Aleksandra Ziembińska-Buczyńska, Jarosław Wiszniowski and Sławomir Ciesielski
 Hoefel, D., Monis, P.T., Grooby, W.L., Andrews, S. & Saint, C.P. (2005). Culture-Independent Techniques
for Rapid Detection of Bacteria Associated with Loss of Chloramine Residual in a Drinking Water
System, Applied and Environmental Microbiology, 71, 11, 6479–6488.
 Jacquot, E., van Tuinen, D., Gianinazzi, S. & Gianinazzi-Pearson, V. (2000). Monitoring species of
arbuscular mycorrhizal fungi in planta and in soil by nestedPCR: application to the study of the impact
of sewage sludge, Plant and Soil, 226, 179–188.
 Jurczyk, Ł., Koc
(Glomales) in roots by nestedPCR and SSCP (Single Stranded Conformation Polymorphism). Plant and Soil 226: 189-196.
Krüger M., Stockinger H., Krüger C., Schüßler A. 2009. DNA-based species level detection of Glomeromycota: one PCR primer set for all arbuscular mycorrhizal fungi. New Phytol. 183: 212-213.
Merryweather J., Fitter A. 1998. The arbuscular mycorrhizal fungi of Hyacinthoides non-scripta . I. Diversity of fungal taxa. New Phytol. 138: 117-129.
Porcel R., Ruiz-Lozano J. M. 2004
arbuscular mycorrhizal fungi (Glomales) in roots by nestedPCR and SSCP (Single Stranded Conformation Polymorphism). Plant and Soil 226: 189-196.
Krüger M., Stockinger H., Krüger C., Schüβler A. 2009. DNA-based species level detection of Glomeromycota: one PCR primer set for all arbuscular mycorrhizal fungi. New Phytol. 183: 212-213. [DOI:10.1111/j.1469 - 8137.2009.02835.x]
Lee J., Lee S., Young J.P. W. 2008. Improved PCR primers for the detection and identification of arbuscular mycorrhizal fungi. Microbiol. Ecol. 65: 339-349. [DOI:10
Nemanja Jezdimirović, Branislav Kureljušić, Vojin Ivetić, Dejan Krnjaić, Oliver Radanović, Jadranka Žutić, Ljiljana Spalević and Milijan Jovanović
The aim of this study was to determine the pathological, mycological and molecular findings in turkey poults with different immunological status experimentally infected with Aspergillus fumigatus. The investigation was carried out 1, 3, 7, 14 and 21 days after intratracheal inoculation of 5.056×107 spores of A. fumigatus to 14-day-old turkey poults in group G-1, as well as to turkey poults in group G-2 which were treated prior to infection with dexamethasone. A. fumigatus was isolated on day 1 p.i. in both groups, but the number of positive samples was bigger in group G-1. A. fumigatus was isolated from the respiratory organs of group G-1as early as on day 1 and 3 p.i. in 4 out of 12 examined specimens (33%). On day 7 p.i. A. fumigatus was possible to isolate from the respiratory organs of 50% of infected birds, on day 14 in 83.33% and on day 21 p.i. A. fumigatus was isolated in 6 out of 6 sacrificed turkey poults (100%). In dexamethasone-treated group A. fumigatus isolates from the respiratory organs on day 1 and 3 p.i. were same as in group G-1, whereas on days 7 and 14 p.i. the number of turkey poults positive to A. fumigatus increased in comparison with the untreated G-1 group. The histopathological lesions in turkey poults treated with dexamethasone developed earlier, were more intensive and extensive. The mycological and nested PCR results revealed a higher number of samples positive for the presence of A. fumigatus DNA in the group G-2, pretreated with dexamethasone.
Sunutcha Suntrarachun, Surasak Akesowan and Thaweesak Tirawatnapong
Background: Equine infectious anemia virus (EIAV) is a lentivirus with an almost worldwide distribution, infecting equids. It causes a persistent infection that is characterized by recurring episodes of fever, anemia, and thrombocytopenia. Most of the horses may control EIAV replication within a year, remaining persistently infected without clinical signs of disease. Objective: Detect EIA nucleic acid from peripheral blood of asymptomatic horses using nested PCR. Materials and method: We used nested PCR, amplifying P26 gag gene of EIAV, for direct detection of viral RNA in plasma and proviral DNA from PBMC in asymptomatic carrier horses in comparison with the Coggins test. EIA nucleic acid was prepared from 20 seropositive and five EIAV seronegative horses. Amplification of 246 bp expected size fragments was obtained using two different sets of primers targeting the P26 gag gene. Results: Among 20 seropositive horses, nine samples were positive for RNA and DNA. The five samples were positive for DNA but not for RNA, which indicates that the virus integrated into the host cell genome with a low level of viral replication. However, six samples were negative for both DNA and RNA. False negative could result due to primer failure caused by gag sequences variation among strains circulating in Thailand when compared with various strains from other parts of the world. EIAV antigens may also be prepared from cell cultures contaminated with other retroviruses thus causing false positives with the Coggins test. Conclusion: Nested PCR can be a useful tool for detecting the presence of EIAV in asymptomatic carrier horses. This may be especially true during the acute stage of the disease where the viremia levels are usually at the highest levels before detectable antibodies appear.
Svetlana Kozireva, Dmitrijs Užameckis, Mihails Bariševs and Modra Murovska
Sensitivity and Reproducibility of Polymerase Chain Reaction Assays for Detection of Human Herpesviruses 6 and 7
Human herpesvirus 6 (HHV-6) and 7 (HHV-7) are ubiquitous viruses that undergo latency and may become reactivated leading to cytomegalovirus reactivation, bone marrow suppression, nervous system dysfunction, graft-versus-host disease and increased mortality. The aim of this study was to identify the most sensitive and reproducible nPCR for detection of HHV-6 and HHV-7 infection and to evaluate the reproducibility of these assays in different laboratories. The sensitivity of the six previously published HHV-6 (one targeting hypothetical protein Bgp009 gene, two — large tegument protein gene, one — major binding protein gene and two targeting hypothetical Bgp071 protein gene) and four HHV-7 (targeting nuclear phosphoprotein, tegument phosphoprotein, large tegument protein and immediately early A transactivator gene) nPCRs was determined. The most sensitive HHV-6 nPCR was targeted Bgp071 protein gene, which could detect 5 genomic copies of HHV-6. The most sensitive and reproducible HHV-7 nPCR assay, targeting nuclear phosphoprotein gene, could detect 1 genomic copy of HHV-7. The reproducibility of the selected HHV-6 and HHV-7 nPCRs was evaluated in five different laboratories. The results obtained in all laboratories were identical to our results, confirming that these nPCRs are useful as assays for molecular diagnostics of HHV-6 and HHV-7 infection.
.: Distribution of bovine leukemia virus proviral sequences in tissues of bovine, ovine and human origin, Ann Rech Vet 1978, 9, 837-844.
12. Klintevall K., Ballagi-Porda’ny A., Naslund K., Belák S.: Bovine leukemia virus: rapid detection of proviral DNA by nestedPCR in blood and organs of experimentally infected calves. Vet Microbiol 1994, 42, 191-204.
13. Kubiś P., Szczotka M., Kuźmak J.: Detection of bovine leukaemia virus proviral DNA in paraffin sections, tissue smears, and peripheral blood leukocytes by in situ PCR. Bull Vet Inst 2007, 51, 337-342.
14. Lagarias D
). The quality of the isolated DNA was assessed by NanoDrop spectrophotometer (NanoDrop Technologies, Thermo Fisher Scientific Inc, Waltham, MA, USA). The optical density values of all samples ranged from 1.8 to 2.0.
Detection of the Mitochondrial DNA 4977 bp Deletion
To screen the 4977 bp deletion in mtDNA, nested-polymerase chain reaction (nested-PCR) analysis was performed; we used the DNA samples extracted from the tissues as templates. In brief, two pairs of nested primers for detection of the 4977 bp deletion were as follows: 1F: 5’-AAC CAC AGT TTC ATG CCC
-Terminal Domain of Clinical Samples and C-Terminal Domain Sequencing . Nested polymerase chain reaction (nested-PCR) was applied to amplify the C-terminal region of EBNA-1. The sequence and position of primers used in two stages of nested-PCR are shown in Table 1 and Figure 1 , respectively. The EBNA-1-1 and EBNA-1-2 primers were used for stage 1 of nested-PCR, and EBNA-1-3 and EBNA-1-4 primers were used in stage 2 of nested-PCR. Both stages of nested-PCR were performed in a total of 15 μL, containing 100 ng genomic DNA template (stage 1) or 100 ng stage 1 PCR product (stage