Optimized method for RNA extraction from leaves of forest tree species

Extraction of ribonucleic acid (RNA) from woody plants is a difficult task due to the peculiarities of plant material rich in polysaccharides and starch. The available techniques are often ineffective, since they result in the absence/reduced quality/reduced amount of RNA in the final preparation. The method we have optimized is based on the use of cethyltrimethyl ammonium bromide (CTAB), purification by phenol-chloroform extraction, use of lithium chloride and ammonium acetate. The method showed high efficiency for the extraction of RNA from the leaves of birch and poplar samples, in vitro and mature plants, in comparison with previously used methods (extraction using NucleoSpin® RNA Plant (Macherey-Nagel, Germany) columns, Su (2009) method, standard guanidine thiocyanate method). Electropherograms of RNA preparations showed its high integrity and concentration (up to 85 ng/μl), significantly higher purity of the preparation (up to 2.7 times). Purification of the preparation in the process of extraction can significantly reduce the yield of desoxyribonucleic acid (DNA). The optimized method is highly reproducible and can be used for further research, complementary DNA (cDNA) synthesis, qualitative and quantitative PCR analysis. The method allows obtaining high-quality RNA from other objects of agricultural and forest plants.

Detection of genetically modified organisms in soy feed and food in Romania

The paper describes the diagnostic methods used in Romania to detect the genetically modified organisms in soy feed and food and the results recorded between 2013 and 2019. The retrospective analyses of surveillance and diagnostic methods (qualitative and quantitative PCR techniques) developed for the genetically modified organisms detection in soy feed and food and of the transgenic Roundup Ready soybean line event 40-3-2 in Romania covered a total of 687 tests between 2013 and 2019 and revealed 32/131 GM events in 2013, 25/142 GM events in 2014, 24/117 GM events in 2015, 16/115 GM events in 2016, 6/97 GM events in 2017, 8/53 GM events in 2018, and 2/32 GM events in 2019.

Identification of the Pol Gene as a Species-Specific Diagnostic Marker for Qualitative and Quantitative PCR Detection of Tricholoma matsutake

Tricholoma matsutake is a rare, precious, and wild edible fungus that could not be cultivated artificially until now. This situation has given way to the introduction of fake T. matsutake commodities to the mushroom market. Among the methods used to detect food adulteration, amplification of species-specific diagnostic marker is particularly important and accurate. In this study, the Pol gene is reported as a species-specific diagnostic marker to identify three T. matsutake varieties and 10 other types of edible mushrooms through qualitative and quantitative PCR. The PCR results did not reveal variations in the amplified region, and the detection limits of qualitative and quantitative PCR were found to be 8 ng and 32 pg, respectively. Southern blot showed that the Pol gene exists as a single copy in the T. matsutake genome. The method that produced the purest DNA of T. matsutake in this study was also determined, and the high-concentration salt precipitation method was confirmed to be the most suitable among the methods tested. The assay proposed in this work is applicable not only to the detection of raw materials but also to the examination of processed products containing T. matsutake.

Autoimmune manifestation of hepatitis C virus infection as a risk for late virological relapse after pegylated interferon and ribavirin therapy

Introduction. We are aware of the risk of late virological relapse (LVR) years after sustained viral response (SVR) by pegylated interferon and ribavirin alfa (PegIFN? + RBV) of chronic hepatitis C viral (HCV) infection. We presented three patients with LVR, treated by PegIFN? and ribavirin 5 years after the SVR was established. Case report. We analysed 129 (38.8% female, 61.2% male, mean age 37.02 ? SD 11.99) patients treated for chronic HCV with PegIFN? + RBV, with at least 5 years from the establishment of SVR. In addition to the biochemical parameters of liver function, the qualitative HCV RNA polymerase chain reaction (PCR) and the quantitative PCR HCV RNA test were made. Five years after establishing SVR in 2.3% (3/129) of patients, the relapse of HCV infection was registered by qualitative and quantitative PCR HCV RNA assay and all of these patients had additional autoimmune diseases: vasculitis, autoimmune hepatitis, and vasculitis of central nervous system. Conclusion. The existence, but low rate of LVR HCV infection was confirmed, dominantly in patients with additional autoimmune diseases. Due to this SVR after therapy by PegIFN? + RBV should be considered as an indicator of successful HCV suppresion, not its complete eradication.