Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe

The combination of recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a strong diagnostic tool for rapid pathogen detection in resource-limited conditions. Here, we compared two methods generating labeled RPA amplicons following their detection by LFT: (1) the basic one with primers modified with different tags at the terminals and (2) the nuclease-dependent one with the primers and labeled oligonucleotide probe for nuclease digestion that was recommended for the high specificity of the assay. Using both methods, we developed an RPA-LFT assay for the detection of worldwide distributed phytopathogen—alfalfa mosaic virus (AMV). A forward primer modified with fluorescein and a reverse primer with biotin and fluorescein-labeled oligonucleotide probe were designed and verified by RPA. Both labeling approaches and their related assays were characterized using the in vitro-transcribed mRNA of AMV and reverse transcription reaction. The results demonstrated that the RPA-LFT assay based on primers-labeling detected 103 copies of RNA in reaction during 30 min and had a half-maximal binding concentration 22 times lower than probe-dependent RPA-LFT. The developed RPA-LFT was successfully applied for the detection of AMV-infected plants. The results can be the main reason for choosing simple labeling with primers for RPA-LFT for the detection of other pathogens.

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called ‘Tiled-ClickSeq’, which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5’UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for Next-Generation Sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called ‘Tiled-ClickSeq’. Tiled-ClickSeq uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, obviating the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended using click-chemistry and a PCR reaction using Illumina adaptors generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5’UTR, at high depth and specificity to virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

OVERCOMING FALSE POSITIVES OF REVERSE TRANSCRIPTION AT THE DETECTION OF CHIMERIC RNAS

Работа посвящена исследованию формирования ложных химерных кДНК в результате смены матриц обратной транскриптазой. Показано, что, изменяя ряд параметров реакции обратной транскрипции, можно существенно уменьшить частоту ложноположительных результатов при выявлении истинных химерных РНК. Полученные результаты позволяют улучшить качество анализа транскриптомов и диагностики заболеваний, ассоциированных с образованием химерных РНК. This work is aimed at the study of formation of false chimeric cDNA as a result of template switch by reverse transcriptase. It is shown that by manipulating a number of parameters of the reverse transcription reaction, it is possible to significantly reduce the frequency of false-positives in the detection of true chimeric RNAs. The results allow to improve the quality of the analysis of transcriptomes and of the diagnostics of diseases associated with the formation of chimeric RNAs.

Evaluation of the Influence of Adalimumab on the Expression Profile of Leptin-Related Genes and Proteins in Keratinocytes Treated with Lipopolysaccharide A

Psoriasis is a disease with a proinflammatory base, in which an increased expression of leptin, tumor necrosis factor alpha (TNF-α), interleukin (IL) IL-12/23, IL-6, is observed. A drug used in the treatment of psoriasis of moderate and acute strength is the monoclonal antibody anti-TNF–adalimumab. The goal of this study was to evaluate the influence of adalimumab on changes in the expression profile of leptin-related genes in human keratinocyte cells exposed to lipopolysaccharide A and analyze if adalimumab acts via leptin pathways. The evaluation of changes of the pattern of genes connected with leptin and proteins coded by them was marked in a culture of human keratinocytes (HaCaT) exposed to 1 µg/mL lipopolysaccharide A (LPS) for 8 h in order to induce the inflammatory process, then to 8 µg/mL of adalimumab for 2.8 and 24 h in comparison with the control (cells not treated with the substances). The techniques used were mRNA microarray, Real-Time Quantitative Reverse Transcription Reaction (RTqPCR), Enzyme-Linked Immunosorbent Assay (ELISA), as well as transfections of HaCaT culture with leptin small interfering RNA (siRNA) in order to see whether adalimumab works through pathways dependent on leptin. A statistically lower expression of leptin and its receptors was observed under the influence of the drug, independent of the exposition time of keratinocytes to adalimumab. In the cells transfected with leptin siRNA, a lower concentration of JAK2 and STAT3 proteins was observed, which confirms that adalimumab works through pathways dependent on leptin. Adalimumab has a modulatory effect on the gene expression pattern and the proteins coded by them connected with leptin in keratinocytes treated with LPS in vitro.

Genomic Investigation of Hepatitis E Virus in Persian Gazelle

Background: Numerous studies have shown that a variety of animal species can be the hosts of the hepatitis E virus. In addition to pigs, wild boars, deer, and rats, new types of hepatitis E virus have been found in ferrets and bats. Objectives: Due to the limited reports of virus identification in deer and the potential role of this animal as a reservoir in maintaining the virus, in the present study, the genome of the virus was investigated in the samples of feces and gastrointestinal swabs of Gazelle. Methods: Samples were collected from 50 Gazelle in the protected area of Moteh and the lands around Maymeh City from winter, 2017 to winter, 2019. After RNA extraction and reverse transcription reaction, the genomic identification of the virus was performed by RT-PCR. Results: The results of the present study showed that out of 50 samples taken, three samples were positive for the hepatitis E virus, including one sample from female Gazelle under one year of age and two samples from female animals over one year of age. Conclusions: No statistically significant relationship was found between hepatitis E infection, age, and sex using statistical tests. The present study indicated the contamination of Iranian wildlife animals and the importance of these animals as the potential reservoirs of the disease.

Differences in the Expression Pattern of mRNA Protein SEMA3F in Endometrial Cancer in vitro under Cisplatin Treatment

Background: Semaphorin 3F (SEMA3F) plays a substantial role in carcinogenesis, because of its role in inducing angiogenesis, and creating a microenvironment for the developing tumor. Objective: The purpose of this work was to assess the impact of cisplatin, depending on the concentration and exposure time on the expression pattern of SEMA3F in an endometrial cancer cell line. Materials and Methods: Cultures of the Ishikawa endometrial cancer cells were incubated with cisplatin with the following concentrations: 2.5μM; 5μM; and 10μM and for the following periods of time: 12; 24; and 48 hours. Cells not incubated with the drug constituted the control in the experiment. To determine the effect of cisplatin on the expression of SEMA3F, the real-time quantitative reverse transcription reaction (RtqPCR; mRNA) was used, as well as the ELISA assay (protein). The statistical analysis was done with the admission of p<0.05. Results: The silencing of SEMA3F expression on the transcriptome and proteome levels in a culture unexposed to the effects of cisplatin in comparison to endometrial cancer cells under the influence of cisplatin (p<0.05) were noted. Along with an increase in the concentration of the drug used, the number of copies of the gene transcript, during the shortest incubation period had a gradual increase. Only for the highest concentration of the drug, substantial statistical differences in the expression of the SEMA3F protein between 24 and 48 hour incubation periods (p<0.05) were determined. Conclusions: Using cisplatin in an endometrial cancer cell culture results in an increased expression of SEMA3F, which advantageously affects the normalization of the neoplastic angiogenic process and lowers the proliferation of the cells making up the mass of the tumor.

Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing

Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq™ SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq™ SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer’s instructions.

SYBR green one-step qRT-PCR for the detection of SARS-CoV-2 RNA in saliva

We describe our efforts at developing a one-step quantitative reverse-transcription (qRT)-PCR protocol to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA directly from saliva samples, without RNA purification. We find that both heat and the presence of saliva impairs the ability to detect synthetic SARS-CoV-2 RNA. Buffer composition (for saliva dilution) was also crucial to effective PCR detection. Using the SG2 primer pair, designed by Sigma-Aldrich, we were able to detect the equivalent of 1.7×106 viral copies per mL of saliva after heat inactivation; approximately equivalent to the median viral load in symptomatic patients. This would make our assay potentially useful for rapid detection of high-shedding infected individuals. We also provide a comparison of the PCR efficiency and specificity, which varied considerably, across 9 reported primer pairs for SARS-CoV-2 detection. Primer pairs SG2 and CCDC-N showed highest specificity and PCR efficiency. Finally, we provide an alternate primer pair to use as a positive control for human RNA detection in SARS-CoV-2 assays, as we found that the widely used US CDC primers (targeting human RPP30) do not span an exon-exon junction and therefore does not provide an adequate control for the reverse transcription reaction.

Analysis of histaminergic system genes expression level in the early symptom stage of Parkinson’s disease

Целью данной работы было изучение изменений экспрессии генов гистаминергической системы в развитии патологических процессов при болезни Паркинсона (БП) на ранней симптомной стадии. На МФТП-индуцированной модели ранней симптомной стадии БП (мыши-самцы линии C57BL/6 по 10 шт. в контрольной и опытной группах) проанализирована экспрессия 9 генов, имеющих отношение к гистаминергической системе, методом обратной транскрипции и ПЦР в реальном времени (TaqMan). В результате были выявлены изменения на уровне мРНК для ряда генов в тканях мозга мышей, которые могут указывать на вовлеченность гистаминергической системы в патогенез БП на ранней стадии. The goal of this work was to study changes of histaminergic system genes expression level in the early symptom stage of PD. We were use MPTP mouse model of early symptom stage of PD in this study. Male mice C57BL/6 were used (n=10 in the control and experimental groups). For analysis we were choose nine genes, which associated with histaminergic system. Measuring of expression levels was carried out using reverse transcription reaction and real-time PCR (TaqMan). Changes at the mRNA level for a number of genes in the brain tissues of mice with MPTP-induced PD were founded. Based on these data, it can be suggested that histaminergic system is involve in early stage PD pathogenesis.