Search results

1

Physical state of human papillomavirus type 16 in cervical intraepithelial lesions and cancers determined by two different quantitative real-time PCR methods

100%

Szostek S., Biesaga B., Zawilinska B., Klimek M., Kosz-Vnenchak M.

Acta Biochimica Polonica

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2015

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vol. 62

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issue 4

923-928

EN

The aim of this study was to analyse the correlation between a new multiplex qPCR assay and a reference qPCR assay for assessment of the human papillomavirus (HPV16) load and the viral genome status. The study was performed on 100 HPV16 positive samples containing premalignant lesions and carcinomas. HPV16 E2 and E6 gene loads were assessed by two PCR methods. The load of E2 and E6 was normalized to the cell number by qPCR targeting the RNase P open reading frame. The physical state of the viral genome was determined as a ratio of E2/E6 copies number per cell. Among 100 samples analysed, there were no statistically significant differences in the E2 and E6 viral load evaluated by multiplex qPCR and qPCR, the correlation coefficients were 0.98 and 0.97, respectively. There were 19% of samples with the integrated, 73% with mixed and 8% with episomal state of viral genome detected by multiplex qPCR and 17%, 79%, 4%, respectively, found by qPCR. Prevalence of integrated and episomal forms estimated by multiplex qPCR was higher than the one obtained by qPCR (Chi2, p < 0.0001), but in samples with premalignant and malignant diagnoses no significant differences were demonstrated regardless of the methods used. Sensitivity and specificity of multiplex qPCR were 93.7% and 100% as compared with qPCR, the positive predictive value was 100%. In summary, the multiplex qPCR assay in respect of HPV16 load and the frequency of viral genome status was shown to be a sensitive and specific reference method. Simultaneous estimation of E2 and E6 genes in one reaction tube reduces the cost of testing.

2

Optimization of the Y831C mutation detection in human DNA polymerase gamma by allelic discrimination assay

100%

Stopińska K., Grzybowski T., Malyarchuk B. A., Derenko M. V., Miścicka-Śliwka D.

Acta Biochimica Polonica

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2006

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vol. 53

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issue 3

591-595

EN

Many well-defined mutations in the gene for the catalytic subunit of polymerase γ (POLG1) have been found to be associated with disease, whereas the status of several mutations remains unresolved due to the conflicting reports on their frequencies in populations of healthy individuals. Here, we have developed a highly sensitive, real-time allelic discrimination assay enabling detection of the Y831C mutation in the POLG1 gene. The Y831C mutation is present in the Polish population at a frequency of 2.25%. The new assay is well suited to both extensive population studies and molecular diagnostics of POLG1.

3

Usefulness of real-time PCR in long-term follow-up of follicular lymphoma patients

100%

Tysarowski A., Fabisiewicz A., Paszkiewicz-Kozik E., Kulik J., Walewski J., Siedlecki J. A.

Acta Biochimica Polonica

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2007

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vol. 54

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issue 1

135-142

EN

The aim of this study was to evaluate the usefulness of quantitative real-time PCR (RQ-PCR) for the monitoring of molecular remission in follicular lymphoma (FL) patients during long-term follow-up. RQ-PCR by the use of TaqMan® detection system is a sensitive tool to monitor minimal residual disease (MRD) in FL through amplification of the t(14;18) fusion gene during and post-therapy. In most cases the breakpoint region occurs within the major breakpoint region (MBR). Among 75 patients diagnosed with FL, cells harboring the fusion gene BCL2/JH were found in peripheral blood of 31 patients (41%). We further monitored 30 of these patients in a period varying from 6 months to 5 years by RQ-PCR. In our study the level indicating the possibility of the presence of MRD was established at more than five t(14;18)-positive cells in the background of 83000 normal cells. The results of this work also confirmed that the presence of MRD detected by RQ-PCR is an indication for careful observation of patients because of a higher risk of disease recurrence.

4

Real-time PCR approach in dermatophyte detection and Trichophyton rubrum identification

100%

Kobylak N., Bykowska B., Nowicki R., Brillowska-Dąbrowska A.

Acta Biochimica Polonica

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2015

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vol. 62

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issue 1

119-122

EN

Dermatophytes are keratinophilic molds that infect human hair, nails and skin. Diagnosis of dermatophytosis is based on morphological, serological and biochemical features. However, identification is difficult and laborious due to similarities between microorganisms. Thus, there is considerable interest to develop mycological diagnostic procedures based on molecular biology methods. In this study, fast, two-step DNA extraction method and real-time PCR was used for detection of dermatophytes DNA using pan-dermatophyte primers and identification of Trichophyton rubrum from pure cultures. The applied method allowed correct detection of all dermatophytes and correct identification of Trichophyton rubrum in less than 2 hours.

5

Anticancer activity of new molecular hybrids combining 1,4-naphthalenedione motif with phosphonic acid moiety in hepatocellular carcinoma HepG2 cells

100%

Długosz A., Gach K., Szymański J., Modranka J., Janecki T., Janecka A.

Acta Biochimica Polonica

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2017

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vol. 64

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issue 1

41-48

EN

Structural motifs found in naturally occurring compounds are frequently used by researchers to develop novel synthetic drug candidates. Some of these new agents are hybrid molecules which are designed through a concept of combining more than one functional element. In this report, anticancer activity of new synthetic molecular hybrids, substituted 3-diethoxyphosphorylnaphtho[2,3-b]furan-4,9-diones and 3-diethoxyphosphorylbenzo[f]indole-4,9-diones, which integrate natural 1,4-naphtalenedione scaffold, present in several anticancer agents, with pharmacophoric phosphonate moiety, were tested against hepatocellular cell line HepG2. Cytotoxicity was examined using MTT assay. Two most potent compounds, furandione 8a and benzoindoldione 12a, which reduced the number of viable HepG2 cells with the IC50 values of 4.13 µM and 5.9 µM, respectively, were selected for further research. These compounds decreased the mRNA expression levels of several genes: Bcl-2, angiogenic vascular endothelial growth factor (VEGF), c-Fos, caspase-8 and increased the expression of Bax, caspase-3 and -9, c-Jun, p21, p53, as determined by quantitative real-time PCR. The ability of these compounds to induce apoptosis and DNA damage was studied by flow cytometry. The obtained data showed that the new compounds inhibited cell viability by increasing apoptosis and decreasing angiogenesis. Compound 8a was a much stronger apoptosis inducer as compared with 12a and strongly activated the intrinsic pathway of apoptosis, associated with the loss of mitochondrial membrane potential and changes in Bax/Bcl-2 ratio. These findings show that the synthetic hybrids combining 1,4-naphthalenedione system and phosphonic acid moiety display potential to be further explored in the development of new anticancer agents.

6

PCR and real-time PCR assays to detect fungi of Alternaria alternata species

88%

Kordalewska M., Brillowska-Dąbrowska A., Jagielski T., Dworecka-Kaszak B.

Acta Biochimica Polonica

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2015

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vol. 62

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issue 4

707-712

EN

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.

7

HPV16 E6 polymorphism and physical state of viral genome in relation to the risk of cervical cancer in women from the south of Poland

88%

Szostek S., Zawilinska B., Klimek M., Kosz-Vnenchak M.

Acta Biochimica Polonica

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2017

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vol. 64

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issue 1

143-149

EN

The aim of this study was to analyse the correlation between HPV16 E6 variants and the physical status of viral genome (integrated, mixed, episomal) among patients with cervical cancer (n=40) and low-grade squamous intraepithelial lesions - LSIL (n=40). The study was performed on 80 HPV16 positive samples. HPV16 E6 variants were identified using PCR and DNA sequencing. Nucleotide sequences of E6 were compared with the prototype sequence (EUR-350T). The physical state of HPV DNA was determined as the ratio of E2/E6 copy number per cell. Twelve different intratypic variants were identified as belonging to European (in 77 samples) and North-American 1 (in 3 samples) sublineages. The most prevalent non-synonymous variant was EUR-350G, which occurred with similar frequency in cervical cancer and LSIL. The frequencies of additional mutations in variants with EUR-350T or EUR-350G sequences differed significantly. For the first time, missense mutations G122A, C153T and G188A were discovered in EUR-350G variant. The integrated viral genome was predominant in women with cervical cancer. The EUR-350T prototype and EUR-350G without additional mutations variants were prevalent in cervical cancer samples with the HPV16 characterized by integrated DNA. In summary, European variants of HPV16 E6 dominated in both cancer and LSIL group. The presence of EUR-350G favoured the occurrence of additional nucleotide changes. We showed that nucleotide changes occur significantly more often in the mixed form of viral DNA and in LSIL group and that the variants without additional mutations may promote integration of HPV16 genome.

8

Reference genes for gene expression studies on non-small cell lung cancer

88%

Gresner P., Gromadzinska J., Wasowicz W.

Acta Biochimica Polonica

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2009

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vol. 56

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issue 2

307-316

EN

Study Objective: The aim of this study was to test a panel of 6 reference genes in order to identify and validate the most suitable reference genes for expression studies in paired healthy and non-small cell lung cancer tissues. Method: Quantitative real-time PCR followed by the NormFinder- and geNorm-based analysis was employed. The study involved 21 non-small cell lung cancer patients. Results: The analysis of experimental data revealed HPRT1 as the most stable gene followed by RPLP0 and ESD. In contrast, GAPDH was found to be the least stable gene. HPRT1 together with ESD was revealed as the pair of genes introducing the least systematic error into data normalization. Validation by bootstrap random sampling technique and by normalizing exemplary gene expression data confirmed the results. Conclusion: Although HPRT1 and ESD may by recommended for data normalization in gene expression studies on non-small cell lung cancer, the suitability of selected reference genes must be unconditionally validated prior to each study.

9

New Primers for Fast Detection of Giardia duodenalis Assemblages A and B Using Real-time PCR

88%

Solarczyk P., Wojtkowiak-Giera A., Hołysz M., Słodkowicz-Kowalska A., Jagodziński P. P., Stojecki K., Rocka A., Majewska A. C., Skrzypczak Ł.

Acta Protozoologica

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2018

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vol. 57

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issue 1

43-48

EN

Giardia duodenalisis one of the six Giardia species and itis the most common, cosmopolitan flagellate that infects humans and many species of animals. This species exhibits considerable genetic diversity; to date, eight assemblages (A–H) have been defined. These assemblages differ in host specificity: assemblages A and B have been found in both humans and in many animal species. Mixed infections with Giardia (A and B) assemblages have been reported in humans and in animals. Many molecular techniques are effective and rapid for the detection of G. duodenalis and also for the determination of genetic variability of isolates in clinical and environmental samples. In this context, the aim of this study was to design new assemblage-specific primers for rapid detection and identification of G. duodenalis assemblages A and B and both of these assemblages simultaneously using quantitative real-time polymerase chain reaction (qPCR). Fragments of glutamate dehydrogenase and triose phosphate isomerase were used as targets in the design of primers. In conclusion, the use of G. duodenalis assemblage-specific primers designed in this study allows quick identification of human infectious G. duodenalis assemblages A and B as well as mixed AB assemblages in a sample without further sequencing of the amplification products, which reduces the cost of study and the waiting time for the results.

10

Detection and differentiation of Newcastle disease virus and influenza virus by using duplex real-time PCR

75%

Nidzworski D., Wasilewska E., Smietanka K., Szewczyk B., Minta Z.

Acta Biochimica Polonica

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2013

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vol. 60

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issue 3

475-480

EN

Newcastle disease virus (NDV), member of the Paramyxoviridae family and avian influenza virus (AIV), member of the Orthomyxoviridae family, are two main avian pathogens causing serious economic problems in poultry health. Both are enveloped, single-stranded, negative-sense RNA viruses and cause similar symptoms, ranging from sub-clinical infections to severe diseases, including decrease in egg production, acute respiratory syndrome, and high mortality. Similar symptoms hinder the differentiation of infection with the two viruses by standard veterinary procedures like clinical examination or necropsy. To overcome this problem, we have developed a new duplex real-time PCR assay for the detection and differentiation of these two viruses. Eighteen NDV strains, fourteen AIV strains, and twelve other (negative control) strains viruses were isolated from allantoic fluids of specific pathogen-free (SPF), embryonated eggs. Four-weeks-old SPF chickens were co-infected with both viruses (NDV - LaSota and AIV - H7N1). Swabs from cloaca and trachea were collected and examined. The results obtained in this study show that by using duplex real-time PCR, it was possible to detect and distinguish both viruses within less than three hours and with high sensitivity, even in case a bird was co-infected. Additionally, the results show the applicability of the real-time PCR assay in laboratory practice for the identification and differentiation of Newcastle disease and influenza A viruses in birds.

Refine search results

Physical state of human papillomavirus type 16 in cervical intraepithelial lesions and cancers determined by two different quantitative real-time PCR methods

Optimization of the Y831C mutation detection in human DNA polymerase gamma by allelic discrimination assay

Usefulness of real-time PCR in long-term follow-up of follicular lymphoma patients

Real-time PCR approach in dermatophyte detection and Trichophyton rubrum identification

Anticancer activity of new molecular hybrids combining 1,4-naphthalenedione motif with phosphonic acid moiety in hepatocellular carcinoma HepG2 cells

PCR and real-time PCR assays to detect fungi of Alternaria alternata species

HPV16 E6 polymorphism and physical state of viral genome in relation to the risk of cervical cancer in women from the south of Poland

Reference genes for gene expression studies on non-small cell lung cancer

New Primers for Fast Detection of Giardia duodenalis Assemblages A and B Using Real-time PCR

Detection and differentiation of Newcastle disease virus and influenza virus by using duplex real-time PCR