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A highly reproducible, dominant, monomorphic fragment of 473 base pair (bp) amplified from the genome of Trypanosoma evansi by arbitrary primer — polymerase chain reaction (AP-PCR) was labelled with digoxigenin and investigated for its potential as DNA probe. Dot-blot hybridisation of total genomic DNA with the probe proved useful in detecting bubaline, cameline and equine strains of T. evansi down to 10 pg of parasite template DNA. No cross-hybridisation was seen with Babesia bigemina, Theileria annulata and the bubaline host DNA. This probe may facilitate laboratory identification of T. evansi in developing countries, without the inherent risk associated with radioisotopes.

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Acta Veterinaria Hungarica
Authors:
Orsolya Erdősi
,
Katalin Szakmár
,
Zsuzsanna Szili
,
Géza Szita
,
Sándor Bernáth
,
József Sövényi
, and
Péter Laczay

The rapid detection of Campylobacter spp. is of utmost importance for the reduction of infections in humans by contaminated food products. The standard culturing method (ISO 10272-1:2006) involves a high time and labour demand. In this paper, we present a method that reduces the detection time of Campylobacter spp. to or below one third as compared to the ISO method, at a reduced cost per test. We used redox potential change of enrichment cultures (Bolton broth with Bolton selective supplement) for reliably selecting Campylobacter-contaminated raw milk and broiler meat samples. Identification of Campylobacter spp. in the contaminated samples was done by real-time PCR method. Culturing time to conclusive redox monitoring varied between 6 and 24 h for positive samples, depending on the contamination rate, in contrast to 136 h with the standard culturing process. However, now the Campylobacter-negative majority of food samples will not need to be tested by real-time PCR because redox potential monitoring can identify them in the selective enrichment phase. This method could be potentially used as a faster alternative to the current standard ISO 10272-1:2006, for nonregulatory monitoring purposes.

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Acta Veterinaria Hungarica
Authors:
Ádám Bálint
,
Miklós Tenk
,
Zoltán Deim
,
Thomas Rasmussen
,
Åse Uttenthal
,
Attila Cságola
,
Tamás Tuboly
,
Attila Farsang
,
Caroline Fossum
,
Sirje Timmusk
,
Mikael Berg
, and
Sándor Belák

A real-time PCR assay, based on Primer-Probe Energy Transfer (PriProET), was developed to improve the detection and quantification of porcine circovirus type 2 (PVC2). PCV2 is recognised as the essential infectious agent in post-weaning multisystemic wasting syndrome (PMWS) and has been associated with other disease syndromes such as porcine dermatitis and nephropathy syndrome (PDNS) and porcine respiratory disease complex (PRDC). Since circoviruses commonly occur in the pig populations and there is a correlation between the severity of the disease and the viral load in the organs and blood, it is important not only to detect PCV2 but also to determine the quantitative aspects of viral load. The PriProET real-time PCR assay described in this study was tested on various virus strains and clinical forms of PMWS in order to investigate any correlation between the clinical signs and viral loads in different organs. The data obtained in this study correlate with those described earlier; namely, the viral load in 1 ml plasma and in 500 ng tissue DNA exceeds 10 7 copies in the case of PMWS. The results indicate that the new assay provides a specific, sensitive and robust tool for the improved detection and quantification of PCV2.

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We designed a multiplex real time PCR for rapid, sensitive and specific detection of Streptococcus pneumoniae, Legionella pneumophila, Chlamydophila pneumoniae and Mycoplasma pneumoniae. The study cases consisted of 129 patients with community acquired pneumonia (CAP). Bacteriological techniques were implemented for detection of the cultivable organisms. DNA were extracted from sputa, throat swabs, bronchoalveolar lavages and tracheal aspirates and used as templates in real time PCR. The primers and probes were designed for cbpA (S. pneumoniae), p1adhesin (M. pneumoniae), mip (L. pneumophila) and ompA (C. pneumoniae). After optimization of real time PCR for every organism, the experiments were continued in multiplex in a single tube. Of 129 CAP specimens, the positive cultures included 14 (10.85%) for S. pneumoniae, 9 (6.98%) for L. pneumophila and 3 (2.33%) for M. pneumoniae. Four specimens (3.10%) were positive for C. pneumoniae by real time PCR. The sensitivity of our real time PCR was 100% for all selected bacteria. The specificity of the test was 98.26%, 98.34%, 100% and 100% for S. pneumoniae, L. pneumophila, M. pneumoniae and C. pneumoniae, respectively. This is the first report on the use of multiplex real time PCR for detection of CAP patients in the Middle East. The method covers more than 90% of the bacterial pathogens causing CAP.

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Acta Microbiologica et Immunologica Hungarica
Authors:
Somayeh Fard
,
Bizhan Nomanpour
,
Bahram Fatolahzadeh
,
Ashraf Mobarez
,
Davood Darban-Sarokhalil
,
Abbas Fooladi
,
Willem Leeuwen
, and
Mohammad Feizabadi

Legionella pneumophila is an important etiological agent in both hospital and community acquired pneumonia. The sensitivity of culture for isolation of L. pneumophila from clinical specimens is low and time consuming. Similar problem also exists when the method of direct immunofluorescence is used. To detect this organism quantitatively from respiratory specimens, a Taq Man based real-time PCR targeting the mip sequence was developed. Both real-time PCR and culture methods were applied on 262 respiratory specimens from 262 ICU patients with pneumonia admitted to 5 different hospitals in Tehran. The results of real-time PCR were compared with those obtained by culture. Real-time PCR and culture found 12 and 4 specimens, respectively, as positive for L. pneumophila. Its technical specificity (100%) was checked against a panel of microorganisms consisting of both Gram-positive and Gram-negative bacteria. Our real-time PCR assay showed high sensitivity (100%) and specificity (96.9%) and could detect 200 organisms per ml from respiratory specimens. Using real-time PCR as a screening method, the frequency of nosocomial pneumonia with L. pneumophila at Tehran hospitals was estimated as 4.58%.

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Granulomatous lesions in bovine and especially swine lymph nodes are still frequently observed during routine veterinary meat inspections even though Mycobacterium bovis infections are no longer detected in domestic animals in Slovenia. Different lymph nodes of pigs (n = 260) were investigated using classical bacteriological and molecular methods. Mycobacterium avium alone was isolated in 47.3% of pigs and in mixed infection with Rhodococcus equi in 3.9% of pigs. R. equi alone was isolated in 27.3% and in mixed infection with mycobacteria other than M. avium in 1.5% of pigs. A total of 133 M. avium isolates were typed using the IS1245, IS901 and FR300 PCR. Almost two thirds (60.9%) of isolates belonged to M. avium hominissuis (IS901-, IS1245+ genotype), 33.8% of isolates belonged to M. avium avium (IS901+, IS1245+ genotype) and 5.3% of isolates remained non-typed. Fifty out of 85 R. equi isolates were tested for the virulence-associated antigens (VapA and VapB). Nearly two thirds (60.0%) were positive for VapB while all the other isolates were VapA- and VapB-negative.

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The integron content of 52 DT104/U302 phage type strains and 53 non-DT104/U302 strains of Salmonella enterica serotype Typhimurium (S. Typhimurium) was studied in PCR experiments using a 5'-CS/3'-CS primer pair (Lévesque et al., 1995). Forty-three out of 44 streptomycin- and/or ampicillin-resistant DT104 and related phage type strains were found to carry a 1 kb and/or 1.2 kb long integron. The other resistance markers did not affect the number and size of integrons; no integron-free multidrug-resistant (MDR) DT104 strains were found. The two large groups of DT104 strains (Felix-Callow's phage types 2 and 2c) proved to be identical in respect of integron patterns (IPs), supporting the views of those authors who consider DT104 a single clone. Strains of human and animal origin did not differ from each other in their IPs. Within the non-DT104 phage types, ampicillin- and/or streptomycin-resistant, integron-free MDR strains were also found. Based on amplicons varying between 290 and 3500 bp an IP system was suggested. The commonest amplicon sizes in non-DT104 strains were 1450 and 2050 bp. The IPs of DT104 strains and of non-DT104 strains containing an integron of 1 and 1.2 kb size were stable. In contrast, the IPs of other non-DT104 strains showed a varying degree of instability. Integron loss was frequently associated with spontaneous plasmid elimination and changes of R-type among the descendants of a given strain.

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Humán papillomavírus (HPV-16) kimutatása a colon laphámsejtes carcinomájából és annak nyirokcsomó-metastasisaiból

Detection of human papilloma virus type 16 in squamous cell carcinoma of colon and its lymph node metastases

Magyar Sebészet
Authors:
Gábor Bognár
,
Gábor István
,
György Ledniczky
, and
Pál Ondrejka

Absztrakt

A humán papillomavírus (HPV) etiológiai szerepe számos laphámeredetű malignomában ismert. A colon laphámsejtes rákja (SCC) ritka betegség, etiológiája bizonytalan. Egy általunk operált SCC-eset kapcsán célunk volt kimutatni egy esetleges HPV-fertőzést. Egy 94 éves nőbeteget operáltunk passage-zavart okozó colon descendens tumor miatt. A szövettani vizsgálat SCC-t igazolt. Korábban kidolgozott módszerrel PCR- és Southern blot hybridisatiós technikával vizsgáltuk meg a daganatszövetet és az eltávolított nyirokcsomókat. Az eset kapcsán átnéztük az SCC-re vonatkozó irodalmat. A malignomákban leggyakrabban előforduló HPV-típusok (16, 18) közül a HPV-16 jelenlétét tudtuk igazolni a SCC-ben és a környező 9 nyirokcsomóból 4-ben. Ezek közül 2 metastaticus volt. Egy SCC-s betegnél sikerült HPV-16-fertőzöttséget kimutatni, a primer tumorból és a metastaticus nyirokcsomók mellett további környező nyirokcsomókból. Ilyen tanulmányt tudomásunk szerint még senki nem közölt.

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A real-time RT-PCR assay utilising light upon extension fluorogenic primer (LUX RT-PCR) was developed for the rapid and efficient detection of avian influenza viruses (AIV). The assay detected each of the AIV isolates tested (16/16) and gave negative results with heterologous pathogens (17/17). The detection limit of the assay proved to be 10-0.5 EID50/0.2 ml and 101.5 EID50/0.2 ml in allantoic fluid of virus-infected embryonated chicken eggs and in spiked chicken faeces samples, respectively. Based on its specificity, sensitivity and relative simplicity, the LUX RT-PCR assay provides a novel, rapid and cost-effective diagnostic tool for avian influenza surveillance and monitoring programs.

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Acta Veterinaria Hungarica
Authors:
Orsolya Erdősi
,
Katalin Szakmár
,
Olivér Reichart
,
Zsuzsanna Szili
,
Noémi László
,
Péter Székely Körmöczy
, and
Péter Laczay

The incidence of outbreaks of foodborne listeriosis has indicated the need for a reliable and rapid detection of the microbe in different foodstuffs. A method combining redox potential measurement and real-time polymerase chain reaction (PCR) was developed to detect Listeria monocytogenes in artificially contaminated raw milk and soft cheese. Food samples of 25 g or 25 ml were homogenised in 225 ml of Listeria Enrichment Broth (LEB) with Oxford supplement, and the redox potential measurement technique was applied. For Listeria species the measuring time was maximum 34 h. The absence of L. monocytogenes could reliably be proven by the redox potential measurement method, but Listeria innocua and Bacillus subtilis could not be differentiated from L. monocytogenes on the basis of the redox curves. The presence of L. monocytogenes had to be confirmed by real-time PCR. The combination of these two methods proved to detect < 10 cfu/g of L. monocytogenes in a cost- and time-effective manner. This method can potentially be used as an alternative to the standard nutrient method for the rapid detection of L. monocytogenes in food.

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