RNA Purification Step Research Articles

Development and validation of a new and rapid molecular diagnostic tool based on RT-LAMP for Hepatitis C virus detection at point-of-care

PurposeGlobally, it is estimated that 1.0 million individuals are newly infected by Hepatitis C virus (HCV) every year, and nearly 50 million people live with a chronic infection, according to World Health Organization. To overcome underdiagnosis of HCV infection among hard-to-reach populations, it is essential to develop new rapid and easy-to-use molecular diagnostic systems. In this work, we have developed a pangenotypic diagnostic tool based on Loop-Mediated Isothermal Amplification (LAMP), coupled to a direct sample lysis procedure for molecular detection of HCV at point-of-care (POC). MethodsProcedure validation was performed using 129 different samples from HCV infected patients (116 serum samples, and 13 fresh blood samples), 27 individuals who tested negative for HCV but positive for HIV, and 11 healthy donors. Serum was collected, lysed for 10 min at room temperature, and assayed by RT-LAMP. To achieve this, a set of 9 LAMP-primers was used for the first time. Parallel RT-qPCR assays were conducted for HCV to both validate the procedure and quantify viral loads. ResultsHCV was detected by RT-LAMP in 109/116 HCV positive serum samples, and in 11/13 positive blood samples in less than 40 min. Compared to RT-qPCR results, our RT-LAMP procedure showed a sensitivity of 94 %, 100 % specificity, and a limit of detection of 3.26 log10 IU/mL (10–20 copies per reaction). ConclusionsWe have developed an accurate system, more affordable than the current available rapid tests for HCV. Since no prior RNA purification step from capillary blood is required, we strongly recommend our RT-LAMP system as a valuable and rapid tool for the molecular detection of HCV at POC.

Development of a Diagnostic Microfluidic Chip for SARS-CoV-2 Detection in Saliva and Nasopharyngeal Samples.

The novel coronavirus SARS-CoV-2 was first isolated in late 2019; it has spread to all continents, infected over 700 million people, and caused over 7 million deaths worldwide to date. The high transmissibility of the virus and the emergence of novel strains with altered pathogenicity and potential resistance to therapeutics and vaccines are major challenges in the study and treatment of the virus. Ongoing screening efforts aim to identify new cases to monitor the spread of the virus and help determine the danger connected to the emergence of new variants. Given its sensitivity and specificity, nucleic acid amplification tests (NAATs) such as RT-qPCR are the gold standard for SARS-CoV-2 detection. However, due to high costs, complexity, and unavailability in low-resource and point-of-care (POC) settings, the available RT-qPCR assays cannot match global testing demands. An alternative NAAT, RT-LAMP-based SARS-CoV-2 detection offers scalable, low-cost, and rapid testing capabilities. We have developed an automated RT-LAMP-based microfluidic chip that combines the RNA isolation, purification, and amplification steps on the same device and enables the visual detection of SARS-CoV-2 within 40 min from saliva and nasopharyngeal samples. The entire assay is executed inside a uniquely designed, inexpensive disposable microfluidic chip, where assay components and reagents have been optimized to provide precise and qualitative results and can be effectively deployed in POC settings. Furthermore, this technology could be easily adapted for other novel emerging viruses.

An Optimal Transport Medium for SARS-CoV-2 Detection in the Direct Method of Rapid Microfluidic PCR System.

Recently developed rapid real-time reverse transcription PCR (RT-PCR) systems adopting microfluidic thermal cycling technology are ideal for point-of-care (POC) testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Because the RNA extraction step before real-time RT-PCR is rate-limiting, a direct RNA extraction method (direct method) that adopts chemical viral lysis and eliminates RNA purification steps is preferable for rapid real-time RT-PCR. In the direct method, selecting the transport medium is essential because it may be introduced into subsequent real-time RT-PCR steps, but might inhibit PCR. However, the influence of transport medium on the combination of the direct method and rapid real-time RT-PCR has been yet unstudied. In the present study, we examined the influence of various transport mediums when combining the direct method and rapid real-time RT-PCR of GeneSoC® (GeneSoC® RT-PCR), the recently developed compact PCR system that adapts novel microfluidic thermal cycling technology. To explore the influence of the transport medium on the GeneSoC® RT-PCR, the concordance of the RNA extraction and direct method was evaluated in the clinical samples collected in viral transport medium (VTM) or eSwab®. The sensitivity of GeneSoC® RT-PCR combined with the direct method was assessed using spiked samples in generic (H2O and PBS) or commercially available transport media (VTM and eSwab®). Analytical sensitivity was examined using clinical specimens collected from the VTM and eSwab®. The inhibitory effect of PCR inhibitors on clinical specimens was assessed using clinical samples diluted 1,000 times. While only 1 copy/reaction of RNA was detected in H2O and eSwab® of the spiked samples, a minimum of 5 copies/reaction was detected in PBS (-) and VTM. Among the clinical specimens tested using the direct method, the detection of viral RNA was unstable in the samples containing less than 100 copies/reaction viral RNA in VTM, whereas less than 10 copies/reaction viral RNA were detected in eSwab®. The positive, negative, and overall concordance between the RNA extraction and the direct method was 84%, 100%, and 85%, respectively, in eSwab® samples, whereas the values were 35%, 100%, and 38%, respectively, in VTM samples. When the clinical samples were diluted 1,000 times, GeneSoC® RT-PCR could detect as low as 1.15 copies/reaction RNA using direct method, and the sensitivity was comparable to that of RNA extraction. The combination of the direct method and microfluidic rapid PCR machine GeneSoC® has a high sensitivity for detecting SARS-CoV-2 RNA in clinical samples with eSwab® transport medium.

A Multiplex Gene Expression Assay for Direct Measurement of RNA Transcripts in Crude Lysates of the Nematode Caenorhabditis elegans Used as a Bioanalytical Tool.

Gene expression profiling in Caenorhabditis elegans has been demonstrated to be a potential bioanalytical tool to detect the toxic potency of environmental contaminants. The RNA transcripts of genes responding to toxic exposure can be used as biomarkers for detecting these toxins. For routine application in environmental quality monitoring, an easy-to-use multiplex assay is required to reliably quantify expression levels of these biomarkers. In the present study, a bead-based assay was developed to fingerprint gene expression in C. elegans by quantitating messenger RNAs (mRNAs) of multiple target genes directly from crude nematode lysates, circumventing RNA extraction and purification steps. The assay uses signal amplification rather than target amplification for direct measurement of toxin-induced RNA transcripts. Using a 50-gene panel, the expression changes of four candidate reference genes and 46 target mRNAs for various contaminants and wastewaters were successfully measured, and the expression profiles indicated the type of toxin present. Moreover, the multiplex assay response was in line with previous results obtained with more time-consuming reverse-transcription quantitative polymerase chain reaction and microarray analyses. In addition, the transcriptomic profiles of nematodes exposed to wastewater samples and extracts prepared from tissues of swimming crabs were evaluated. The profiles indicated the presence of organic pollutants. The present study illustrates the successful development of a multiplex fluorescent bead-based approach using nematode C. elegans crude lysates for gene expression profiling of target RNAs. This method can be used to routinely fingerprint the presence of toxic contaminants in environmental samples and to identify the most biologically active fraction of the contaminant mixture in a toxicity identification and evaluation approach. Environ Toxicol Chem 2023;42:130-142. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Diagnostics of Infections Produced by the Plant Viruses TMV, TEV, and PVX with CRISPR-Cas12 and CRISPR-Cas13.

Viral infections in plants threaten food security. Thus, simple and effective methods for virus detection are required to adopt early measures that can prevent virus spread. However, current methods based on the amplification of the viral genome by polymerase chain reaction (PCR) require laboratory conditions. Here, we exploited the CRISPR-Cas12a and CRISPR-Cas13a/d systems to detect three RNA viruses, namely, Tobacco mosaic virus, Tobacco etch virus, and Potato virus X, in Nicotiana benthamiana plants. We applied the CRISPR-Cas12a system to detect viral DNA amplicons generated by PCR or isothermal amplification, and we also performed a multiplexed detection in plants with mixed infections. In addition, we adapted the detection system to bypass the costly RNA purification step and to get a visible readout with lateral flow strips. Finally, we applied the CRISPR-Cas13a/d system to directly detect viral RNA, thereby avoiding the necessity of a preamplification step and obtaining a readout that scales with the viral load. These approaches allow for the performance of viral diagnostics within half an hour of leaf harvest and are hence potentially relevant for field-deployable applications.

Evaluation of Using Direct Viral Transport Medium Samples without Nucleic Acid Isolation for SARS-CoV-2 Diagnosis by RT-PCR.

Diagnosis with reverse transcriptase polymerase chain reaction (RT-PCR) test is a very important step for the control of the COVID-19 pandemic. The aim of this study is to compare the RT-PCR results of the samples taken directly from the viral transport medium (VTM) without extraction with the RT-PCR results of two different extraction methods, one automated and the other manual, in the diagnosis of COVID-19. Among the respiratory tract samples sent to Sakarya Training and Research Hospital Microbiology Laboratory for COVID-PCR study, 20 negative and 43 positive samples with different cycle threshold (CT) values were included in the study. Both manual nucleic acid isolation with the vNAT isolation kit (Bioeksen, Turkey) and automatic nucleic acid isolation with the EZ1 Virus Mini Kit v2.0 in the isolation device were performed simulta-neously from the patient samples included in the study and the results were compared. The mean Ct values of the samples were found to be 21.58 using manual vNAT as the extraction method, 17.63 using the automated magnetic bead method, and 21.45 in PCR from direct VTM without extraction. When the automatic magnetic beads extraction method was taken as the reference method, the sensitivity of direct PCR was 97.3%, the specificity was 95%, the positive predictive value was 97.3%, and the negative predictive value was 95%. Phi coefficients were found to be 0.927 between vNAT and direct PCR, 1 between vNAT and EZ1, and 0.922 between direct PCR and EZ1. Direct PCR has advantages such as eliminating RNA extraction and purification steps, providing a shorter detection time, and using less labor and less consumables without reducing the diagnostic accuracy. It is thought that this method can help as a useful process management for the control of the epidemic in countries with limited resources.

Direct Detection of Extracellular Vesicle miRNAs Using a Single-Step RT-qPCR Assay.

Extracellular vesicles (EVs) are biological carriers, and EV-associated miRNAs (EV-miRNAs) are considered as a novel biomarker in multiple diseases. Currently, the column-based purification method is used to purify miRNAs from EVs. However, this method of purification is complex, time-consuming, and expensive. Therefore, a simple and cost-effective single-step quantitative reverse transcription-polymerase chain reaction (RT-qPCR) method is required to detect the expression of EV-miRNAs. This chapter describes a protocol for directly analyzing the EV-miRNAs expression from mouse bronchoalveolar lavage fluid (BALF) and serum without going for an RNA isolation and purification step from EVs. It is an efficient method in several terms such as cost-wise, time, low expertise, and accuracy in results. This method may be helpful in diagnostic blood tests used in medical centers or research laboratories.

A Simple Multiplex Reverse Transcription-PCR Method for the Diagnosis of L-A and M Totiviruses in Saccharomyces cerevisiae.

Killer yeasts and their toxins have many potential applications in environmental, medical, and industrial biotechnology. The killer phenotype in Saccharomyces cerevisiae relies on the cytoplasmic persistence of two dsRNA viruses, L-A and M. M encodes the toxin, and L-A provides proteins for expression, replication, and capsids for both viruses. Yeast screening and characterization of this trait are usually performed phenotypically based on their toxin production and immunity. In this study, we describe a simple and specific reverse transcription (RT) multiplex PCR assay for direct diagnosis of the dsRNA totivirus genomes associated with the killer trait in the S. cerevisiae yeast. This method obviates RNA purification steps and primer addition to the RT reaction. Using a mixture of specific primers at the PCR step, this multiplex RT-PCR protocol provided an accurate diagnosis of both L-A and M totivirus in all its known variants, L-A-1/M1, L-A-2/M2, L-A-28/M28, and L-A-lus/Mlus, found in infected killer yeasts. Using this method, the expected L-A-2/M2 totivirus associations in natural wine yeasts cells were identified but, importantly, asymptomatic L-A-2/M2 infected cells were found in addition to unexpected L-A-lus/M2 totiviral associations. IMPORTANCE The killer phenomenon in S. cerevisiae yeast cells provides the opportunity to study host-virus interactions in a eukaryotic model. Therefore, the development of simple methods for their detection significantly facilitates their study. The simplified multiplex RT-PCR protocol described here provides a useful and accurate tool for the genotypic characterization of yeast totiviruses in killer yeast cells. The killer trait depended on two dsRNA totiviruses, L-A and M. Each M dsRNA depends on a specific helper L-A virus. Thus, direct genotyping by the described method also provided valuable insights into L-A/M viral associations and their coadaptational events in nature.

Clinical performance and potential of a SARS-CoV-2 detection kit without RNA purification steps

Abstract Objectives Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is rapidly spreading globally. Early diagnosis plays an essential role in controlling the infection. Therefore, early and accurate SARS-CoV-2 detection assays along with easy operation are required. The aim of this study was to compare the clinical performance of the Ampdirect™ 2019-nCoV Detection Kit (SHIMADZU assay), which does not require RNA purification steps, with that of the preexisting SARS-CoV-2 detection assays, which use a purified RNA template. Methods A total of 71 samples (65 nasopharyngeal specimens and 6 sputum specimens) were collected from 32 individuals, including patients infected with SARS-CoV-2 and those with suspected infection. The sensitivity and kappa (κ) coefficient were assessed between the SARS-CoV-2 detection assays using the reference standard, which was defined as a true positive result by any one of the four SARS-CoV-2 detection assays. Results The overall sensitivity and κ coefficient of the SHIMADZU assay were 86.0% (95% confidence interval [CI]: 77.9–94.2) and 0.83 (95% CI: 0.69–0.96), respectively. In particular, among the 18 samples collected within 10 days from symptom onset, the sensitivity and κ coefficient of the SHIMADZU assay were 100% and 1.0, respectively. Conclusions Although a relatively small number of samples was evaluated, the SHIMADZU assay showed good analytical performance and as such would be highly useful for the detection of SARS-CoV-2. The test can be performed easily and quickly and has the potential for future applications in situations where a highly sensitive diagnosis is required.

Monitoring SARS-CoV-2 in sewage: Toward sentinels with analytical accuracy

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemia has been one of the most difficult challenges humankind has recently faced. Wastewater-based epidemiology has emerged as a tool for surveillance and mitigation of potential viral outbreaks, circumventing biases introduced by clinical patient testing. Due to the situation urgency, protocols followed for isolating viral RNA from sewage were not adapted for such sample matrices. In parallel to their implementation for fast collection of data to sustain surveillance and mitigation decisions, molecular protocols need to be harmonized to deliver accurate, reproducible, and comparable analytical outputs. Here we studied analytical variabilities linked to viral RNA isolation methods from sewage. Three different influent wastewater volumes were used to assess the effects of filtered volumes (50, 100 or 500 mL) for capturing viral particles. Three different concentration strategies were tested: electronegative membranes, polyethersulfone membranes, and anion-exchange diethylaminoethyl cellulose columns. To compare the number of viral particles, different RNA isolation methods (column-based vs. magnetic beads) were compared. The effect of extra RNA purification steps and different RT-qPCR strategies (one step vs. two-step) were also evaluated. Results showed that the combination of 500 mL filtration volume through electronegative membranes and without multiple RNA purification steps (using column-based RNA purification) using two-step RT-qPCR avoided false negatives when basal viral load in sewage are present and yielded more consistent results during the surveillance done during the second-wave in Delft (The Hague area, The Netherlands). By paving the way for standardization of methods for the sampling, concentration and molecular detection of SARS-CoV-2 viruses from sewage, these findings can help water and health surveillance authorities to use and trust results coming from wastewater based epidemiology studies in order to anticipate SARS-CoV-2 outbreaks.

Validation Study for VERIPRO® SARS-CoV-2 Env Assay for the Detection of SARS-CoV-2 from Stainless-Steel Environmental Surface Swabs: Emergency Response Validation—AOAC Performance Tested MethodSM 122001

BackgroundThe VERIPRO SARS-CoV-2 Env assay uses reverse transcriptase PCR (RT-PCR) to detect SARS-CoV-2, the causative agent of COVID-19, from stainless steel environmental sample swabs.ObjectiveTo validate the VERIPRO SARS-CoV-2 Env assay as part of the AOAC Research Institute’s Emergency Response Validation Performance Tested MethodSM program.MethodsThe VERIPRO SARS-CoV-2 Env assay was evaluated for specificity using in silico analysis of 15 764 SARS-CoV-2 sequences and 65 exclusivity organisms (both near neighbors and background organisms). The candidate method was evaluated in an unpaired study design for one environmental surface (stainless steel) and compared to the U.S. Centers for Disease Control and Prevention 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel, Instructions for Use (Revision 4, Effective 6/12/2020).ResultsResults of the in silico analysis demonstrated the specificity of the method in being able to detect target sequences and discriminate them from near neighbors. In the matrix study, the candidate method demonstrated statistically significant better recovery of the target analyte than the reference method.ConclusionsThe VERIPRO SARS-CoV-2 Env assay is a rapid and accurate method that can be utilized by food producers to detect the causative agent of COVID-19 on food contact surfaces.HighlightsThe VERIPRO SARS-CoV-2 Env assay can be performed without the need for an optional RNA purification step to detect SARS-CoV-2 from environmental surfaces.

Rapid processing of SARS-CoV-2 containing specimens for direct RT-PCR.

Widespread diagnostic testing is needed to reduce transmission of COVID-19 and manage the pandemic. Effective mass screening requires robust and sensitive tests that reliably detect the SARS-CoV-2 virus, including asymptomatic and pre-symptomatic infections with a low viral count. Currently, the most accurate tests are based on detection of viral RNA by RT-PCR. We developed a method to process COVID-19 specimens that simplifies and increases the sensitivity of viral RNA detection by direct RT-qPCR, performed without RNA purification. In the method, termed Alkaline-Glycol Processing (AG Processing), a SARS-CoV-2-containing biological specimen, such as saliva or a swab-collected suspension, is processed at pH 12.2 to 12.8 for 5 min at room temperature. An aliquot of the AG-processed specimen is used for detection of SARS-CoV-2 RNA by direct RT-qPCR. AG processing effectively lyses viruses and reduces the effect of inhibitors of RT-PCR that are present in biological specimens. The sensitivity of detecting viral RNA using AG processing is on par with methods that include a viral RNA purification step. One copy of SARS-CoV-2 virus per reaction, equivalent to 300 copies per ml of saliva, is detectable in the AG-processed saliva. The LOD is 300 viral copies per ml of initial saliva specimen. AG processing works with saliva specimens or swab specimens collected into Universal Transport Medium, is compatible with heat treatment of saliva, and was confirmed to work with a range of CDC-approved RT-qPCR products and kits. Detection of SARS-CoV-2 RNA using AG processing with direct RT-qPCR provides a reliable and scalable diagnostic test for COVID-19 that can be integrated into a range of workflows, including automated settings.

Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification.

Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44-104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

Direct on-the-spot detection of SARS-CoV-2 in patients.

Many countries are currently in a state of lockdown due to the SARS-CoV-2 pandemic. One key requirement to safely transition out of lockdown is the continuous testing of the population to identify infected subjects. Currently, detection is performed at points of care using quantitative reverse-transcription PCR, thus requiring dedicated professionals and equipment. Here, we developed a protocol based on reverse transcribed loop-mediated isothermal amplification for the detection of SARS-CoV-2. This protocol is applied directly to SARS-CoV-2 nose and throat swabs, with no RNA purification step required. We tested this protocol on over 180 suspected patients, and compared the results to those obtained using the standard method. We further succeeded in applying the protocol to self-collected saliva samples from confirmed cases. Since the proposed protocol can detect SARS-CoV-2 from saliva and provides on-the-spot results, it allows simple and continuous surveillance of the community.Impact statementHumanity is currently experiencing a global pandemic with devastating implications on human health and the economy. Most countries are gradually exiting their lockdown state. We are currently lacking rapid and simple viral detections, especially methods that can be performed in the household. Here, we applied RT-LAMP directly on human clinical swabs and self-collected saliva samples. We adjusted the method to allow simple and rapid viral detection, with no RNA purification steps. By testing our method on over 180 human samples, we determined its sensitivity, and by applying it to other viruses, we determined its specificity. We believe this method has a promising potential to be applied world-wide as a simple and cheap surveillance test for SARS-CoV-2.

Protocol for efficient fluorescence 3′ end-labeling of native noncoding RNA domains

Noncoding RNAs (ncRNAs) comprise a class of versatile transcripts that are highly involved in the regulation of a wide range of biological processes. Functional long ncRNAs (> 200 nts in length) often adopt secondary structures that arise co-transcriptionally. To maintain the secondary structure elements as well as preparation homogeneity of such transcripts, native-like conditions should be maintained throughout the in vitro synthesis, purification and chemical tagging processes. In this optimized protocol, we describe a simple method for obtaining homogenous samples followed by chemically tagging the 3′ termini of natively-purified structured ncRNA domains that are longer than 200 nts. This protocol replaces traditional hazardous radioactive labeling with fluorescence tagging, and eliminates laborious and time consuming RNA purification and concentration steps and replaces them with straightforward recovery of RNA through centrifugal filtration, preserving the homogeneity and mono-dispersion of the preparations. The protocol provides:•An integrative, simple and straightforward approach for synthesis, purification and labeling of structured ncRNAs whilst maintaining their secondary structure intact.•Replacing hazardous, laborious and time-consuming radioactive labeling of RNA with much simpler fluorescence tagging, thereby facilitating potential downstream applications such as electrophoretic mobility shift assay (EMSA).•A versatile protocol that could be applicable to a wide-range of chemical tags and in principle could be used to label DNA or RNA.

Optimization of a Quantitative Real-Time RT-PCR Technique for Evaluation of Concentration of Genomic +RNA of Tick-Borne Encephalitis Virus

Background. The specific detection of genomic/template +RNA and replicative –RNA of tick-born encephalitis virus (TBEV) is necessary to study the mechanisms of viral replication in the cells of reservoir and accidental hosts. However, the current approaches of quantitative reverse transcription – polymerase chain reaction (qRT-PCR) are rather focused on the detection of total viral RNA load in the sample. Thus, the significant optimization is necessary both for RT-PCR and for RNA copy number standard preparation.Aims. To develop the set of standard samples of synthetic +RNA of TBEV and to optimize qRT-PCR for quantification of genomic +RNA of the virus.Materials and methods. Fragment of the genomic +RNA of TBEV was synthesized using pTZ57R-T\A plasmid vector with embedded T7 promoter and T7 RNA polymerase. The DNA contamination was removed using RNase-free DNase I treatment followed by additional RNA purification step. Reverse transcription was performed using specific antisense primer 11154R 5`- AGCGGGTGTTTTTCCG-3` and qPCR detection was used according to the modified procedure of M. Schwaiger and P. Cassinotti (2003).Results. As a result of the amplification of standard samples, the concentration of positive polarity ТBEV RNA, carried out in five independent repetitions on different days, the correlation coefficient R2 between the quantification cycle and the concentration of the standard sample was 0.99, and the efficiency of PCR was 100 %. The coefficient of variation in assessing the inter-test accuracy of determination averaged 2.8 %.Conclusions. Optimized qRT-PCR procedure and set of +RNA standards allow to determine the concentration of genomic +RNA of TBEV in routine laboratory practice.

A Simple, Rapid and Efficient Method of Pepino mosaic Virus RNA Isolation from Tomato Fruit

The main concern in molecular detection of RNA viral pathogens in plants is the achievement of good quality of the extracted RNA. Various methods of isolating RNAs from both polysaccharide-rich and poor tissues and other recalcitrant plants are available. However, the use of time and reagent consuming methods and those involving hazardous chemicals is somewhat cumbersome and problematic, especially when it is not necessary for specific purposes like isolating viral RNA from tomato fruit, hence the objective of this paper. We describe an alternative, simple and rapid method for preparing viral RNA from tomato fruit without RNA extraction and purification steps, case of Pepino mosaic virus (PepMV). The method employs mechanical treatment and suspension in water. The quality of RNA obtained was judged by spectrometric readings and validated in RT-PCR assays. The used protocol was compared with the usual TRIzol method. The results showed that the yield and the quality of RNA obtained using the proposed method are efficient and highly yielded in comparison with TRIzol method. Moreover, the developed method successfully allowed a sensitive and reproducible detection of PepMV predicted bands in RT-PCR. Thus, molecular detection of PepMV from tomato fruit can be performed routinely without fastidious RNA isolation. As well, this will make the diagnosis of other RNA viruses infecting tomato crops easier and less time-consuming, in comparison with the other methods performed with expensive commercial kits and those involving toxic chemicals. Finally, the described established method will contribute effectively in strategies of phytosanitary and certification programs of tomato crops worldwide.

Comparison and optimization of detection methods for noroviruses in frozen strawberries containing different amounts of RT-PCR inhibitors

Frozen berries have been repeatedly identified as vehicles for norovirus (NoV) transmission causing large gastroenteritis outbreaks. However, virus detection in berries is often hampered by the presence of RT-PCR-inhibiting substances. Here, several virus extraction methods for subsequent real-time RT-PCR-based NoV-RNA detection in strawberries were compared and optimized. NoV recovery rates (RRs) between 0.21 ± 0.13% and 10.29 ± 6.03% were found when five different artificially contaminated strawberry batches were analyzed by the ISO/TS15216-2 method indicating the presence of different amounts of RT-PCR inhibitors. A comparison of five different virus extraction methods using artificially contaminated strawberries containing high amounts of RT-PCR inhibitors revealed the best NoV RRs for the ISO/TS15216 method. Further improvement of NoV RRs from 2.83 ± 2.92% to 15.28 ± 9.73% was achieved by the additional use of Sephacryl®-based columns for RNA purification. Testing of 22 frozen strawberry samples from a batch involved in a gastroenteritis outbreak resulted in 5 vs. 13 NoV GI-positive and in 9 vs. 20 NoV GII-positive samples using the original ISO/TS15216 method vs. the extended protocol, respectively. It can be concluded that the inclusion of an additional RNA purification step can increase NoV detection by the ISO/TS15216-2 method in frozen berries containing high amounts of RT-PCR inhibitors.

A Novel Technology for Multiplex Gene Expression Analysis Directly from Whole Blood Samples Stabilized at Ambient Temperature Using an RNA-Stabilizing Buffer

We describe a novel method, based on target-dependent chemical ligation of probes, which simplifies the multiplexed quantitation of gene expression from blood samples by eliminating the RNA purification step. Gene expression from seven genes was evaluated over a range of sample inputs (16.7 to 0.25 μL of whole blood in serial dilutions) from three healthy donors. Mean CVs were ≤11% for five technical replicates for whole blood inputs ≥2.1 μL. The method showed a limit of detection of 300 copies of RNA by using titration of in vitro transcripts for four genes. Gene expression measured on stabilized blood samples was highly correlated (Spearman rank correlation method, ρ = 0.80) to gene expression results obtained with RNA isolated from matched samples (three donors, five technical replicates). Gene expression changes determined with seven radiation-responsive genes on six healthy donor blood samples before and after ex vivo irradiation were highly correlated (ρ = 0.93) to those measured with a TaqMan quantitative real-time RT-PCR assay on RNA purified from matched samples. Thus, this method is reproducible, sensitive, and correlated to quantitative real-time RT-PCR and may be used to streamline the multiplex gene expression analysis of large numbers of stabilized blood samples without RNA purification.

A comparison of sperm RNA-seq methods

A significant challenge to the effective application of RNA-seq to the complete transcript analysis of low quantity and/or degraded samples is the amplification of minimal input RNA to enable sequencing library construction. Several strategies have been commercialized in order to facilitate this goal. However, each strategy has its own specific protocols and methodology, and each may introduce unique bias and in some cases show specific preference for a collection of sequences. Our wider investigation of human spermatozoal RNAs was able to reveal their complexity despite being generally characterized by low quantity and high fragmentation. In this study, the following four commercially available RNA-seq amplification and library protocols for the preparation of low quantity/highly fragmented samples, SMARTer™ Ultra Low RNA (SU) for Illumina® Sequencing, SeqPlex RNA Amplification (SP), Ovation® RNA-Seq System V2 (OR), and Ovation® RNA-Seq Formalin Fixed Paraffin Embedded System (FFPES) were assessed using human sperm RNAs. Further investigation analyzed the effects on the end results of two different library preparation methods, Encore NGS Multiplex System I (Enc) and Ovation Ultralow Library Systems (UL), that appeared best suited to this type of RNA, along with other potential confounding factors such as FFPE preservation. Our results indicate that for each library preparation protocol, the differences in the initial amount of input RNA and choice of RNA purification step do not generate marked differences in terms of RNA profiling. However, substantial disparity is introduced by individual amplification methods prior to library construction. These significant differences may be caused by the different priming methods or amplification strategies used in each of the four different protocols examined. The observation of intra-sample variation introduced by the choice of protocol highlights the role that external factors play in planning and subsequent reliable interpretation of results of any RNA-seq experiment.