin (RIF) is one of the most important TB chemotherapeutic agents that act by inhibiting mycobacterial transcription, targeting DNA-dependent RNA polymerase (rpoB). The aim of the present study was to determine rpoB gene polymorphism and its association with Multi Drug Resistance (MDR) pattern of MTB by PCR and to determine the associated risk factors for drug resistance development. In this study rpoB mutations in the hot spot region (511–533 codons) were detected in 32 (69.6%) out of 46 smear positive (non-converged) MDR-TB patients and 5(10.87%) out of 46 smear negative (converged) MDR-TB patients. However, mutant allele was not detected in smear positive susceptible counter parts. The patients’ prior anti TB treatment history, HIV infection, origin of infection and drug misuse were found significant risk factors (p = .000, .000, .004, .000, respectively) for drug resistance development. The detection of mutations at 511–533 codons by PCR
Alexandra Veress 1,∗ , János Kömüves 2 , Tímea Wilk 1 , Edit Zajácz 3 , Zoltán Kerényi 1 , Róbert Kocsis 3 , Ferenc Olasz 4 , Péter Papp 5 1
National Agricultural Research and Innovation Centre, Hungary Agricultural Biotechnology Institute, Hungary 3 Agricultural Biotechnology Center, Hungary 4 Research Centre for Farm Animal Gene Conservation, Hungary 5 Hungarian Dairy Research Institute, Hungary 2
Honey is regarded as valuable in keeping human’s good health and has been used in traditional medicine since ancient times. The beneficial effects of honey are attributed to the metabolic products of bacteria living in the gastrointestinal tract of Apis mellifera. Lactic acid bacteria (LAB), isolated from honey and honeybee stomach may have positive effects on human and animal health, e.g. Lactobacilli are widely utilized as probiotics. The aim of this study was to isolate LAB strains from honey and honeybee stomach samples. The isolates were identified at species level by using their 16S rDNA gene sequences. In order to characterize the strains, antibacterial activity was tested against indicator bacteria. Isolates of Lactobacillus spp. that were cultured from different honeybee stomach samples inhibited the growth of Escherichia coli and Salmonella enterica. Lactobacillus helsingborgensis and L. kunkeei strains could be candidates for probiotic
S175
Fusion DNA polymerase as a useful tool in molecular diagnostic and genetic engineering
(polymerase chain reaction) in 69.6% of non-converged MDR-TB patients indicated that most of drug resistance development is due to mutation at this position and the high prevalence of mutant rpoB allele.
∗ , Beata Krawczyk, Marcin Olszewski ´ Marta Spibida
http://dx.doi.org/10.1016/j.nbt.2016.06.1327
P23-7
Gda´ nsk University of Technology, Poland DNA polymerase is an enzyme which plays crucial role in replication and DNA repair. One of the most important steps in polymerisation activity of this enzyme, which is responsible for their final efficiency, is initiation step connected with binding to matrix DNA. Therefore, it is reasonable to modify well known DNA polymerases in order to facilitate binding to polymerised DNA strand. Example of such modification may be creation of fusion DNA polymerases with proteins which naturally binds to single and double stranded DNA. However there is only few examples described in scientific literature. There is lack of information about fusion of the most frequently used in molecular diagnostic Taq polymerase with single/double stranded binding proteins. The aim of our study was determined the influence on particular properties of Taq DNA polymerase essential in molecular diagnostic and genetic engineering. The polymerase was connected with with NeqSSB – small proteins with ability to bind ds and ssDNA from bacteria Nanoarcheum equitans by 6-amino acides linker. Taq DNA polymerase was produced in E. coli cell, purified with using metal affinity chromatography and concentrated to a dozen units per microliter. Properties of obtained protein was determined using different PCR sets and compared to wild enzyme. Obtained fusion polymerase showed increasing of processivity and resistant to inhibitors form blood sample in comparison to unmodified Taq polymerases. The fusion with DNA binding protein proved to favorably influence on properties useful in molecular diagnostic and genetic engineering.
P23-9 HOOF-print technology assay for rapid differential detection of strains of Brucella melitensis Hui Zhang Shihezi University, China Identifying and discriminating the source of Brucella melitensis from vaccination or natural infection using the HOOF-Prints technology. 8 pairs of primers were respectively designed based on the biological analysis of 8 flanking sequences of Brucella melitensis by the software in present work. Phylogenetic analysis and the determination of vaccine mutation for M5 using HOOF-Prints technology also have been carried out in present work. Take alterable octameric nucleotides as the primers, analyzing and comparing the amplified short polymorphic fragments. On the phylogenetic analysis of short polymorphic fragments, there are significant differences between the vaccine strain M5 and wild type M0217. The result of experiments provides an important insight to identify and discriminate the infection source between vaccine strain and wild type of Brucella. Thus, some losses due to the seropositive elimination of livestock can be avoided. In parallel, it solved the quarantine of importing and exporting which is hard to distinguish the infection between vaccine and wild during the trade circulation of animals. http://dx.doi.org/10.1016/j.nbt.2016.06.1328
http://dx.doi.org/10.1016/j.nbt.2016.06.1326
Genomics P23-8
P24-1
The association of rpo gene polymorphism and multi-drug resistance pattern of clinical isolates of Mycobacterium tuberculosis
Analysis of bacteria isolated from honey and honeybee stomach
Nega Berhane Tessemma University of Gondar, Ethiopia Tuberculosis (TB) is the leading cause of death in the world due to bacterial infection. Despite the use of effective chemotherapy in the past years, drug-resistant TB especially multidrug resistance (MDR). Rifampin (RIF) is one of the most important TB chemotherapeutic agents that act by inhibiting mycobacterial transcription, targeting DNA-dependent RNA polymerase (rpoB). The aim of the present study was to determine rpoB gene polymorphism and its association with Multi Drug Resistance (MDR) pattern of MTB by PCR and to determine the associated risk factors for drug resistance development. In this study rpoB mutations in the hot spot region (511–533 codons) were detected in 32 (69.6%) out of 46 smear positive (non-converged) MDR-TB patients and 5(10.87%) out of 46 smear negative (converged) MDR-TB patients. However, mutant allele was not detected in smear positive susceptible counter parts. The patients’ prior anti TB treatment history, HIV infection, origin of infection and drug misuse were found significant risk factors (p = .000, .000, .004, .000, respectively) for drug resistance development. The detection of mutations at 511–533 codons by PCR
Alexandra Veress 1,∗ , János Kömüves 2 , Tímea Wilk 1 , Edit Zajácz 3 , Zoltán Kerényi 1 , Róbert Kocsis 3 , Ferenc Olasz 4 , Péter Papp 5 1
National Agricultural Research and Innovation Centre, Hungary Agricultural Biotechnology Institute, Hungary 3 Agricultural Biotechnology Center, Hungary 4 Research Centre for Farm Animal Gene Conservation, Hungary 5 Hungarian Dairy Research Institute, Hungary 2
Honey is regarded as valuable in keeping human’s good health and has been used in traditional medicine since ancient times. The beneficial effects of honey are attributed to the metabolic products of bacteria living in the gastrointestinal tract of Apis mellifera. Lactic acid bacteria (LAB), isolated from honey and honeybee stomach may have positive effects on human and animal health, e.g. Lactobacilli are widely utilized as probiotics. The aim of this study was to isolate LAB strains from honey and honeybee stomach samples. The isolates were identified at species level by using their 16S rDNA gene sequences. In order to characterize the strains, antibacterial activity was tested against indicator bacteria. Isolates of Lactobacillus spp. that were cultured from different honeybee stomach samples inhibited the growth of Escherichia coli and Salmonella enterica. Lactobacillus helsingborgensis and L. kunkeei strains could be candidates for probiotic