Stored-product psocids (Psocoptera: Liposcelididae) are cosmopolitan storage pests that can damage stored products and cause serious economic loss. However, because of the body size (~1 mm) of eggs, nymphs, and adults, morphological identification of most stored-product psocids is difficult and hampers effective identification. In this study, 10 economically important stored-product Liposcelis spp. psocids (Liposcelis brunnea, L. entomophila, L. decolor, L. pearmani, L. rufa, L.mendax, L. bostrychophila, L. corrodens, L. paeta, and L. tricolor) were collected from 25 geographic locations in 3 countries (China, Czech Republic, and the United States). Ten species-specific probes for identifying these 10 psocid species were designed based on ITS2 sequences. The microarray method and reaction system were optimized. Specificity of each of the ten probes was tested, and all probes were found suitable for use in identification of the respective10 Liposcelis spp. psocids at 66 °C. This method was also used to identify an unknown psocid species collected in Taian, China. This work has contributed to the development of a molecular identification method for stored-product psocids, and can provide technical support not only to facilitate identification of intercepted samples in relation to plant quarantine, but also for use in insect pest monitoring.
- MeSH
- DNA sondy chemie metabolismus MeSH
- druhová specificita MeSH
- hmyz genetika MeSH
- ribozomální DNA chemie metabolismus MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ongoing evolution of viral pathogens is a significant issue in diagnostic virology employing TaqMan qPCR/RT-qPCR. Specific concerns are related to false negativity due to probe binding failure. One option for compensating for such deficiency is to integrate a second identically labelled probe in the assay. However, how this alteration influences the reaction parameters has not been comprehensively demonstrated. In the present study, we evaluate a TaqMan protocol using two identically labelled hydrolysis probes (simple, LNA (locked-nucleic-acid)) and MGB (minor-groove-binder) modified probes and combinations thereof in a single assay. Our results based on a synthetic amplicon suggest that the second probe does not compromise the TaqMan qPCR/RT-qPCR parameters, which repeatedly and reproducibly remained comparable to those of the corresponding single-probe assays, irrespective of the relative probe orientation, whether opposite or tandem, and probe modifications or combinations thereof. On the other hand, the second probe additively contributed to the overall fluorescence signal. The utility of the dual-probe approach was demonstrated on practical examples by using field specimens. We hope that the present study might serve as a theoretical basis for the development or improvement of TaqMan qPCR/RT-qPCR assays for the detection of highly variable nucleic acid templates.
- MeSH
- barvení a značení * MeSH
- DNA sondy metabolismus MeSH
- fluorescence MeSH
- hydrolýza MeSH
- kalibrace MeSH
- koně MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- nukleové kyseliny metabolismus MeSH
- psi MeSH
- reprodukovatelnost výsledků MeSH
- sekvence nukleotidů MeSH
- zvířata MeSH
- Check Tag
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence.
- MeSH
- DNA sondy chemie metabolismus MeSH
- fluoresceiny chemie MeSH
- fluorescenční barviva chemie MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- nukleové kyseliny metabolismus MeSH
- RNA virová metabolismus MeSH
- Taq-polymerasa metabolismus MeSH
- virus chřipky A genetika MeSH
- virus slintavky a kulhavky genetika MeSH
- změna skupenství MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ten published DNA-based analytical methods aiming at detecting material of almond (Prunus dulcis) were in silico evaluated for potential cross-reactivity with other stone fruits (Prunus spp.), including peach, apricot, plum, cherry, sour cherry and Sargent cherry. For most assays, the analysis of nucleotide databases suggested none or insufficient discrimination of at least some stone fruits. On the other hand, the assay targeting non-specific lipid transfer protein (Röder et al., 2011, Anal Chim Acta 685:74-83) was sufficiently discriminative, judging from nucleotide alignments. Empirical evaluation was performed for three of the published methods, one modification of a commercial kit (SureFood allergen almond) and one attempted novel method targeting thaumatin-like protein gene. Samples of leaves and kernels were used in the experiments. The empirical results were favourable for the method from Röder et al. (2011) and a modification of SureFood allergen almond kit, both showing cross-reactivity <10(-3) compared to the model almond.
The Russsian wheat aphid (RWA), Diuraphis noxia (Kurdjumov), is a worldwide pest of cereals. Despite its economic importance, little is known about its genome. Here we investigated physical genomic features in RWA by karyotype analysis using differential staining with AgNO(3), CMA(3), and DAPI, by chromosomal localization of ribosomal DNA (rDNA), H3 and H4 histone genes, and the "arthropod" telomeric sequence (TTAGG)(n) using fluorescence in situ hybridization (FISH), and by measuring the RWA genome size using flow cytometry. The female karyotype, 2n = 10, is composed of four autosome pairs and a pair of X chromosomes, whereas the male karyotype, 2n = 9, has a single X. The X chromosome is the largest element in the karyotype. All three molecular markers used, i.e., 18S rRNA and both H3 and H4 probes are co-localized at one end of the X chromosome. The FISH probes revealed that the AgNO(3)-positive bridge between two prometaphase X chromosomes of females, which is believed to be responsible for the elimination of one X chromosome in aphid oocytes determined to undergo male development, contains clusters of both histone genes, in addition to an rDNA cluster. Interestingly, RWA lacks the (TTAGG)(n) telomeric sequence in its genome, in contrast to several previously investigated aphid species. Additionally, we compared female and male genome sizes. The female genome size is 2C = 0.86 pg, whereas the male genome size is 2C = 0.70 pg. The difference between the DNA content in the two genders suggests that the RWA X chromosome occupies about 35% of the female haploid genome (1C = 0.43 pg), which makes it one of the largest sex chromosomes in the animal kingdom.
- MeSH
- chromozomy hmyzu genetika MeSH
- DNA sondy genetika metabolismus MeSH
- genom hmyzu MeSH
- histony genetika MeSH
- hybridizace in situ fluorescenční MeSH
- karyotypizace MeSH
- mšice genetika MeSH
- ribozomální DNA genetika MeSH
- RNA ribozomální genetika MeSH
- Southernův blotting MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Quaternary benzo[c]phenanthridine alkaloids (QBAs) are naturally occurring compounds isolated from plants in the Fumariaceae, Papaveraceae, Ranunculaceae, and Rutaceae families. In addition to having a wide range of biological activities, they are also attractive for their fluorescent properties. We observed interesting fluorescent characteristics in the QBAs-macarpine (MA), sanguirubine (SR), chelirubine (CHR), sanguilutine (SL), chelilutine (CHL), sanguinarine (SA) and chelerythrine (CHE) after interaction with living cells. METHODS: Water stock solutions of the alkaloids (10-100 microg/ml) were added to intact cells, and after a brief incubation the cells were observed. Human cell lines HL60 (human promyelocytic leukemia), HeLa (human cervix adenocarcinoma), and LEP (human lung fibroblasts), and piglet blood were used in the experiments. Blood cells were stained with MA in combination with FITC-conjugated anti-CD45 surface marker antibody. Cells were analyzed by fluorescence microscopy and by flow cytometry. RESULTS: All tested alkaloids immediately entered living cells with MA, CHR, and SA binding to DNA. MA showed the best DNA staining properties. Fluorescence microscopy of MA, CHR, and SA stained cells described the nuclear architecture and clearly described chromosomes and apoptotic fragments in living cells. Moreover MA can rapidly represent the cellular DNA content of living cells at a resolution adequate for cell cycle analysis. QBAs were excitable using common argon lasers (488 nm) emitting at a range of 575-755 nm (i.e. fluorescence detectors FL2-5). Spectral characteristics of MA allow simultaneous surface immunophenotyping. CONCLUSIONS: It was shown that MA, CHR, and SA stain nucleic acids in living cells. They can be used as supravital fluorescent DNA probes, both in fluorescence microscopy and flow cytometry, including multiparameter analysis of peripheral blood and bone marrow. MA binds DNA stochiometrically and can provide information on DNA content. Copyright (c) 2007 International Society for Analytical Cytology.
