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The aim of the study was to develop multiplex PCR (mPCR) assays which allow identification of shigatoxigenic Escherichia coli (STEC) strains and a characterization of their virulence properties. As an internal control, a fragment of 16S rRNA gene - two amplicons of 230 bp (stx gene) and 401 bp (16S rRNA) were obtained in the test, designated as mPCR-1 for the amplification of the stx gene, characteristic for all known Shiga toxin variants. The mPCR-2 assay was developed to characterize the stx-positive bacterial colonies, which allowed the detection of Shiga toxin 1 and/or 2, the affiliation of STEC O157:H7 serotype (rfbO157 and fliCн₇ genes), and internal control, resulting in amplicons of 348 bp (stx1), 584 (stx2), 420 bp (rfbO157), 247 bp (fliCн₇) and 798 bp (16S rRNA). The detection of markers O26wzx, rfbO111 and rfbO113, typical for E. coli O26, O111 and O113, respectively, was performed with mPCR-3. The products of molecular masses 153, 406 and 593 bp were observed. All STEC’s were also tested using mPCR-4, for the presence of enterohemolysin (ehlyA) and intimin (eaeA) markers generating specific bands of 837 bp (eaeA gene), 534 bp (ehlyA) and 401 (16S rRNA). The mPCR assays developed allow STEC to be identified and characterized and may be used in routine diagnosis of these bacteria.
The aim of the study was to assess the suitability of multiplex PCR and culture for the detection of virulent R.equi in tracheobronchial aspirate (TBA) and feces of foals from enzootic farms. Fecal and TBA samples were taken randomly from a representative group of foals aged between 1 and 6 months. The solid selective medium NANAT was used for culture examination. Multiplex PCR reaction was performed with the use of two sets of primers complementary to the conservative gene fragment encoding the 16S subunit of ribosomal RNA of R.equi and the plasmid gene encoding virulence associated protein A (VapA), which determines bacterial virulence. During clinical observations three experimental groups (A, B, C), differing in the intensity of respiratory signs, were selected for further studies. In foals from group A, showing no clinical signs from the respiratory tract, the results of the examinations of TBA and fecal samples were negative irrespective of the method used. In group B, showing moderate respiratory signs, 15 TBA and fecal samples were examined. PCR results were positive for 7 TBA samples and 2 fecal samples, whereas culture examinations were positive for only 3 TBA samples from this group. In group C, consisting of 6 foals with severe respiratory signs, positive PCR and culture results were obtained for all TBA samples and for 3 fecal samples.
Celem badań było ustalenie czy modyfikacja metody MLST opracowanej przez Sreedhar i wsp. (2002) do różnicowania szczepów enterokoków pozwala na uzyskanie wyników wystarczających do realizacji powyższego zadania. Wprowadzona modyfikacja polegała na zastąpieniu sekwencjonowania produktu amplifikacji analizą restrykcyjną. Wyniki analizy 32 szczepów Enterococcus faecalis izolowanych w regionie gdańskim potwierdziły przydatność powyższej modyfikacji do różnicowania szczepów enterokoków.
Celem podjętych badań była identyfikacja oraz analiza molekularna 35 szczepów Staphylococcus spp., wyizolowanych z mleka krów z kliniczną postacią mastitis, pod kątem ich potencjalnej zdolności do wytwarzania enterotoksyn gronkowcowych (SEs), najczęściej wywołujących zatrucia pokarmowe ludzi (SEA-SEE).Wytypowane szczepy scharakteryzowano wstępnie na podstawie wzrostu kolonii bakteryjnych na chromogennym podłożu CHROMagar Staph. aureus. Następnie wszystkie izolaty analizowano z użyciem gatunkowo specyficznej reakcji PCR, ukierunkowanej na wykrycie dwóch markerów molekularnych: nuc – specyficznego dla gatunku S. aureus i gehM – specyficznego dla S. xylosus. Dodatkowo oznaczano enterotoksynogenność wykrytych szczepów (obecność genów sea-see, w tym secv), z użyciem metody multipleks PCR. Spośród 35 analizowanych izolatów aż 18 szczepów (51,4%) stanowiło gronkowce koagulazoujemne (CNS) z gatunku S. xylosus, a tylko dwa izolaty (5,7%) należały do koagulazododatnich szczepów S. Aureus. W przypadku siedmiu szczepów S. xylosus (20%) wykryto metodą multipleks PCR geny enterotoksyn (se) zidentyfikowane jako: sea (71,4%), secv (14,3%) oraz sed (14,3%). Ponadto, w DNA pięciu spośród tych szczepów (71,4%), uzyskano amplikony specyficzne dla innych, bliżej nieokreślonych genów se lub ich wariantów, które na podstawie analizy in silico oznaczono jako: seg, seh, sei lub selu. Uzyskane wyniki wskazują na konieczność uwzględniania w diagnostyce gronkowców koagulazoujemnych ich właściwości toksynogennych, ponieważ mogą one stanowić ważną grupę mikroorganizmów obecnych w żywności. Przyczyniły się także do podkreślenia znaczącej roli enterotoksynogennych szczepów S. xylosus w etiologii mastitis.Wykonane badania potwierdziły przydatność metod molekularnych do wykrywania potencjalnie enterotoksycznych szczepów Staphylococcus spp. izolowanych od zwierząt.
Zastosowano metodę multiplex PCR i PCR-RFLP (PCR-restriction fragment length polymorphism) do identyfikacji szczepów Bacillus anthracis. Metoda multiplex PCR pozwalała na wykrycie genów kodujących czynniki wirulencji lef, cya,pag u szczepów B. anthracis obecnych w plazmidzie pXO1, genu cap występującego w plazmidzie pXO2 oraz markera chromosomalnego Ba813. Analiza genotypowa sekwencji SG-749 z zastosowaniem metody PCR- RFLP wykazała, że wszystkie badane szczepy B. anthracis, charakteryzowały się tym samym wzorem restrykcyjnym złożonym z dwóch fragmentów DNA o wielkości 90 i 660 bp, co umożliwiło odróżnienie ich od innych blisko spokrewnionych gatunków bakterii z grupy B. cereus posiadających różne wzory restrykcyjne.
Listeria monocytogenes is an important foodborne pathogen that causes a disease known as listeriosis, which is especially dangerous for pregnant women. Infection with L. monocytogenes may also result in stillbirths, abortions and premature deliveries, as well as meningitis, septicaemia, encephalitis, and meningoencephalitis. Conventional detection methods of these bacteria are time-consuming; therefore, rapid alternative methods, including those based on molecular tests are needed. PCR is sensitive and specific; however, it requires the use of an agarose gel, which increases the time of analysis. A technique that allows the elimination of this step is real-time PCR, which enables the quantitative determination of L. monocytogenes in foods. A modification of PCR is multiplex PCR that allows detection of several genes at the same time and distinguishes between different species of microorganisms. Techniques such as RT-PCR or NASBA, where the target molecule is RNA, are used to detect viable cells and also allow quantitative analyses to be performed. Another rapid and specific method is LAMP, which can be performed in one hour in a water bath or heating block, without the use of a thermocycler. Biosensors and microarrays are examples of new technologies that due to the possibility to use anywhere and immediate interpretation of the results can be routinely used in the future for identification of L. monocytogenes in food.
Laboratory diagnosis of swine dysentery (SD) and proliferative enteropathy (PE) by standard bacteriological methods is time consuming and bears the risk of false negative results. Limitations concerning the isolation of L. intracellularis and difficulties with conventional bacteriological procedures were the primary reasons for developing a PCR for the diagnosis of PE and SD. The aim of this study was to develop a multiplex PCR for the detection of B. hyodysenteriae and L. intracellularis and to determine the usefulness of this technique in diagnosing the above mentioned diseases. The investigations were evaluated on strains of B. hyodysenteriae B204 and the bacterial filtrate of L. intracellularis. In order to determine the sensitivity of multiplex PCR from bacterial suspension (B. hyodysenteriae 2 × 108 cfu/ml) and (L. intracellularis - 1.1 × 106 cfu/ml) 10-fold dilutions were prepared in a Tris-HCl, pH 8.5 buffer or in supernatant of swine feces in Tris-HCl, pH 8.5 buffer. The elaborated multiplex PCR was able to detect 1.1 × 104 cfu/ml L. intracellularis and 2 × 105 cfu/ml B. hyodysenteriae. In analyzing the sensitivity of multiplex PCR in the presence of the two mentioned species of bacteria in feces it was assumed that the presence of a 2000 times greater number of B. hyodysenteriae cells in fecal sample than L. intracellularis did not decrease the sensitivity of multiplex PCR in the detection of L. intracellularis. At the same time about a two times higher amount of L. intracellularis cells than B. hyodysenteriae decreased the sensitivity of multiplex PCR for the detection of B. hyodysenteriae by 200 times. However the presence of porcine feces which contain inhibitory factors decreased the detection of L. intracellularis. Amplification of extracted DNA from feces suspension gave a 100 times lower sensitivity than amplification of extracted DNA from Tris-HCl, pH 8.5 buffer with the same number of CFU/ml L. intracellularis. On the other hand sensitivity of the method was satisfying even if the number of B. hyodysenteriae cells was 2000 times higher. The results proved that multiplex PCR for the detection of B. hyodysenteriae was not susceptible to inhibitory substances, however twice as high a number of L. intracellularis cells than B. hyodysenteriae in a sample hampered the amplification specific to B. hyodysenteriae. The results of this study demonstrated that multiplex PCR is useful for the detection of L. intracellularis. In case of an intensive infection of pigs by both pathogens multiplex PCR has limitations in the diagnosis of B. hyodysenteriae.
Shiga toxin-producing E. coli (STEC) are a serious cause of human diseases. The infections are mainly associated with strains belonging to serogroups O157, O26, O103, O111 and O113, while the main source of infection is contaminated food of animal origin (especially beef). In this study the molecular identification methods for STEC were used which enabled detection of STEC in raw beef using multiplex PCR (mPCR) assays, isolation of individual bacterial colonies with digoxigenin (DIG)-labeled DNA probe and characterization of the virulence markers by the use of mPCR. The molecular method was used to examine 272 raw beef samples obtained from slaughterhouses. After the mPCR-1 analysis 16 stx-positive samples (5.88%) were detected. The STEC isolates were then tested using the mPCR and PCR assays. Eight of them belonged to O157:H7 serotype by the presence of the rfbO157 and fliCн₇ genes. The stx₁ marker was observed in all E. coli O157:H7 and in four of the non-O157:H7 isolates tested. Moreover, the stx₂ gene was detected in all E. coli O157:H7 and in seven of the non-O157:H7, three of them also possessed the stx₁ marker. Eight of the rfbO157-negative STEC were examined with the mPCR-3 assay to detect the rfbO111, rfbO113 and O26wzx markers, three of them produced the characteristic amplicon for the O26 group. These isolates also carried the stx₁ (one strain) or stx₂ genes (two isolates). Among the STEC tested, none belonged to the O111 or O113 groups. The amplification with the mPCR-4 assay showed that all isolates harbored the enterohemolysin (ehlyA) gene whereas the intimin marker (eaeA) was observed in all E. coli O157:H7 and O26 isolates.
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