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Tytuł:
In situ detection of DNA and mRNA of human cytomegalovirus to distinguish different forms of viral infection in leukocytes
Autorzy:
Zawilinska, Barbara
Bulek, Katarzyna
Kopec, Jolanta
Kosz-Vnenchak, Magdalena
Tematy:
in situ PCR
PCR
antigenemia pp65
cytomegalovirus
in situ RT-PCR
Pokaż więcej
Wydawca:
Polskie Towarzystwo Biochemiczne
Powiązania:
https://bibliotekanauki.pl/articles/1041199.pdf  Link otwiera się w nowym oknie
Opis:
In situ PCR and in situ reverse transcription PCR (RT-PCR) were applied to discriminate between latent and productive infection of human cytomegalovirus (HCMV) in leukocytes. We investigated 28 samples, in which viral pp65 antigen was detected only in the cytoplasm of leukocytes. Additionally we assayed 12 specimens lacking pp65 antigen. Using nested PCR (nPCR), viral DNA was detected in 27 samples. In six samples the results of nPCR were unreadable due to the presence of polymerase inhibitors. By application of in situ PCR, we were able to confirm the presence of viral DNA in the nucleus and/or cytoplasm. Productive infection was recognized in 20 samples in which transcripts for late viral genes were detected. Among the 20 samples negative by in situ RT-PCR, we recognized phagocytosis of viral particles in eight and the latent form of HCMV infection in five.
Dostawca treści:
Biblioteka Nauki
Artykuł
Tytuł:
PCR and real-time PCR assays to detect fungi of Alternaria alternata species
Autorzy:
Kordalewska, Milena
Brillowska-Dąbrowska, Anna
Jagielski, Tomasz
Dworecka-Kaszak, Bożena
Tematy:
Alternaria alternata
detection
identification
PCR
real-time PCR
Pokaż więcej
Wydawca:
Polskie Towarzystwo Biochemiczne
Powiązania:
https://bibliotekanauki.pl/articles/1038891.pdf  Link otwiera się w nowym oknie
Opis:
Fungi of the Alternaria genus are mostly associated with allergic diseases. However, with a growing number of immunocompromised patients, these fungi, with A. alternata being the most prevalent one, are increasingly recognized as etiological agents of infections (phaeohyphomycoses) in humans. Nowadays, identification of Alternaria spp. requires their pure culture and is solely based on morphological criteria. Clinically, Alternaria infections may be indistinguishable from other fungal diseases. Therefore, a diagnostic result is often delayed or even not achieved at all. In this paper we present easy to perform and interpret PCR and real-time PCR assays enabling detection of A. alternata species. On the basis of alignment of β-tubulin gene sequences, A. alternata-specific primers were designed. DNA from fungal isolates, extracted in a two-step procedure, were used in PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The assays specificity was confirmed, since positive results were obtained for all A. alternata isolates, and no positive results were obtained neither for other molds, dermatophytes, yeast-like fungi, nor human DNA. The assays developed here enable fast and unambiguous identification of A. alternata pathogens.
Dostawca treści:
Biblioteka Nauki
Artykuł
Tytuł:
Molecular detection, typing, and virulence potential of Salmonella Serotypes isolated from poultry feeds
Autorzy:
Shahbazi, G.
Shayegh, J.
Ghazaei, C.
Ghazani, M.H.M.
Hanifian, S.
Tematy:
Salmonella
feed
BOXAIR-PCR
Rep- PCR
drug resistance
biofilm
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Wydawca:
Polska Akademia Nauk. Czasopisma i Monografie PAN
Powiązania:
https://bibliotekanauki.pl/articles/16647581.pdf  Link otwiera się w nowym oknie
Opis:
Salmonella contamination in poultry feed is one of the main issues in poultry industry and public health. The aim of the present study was molecular detection and typing of Salmonella serotypes isolated from poultry feeds. Moreover, we determined the antibiotic resistance pattern and the ability of biofilm formation in the serotypes. To this end, eighty feed samples were collected from aviculture depots. Salmonella serotypes were identified by culture and PCR methods. For serological identification, a slide agglutination test was used. BOXAIR and rep-PCR methods were applied to evaluate the diversity of serotypes. The disc diffusion method was performed to evaluate the antibiotic susceptibility of serotypes to sixteen antibiotics. Biofilm formation was also assessed by the microtiter-plate test. From a total of 80 feed samples, 30 samples were contaminated with Salmonella spp., which were divided into 5 different serotypes belonging to B, C, and D serogroups. BOXAIR-PCR (D value [DI] 0.985) and rep-PCR (DI 0.991) fingerprinting of isolates revealed 23 and 19 reproducible fingerprint patterns, respectively. A higher antibiotic resistance was observed to ampicillin and doxycycline (100% each), followed by chloramphenicol (83.33%) and tetracycline (73.33%). Multidrug resistance (MDR) was detected in all Salmonella serotypes. Half of the serotypes possessed the ability of biofilm formation with varied adhesion strengths. These results revealed the high and unexpected prevalence of Salmonella serotypes in poultry feed with MDR and biofilm formation ability. BOXAIR and rep-PCR revealed a high diversity of Salmonella serotypes in feeds and subsequently indicated variation in the source of Salmonella spp. The unknown sources harboring high diversity of Salmonella serotypes indicated poor control, which could cause problems for feed manufacturing.
Dostawca treści:
Biblioteka Nauki
Artykuł
Tytuł:
Zastosowanie metod PCR i FISH w szybkiej diagnostyce bakteryjnych zakażeń krwi
Use of PCR and FISH methods for rapid identifi cation of bacterial bloodstream infections
Autorzy:
Gosiewski, Tomasz
Pietrzyk, Agata
Brzychczy-Włoch, Monika
Heczko, Piotr B.
Tematy:
diagnostyka sepsy
hodowla krwi
pcr
inhibicja pcr
fish
diagnostics of sepsis
blood culture
pcr inhibition
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Wydawca:
Śląski Uniwersytet Medyczny w Katowicach
Powiązania:
https://bibliotekanauki.pl/articles/1038423.pdf  Link otwiera się w nowym oknie
Opis:
INTRODUCTION The aim of this study was to evaluate the possibility of applying PCR and FISH methods in rapid diagnostics of bacterial bloodstream infections. MATERIAL AND METHODS 56 samples: 28 venous blood samples and 28 samples of culture media from BACTEC machine after incubation cycle were tested. All blood samples originated from patients with clinical symptoms of sepsis who were diagnosed in the Laboratory of Microbiological Diagnostics at the Chair of Microbiology Medical College Jagiellonian University in Krakow. The blood samples were tested to presence of bacteria in parallel, using PCR, FISH and culture monitored BACTEC system. Additionally, each bottle with medium was analyzed by FISH method. DNA was extracted with QIAamp DNA Blood Mini (Qiagen). To remove PCR inhibitors the step of samples purifi cation with ammonium chloride was added. For PCR reaction a pair of 16SrRNA(+) and 16SrRNA(-) starters which enables detection of all bacteria species was applied. In FISH method a probe specifi c to all bacteria species EUB338 and probes specifi c to Enterobacteriaceae (ENT183) and Staphylococcus genus (STA) were used. RESULTS Percentage of positive venous blood samples for PCR, FISH and BACTEC was: 71.4%, 28.6% and 10.7%, respectively. The diff erences between results obtained by PCR and FISH methods and by PCR and BACTEC were statistically signifi cant. In case of culture media samples analysis a percentage of positive results for FISH was 58.3%, while for culture method 33.3%. This diff erence was not statistically signifi cant. The time needed to receive a results of samples examination using PCR and FISH methods was about 4–5 hours. DNA amplifi cation was obtainable only for these blood samples which were in addition initially purifi ed, and special cellular sediment washing procedure was used. For unpurifi ed samples inhibition eff ect was noted. Conclusions Results obtained by us indicated that PCR and FISH methods: allow to detect bacteria in whole blood samples, are much more sensitive than culture method, shorten waiting time for results to few hours and that the use of additional procedure of blood samples purifi cation is needed to receive a positive result of amplification.
WSTĘP Celem pracy była ocena możliwości zastosowania metod PCR i FISH w szybkiej diagnostyce bakteryjnych zakażeń krwi. MATERIAŁ I METODY Analizie poddano 56 próbek: 28 próbek krwi żylnej i 28 próbek podłoży hodowlanych. Wszystkie próbki krwi pochodziły od pacjentów z klinicznymi objawami sepsy, diagnozowanych w Pracowni Diagnostyki Mikrobiologicznej Katedry Mikrobiologii Collegium Medicum Uniwersytetu Jagiellońskiego w Krakowie. Pobrane próbki krwi badano na obecność bakterii, równolegle w systemie hodowli monitorowanej BACTEC oraz za pomocą metod PCR (polymerase chain reaction) i FISH (fl uorescent in situ hydridization). Dodatkowo, każdą butelkę z podłożem poddawano analizie za pomocą metody FISH. Izolację DNA prowadzono przy użyciu zestawu QIAamp DNA Blood Mini. W celu usunięcia inhibitorów PCR wprowadzono etap oczyszczania próbek chlorkiem amonu. Do reakcji PCR wykorzystano parę starterów 16SrRNA(+) i 16SrRNA(-) umożliwiającą wykrycie wszystkich gatunków bakterii. W metodzie FISH wykorzystano sondę EUB338 swoistą dla wszystkich gatunków bakterii oraz sondy swoiste dla Enterobacteriaceae (ENT183) i rodzaju Staphylococcus (STA). WYNIKI Odsetek wyników dodatnich dla próbek krwi żylnej wynosił: 71,4%, 28,6% i 10,7% odpowiednio dla PCR, FISH i BACTEC. Różnice między wynikami uzyskanymi za pomocą metod PCR i FISH oraz PCR i BACTEC były statystycznie istotne. W przypadku analizy próbek podłóż hodowlanych, odsetek wyników dodatnich dla metody FISH wynosił 58,3%, natomiast dla metody hodowlanej 33,3%. Różnica ta nie była statystycznie istotna. Czas potrzebny do uzyskania wyników badań prowadzonych za pomocą metod PCR i FISH wynosił około 4–5 godzin. Amplifikacja DNA była możliwa jedynie dla próbek krwi poddanych dodatkowo wstępnej procedurze oczyszczania i przepłukiwania osadu komórkowego. W pozostałych przypadkach obserwowano efekt inhibicji. WNIOSKI Uzyskane wyniki wykazały, że metody PCR i FISH pozwalają na wykrycie obecności bakterii w próbkach pełnej krwi, są bardziej czułe od metody hodowlanej, skracają czas oczekiwania na wynik do kilku godzin, zaś reakcja amplifi kacji wymaga zastosowania dodatkowej procedury oczyszczania próbek krwi.
Dostawca treści:
Biblioteka Nauki
Artykuł
Tytuł:
Cloning of two genes encoding Rab7 in Paramecium
Autorzy:
Surmacz, Liliana
Wiejak, Jolanta
Wyroba, Elzbieta
Tematy:
Rab7
hybridization
Paramecium
digoxygenin labeling
cloning
PCR/RT-PCR
Pokaż więcej
Wydawca:
Polskie Towarzystwo Biochemiczne
Powiązania:
https://bibliotekanauki.pl/articles/1041281.pdf  Link otwiera się w nowym oknie
Opis:
Rab7 is a small GTPase that plays a crucial role in the regulation of transport from early to late endosomes and lysosomes, phagosome maturation and in lysosomal biogenesis in mammalian cells. It contains conserved and unique sequence elements that mediate its function. Two Rab7 genes, Rab7a (703 bp) and Rab7b (707 bp) were identified in the unicellular eukaryote Paramecium by PCR amplification. They contain three short introns of different lengths (28-32 bp) and sequence located at identical positions in both genes. The presence of two Rab7 genes in the Paramecium genome was confirmed by Southern hybridization analysis performed with six different restriction enzymes. Expression of both genes was assessed by Northern blot and RT-PCR. Two transcripts of 1.8 and 2.2 kb were identified by hybridization analysis. The cloned complementary DNAs, both of 618 nucleotides in length, encode polypeptides of 206 amino acids that are 97.6% identical and differ in their C-termini. The predicted protein sequences of Rab7a and Rab7b contain all characteristic domains essential for Rab function: the effector domain (YRATVGADF) and four GTP-binding consensus sequences (GDSGVGKT, WDTAGQ, NKLD, SAK) as well as the prenylation motif (-CC) at the C-terminus indispensable for Rab binding to the membrane. Similarity searches revealed 81.6-82.1% homology of Paramecium Rab7 isoforms to human Rab7 and a lack of an insert typical for the Kinetoplastida - the species that appeared earlier in evolution. Paramecium is the first free-living lower eukaryote in which homologues of Rab7 have been identified that exhibit features similar to those of mammalian Rab7.
Dostawca treści:
Biblioteka Nauki
Artykuł
Tytuł:
Molecular identification of Giardia duodenalis isolates from domestic dogs and cats in Wroclaw, Poland
Autorzy:
Piekarska, Jolanta
Bajzert, Joanna
Gorczykowski, Michał
Kantyka, Magdalena
Podkowik, Magdalena
Tematy:
giardia duodenalis
assemblage
dogs
cats
nested-pcr
pcr-rflp
zoonosis
Pokaż więcej
Wydawca:
Instytut Medycyny Wsi
Powiązania:
https://bibliotekanauki.pl/articles/990993.pdf  Link otwiera się w nowym oknie
Opis:
Introduction. Giardia duodenalis (G. intestinalis) is a common protozoan causing gastrointestinal disorders in many species of mammals. The genus of Giardia has high molecular diversity. Dogs and cats, in addition to their typical infection with assemblages C, D and F, may be a reservoir of zoonotic assemblages (A and B). Objective. The aim of this study was a genetic characteristic of Giardia isolates of dogs and cats from the area of Wroclaw (Poland). Materials and method. A total of 128 and 33 faecal samples from dogs and cats, respectively, were analyzed by routine coprological methods. The animals were diagnosed on the presence of G. duodenalis antigens in faeces soluble with the use of SNAP Giardia (IDEXX Laboratories) immunosorbent assay. 27 DNA isolates of Giardia were subjected to molecular identification (PCR-RFLP). Results and conclusions. The prevalence of G. duodenalis was 21.1% (27/128) in dogs and 15.1% (5/33) in cats. In dogs, C assemblage was present in 18 (81%) positive stool samples, D assemblage in 2 (9%) samples, B assemblage present in one (4.5%), and mixed assemblages (C and D) occurred in one (4.5%) sample. F assemblage was found in 4 (80%) cats’ positive stool samples and A assemblage occurred in one case (20%). Confirmation of the presence of A and B zoonotic assemblages suggests that infected pets can be a threat to human health. This study describes for the first time the presence of mixed infections within host-specific C and D assemblages in dogs in Poland.
Dostawca treści:
Biblioteka Nauki
Artykuł
Tytuł:
Qualitative analysis of bacterial biocenoses in two sequencing batch reactors treating reject water under different technological conditions
Autorzy:
Karło, A.
Ziembińska-Buczyńska, A.
Cema, G.
Surmacz-Górska, J.
Tematy:
anammox
CANON
nitrification
PCR-DGGE
RT-PCR-DGGE
nitryfikacja
Pokaż więcej
Wydawca:
Uniwersytet Warmińsko-Mazurski w Olsztynie
Powiązania:
https://bibliotekanauki.pl/articles/363132.pdf  Link otwiera się w nowym oknie
Opis:
Complete nitrogen removal over nitrite (CANON) was used to treat reject water with ammonia concentrations ranging from 70 to 154mg·L-1. Two experimental sequential batch reactors, SBR_A and SBR_B, differed in the time of the reject water inflow (6h40min vs 40min), process temperature (25 vs 29°C), and the number of aeration periods per day (3 vs 6, respectively). Nitrogen removal efficiency was higher in SBR_B (50-90%) than in SBR_A (40-80%). Analysis of total (PCR-DGGE) and active (RT-PCR-DGGE) bacteria revealed that the biodiversity of the bacterial biocenoses, expressed as the Shannon-Wiener Biodiversity Index, was higher in SBR_B (2.75-3.10) than in SBR_A (1.80-2.75).
Dostawca treści:
Biblioteka Nauki
Artykuł
Tytuł:
Metody oparte o amplifikację DNA techniką PCR wykorzystywane w ocenie bioróżnorodności mikroorganizmów glebowych
Methods based on DNA PCR-amplification for evaluation of the soil microbial diversity
Autorzy:
Łyszcz, Małgorzata
Gałązka, Anna
Tematy:
soil microorganism
genetic diversity
molecular markers
PCR based methods
ARDRA
PCR-RFLP
RAPD
REP-PCR
TRFLP
Pokaż więcej
Wydawca:
Polskie Towarzystwo Przyrodników im. Kopernika
Powiązania:
https://bibliotekanauki.pl/articles/1034148.pdf  Link otwiera się w nowym oknie
Opis:
Mikroorganizmy glebowe, pod względem cech genomowych i fenotypowych, stanowią wysoce zróżnicowaną grupę organizmów żywych. Z powodu tak dużej różnorodności ważne jest dobranie odpowiednich metod, dających największy stopień różnicowania mikroorganizmów. Narzędziami umożliwiającym analizę zmienności genetycznej mikroorganizmów są techniki genetyczne, a wśród nich jedną z najważniejszych jest łańcuchowa reakcja polimerazy, czyli PCR (Polymerase Chain Reaction), technika opracowana w latach 1980. Niniejsza praca stanowi przegląd podstawowych zagadnień dotyczących badania zmienności genetycznej mikroorganizmów glebowych w oparciu o markery molekularne z wykorzystaniem technik bazujących na reakcji PCR tj. PCR-RFLP, TRFLP, ARDRA, RAPD.
Soil microorganisms represent a highly diverse group of living organisms in terms of genomic and phenotypic characteristics. Due to such a large diversity, it is important to select appropriate identification methods which would secure its most complete determination. Genetic techniques are proper tools of choice for analyzing genetic variability of microorganism, the most important of which is the polymerase chain reaction (PCR), developed in the 1980s. This work presents an overview of the basic issues concerning studies on genetic variability of soil microorganisms with help of molecular markers and application of PCR techniques such as PCR-RFLP, TRFLP, ARDRA, RAPD.
Dostawca treści:
Biblioteka Nauki
Artykuł
Wyświetlanie 1-10 z 290

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