RT-PCR Reaction Research Articles

Ulcerative dermal necrosis of mature Atlantic salmon in the Kola and Tuloma rivers (Murmansk region): retrospective and prospects for studying the problem

The aim of the work is to analyze the results of virological and immunological studies, to track the dynamics of the entry and changes in external signs of pathology of Atlantic salmon into the Kola and Tuloma rivers.Methods used: fish size and weight indicators were determined using generally accepted methods. Methods for isolating viruses on continuous cell lines and their identification in RT-PCR reaction were used. Bactericidal activity of blood serum (BABS) was assessed using a nephelometric method. The content of non-specific immune complexes (IC) was established using a spectrophotometric method with selective precipitation with PEG-6000.According to the results of the work, it was revealed that in the Kola and Tuloma rivers, clinical manifestations of pathologies in fish have changed. Since 2021, typical symptoms of UDN have been replaced by signs of «red skin disease». The results of immunological studies indicate high activity of innate humoral immunity mechanisms in the examined individuals. The carriage of infectious pancreatic necrosis virus in Atlantic salmon populations living in the Kola and Tuloma rivers has been established. Clinical signs of pathology in Atlantic salmon are described in detail, and fish counting data for the Kola and Tuloma rivers are presented.The novelty of the work: for the first time, photographs of all noted typical signs of pathology, as well as the results of virological and immunological studies of Atlantic salmon in the Kola and Tuloma rivers for 2022–2024 are presented. A retrospective overview is given and prospects for studying the problem are outlined.Practical significance: the conducted research will allow creating a database on the results of comprehensive studies of Atlantic salmon diseases in the Kola and Tuloma rivers. Compare the results obtained in fish with and without clinical signs of pathologies over several years. Determine differences in the immune status of fish with different signs of pathology based on the results of long-term studies. This will help to understand how UDN disease develops over time and what causes it.

Confirmation of highly pathogenic avian influenza H5N1 in skuas, Antarctica 2024.

From December 2023 to March 2024, a surveillance program aiming to detect Highly Pathogenic Avian Influenza (HPAI) H5N1 was conducted on Antarctica territories, specifically at Fildes Peninsula (King George Island, Maritime Antarctic), and James Ross Island. At Fildes Peninsula, samples from marine birds and mammals were collected from four accessible sampling locations with significant animal colonies: Ardley Island, hosting a large concentration of Gentoo penguins (Pygoscelis papua); Ardley Cove, where small groups of likely non-breeding Chinstrap penguins (Pygoscelis antarcticus) were present; seal haul-out sites of Southern elephant (Mirounga leonina) and Weddell (Leptonycotes wedellii); and, a nesting site of Southern giant petrels (Macronectes giganteus). Additionally, six samples were collected from five dead skuas near the Lachman lakes on James Ross Island (63.7989S, 57.8105W) on March 3, 2024. Despite collecting a total of 943 samples from Fildes Peninsula, all results tested negative for HPAI, and no animals displayed clinical signs or behaviors consistent with HPAI infection. However, all skua samples from James Ross Island tested positive for HPAI H5N1 clade 2.3.4.4 by specific real-time RT-PCR reactions, confirming the first recorded HPAI-related mortality event in Antarctica (south of 60°S), specifically in skuas. Further research is necessary to genetically characterize the virus and better understand the role of skuas in viral dissemination in Antarctica.

Expression of thermostable MMLV reverse transcriptase in Escherichia coli by directed mutation

The functionality of Moloney murine leukemia virus reverse transcriptase (MMLV RT) will increase with the improvement of its solubility and thermal stability. Introduce directed mutation at specific positions of the MMLV RT sequence and codon optimization is needed to achieve these properties. The two RT coding sequences with (rRT-K) and without directed mutations (rRT-L) were versatility optimized and expressed to analyze the ribonuclease H (RNase H) inactivity and thermostable polymerase activity. For this purpose, the five-point mutations (438–442aa) and three-point mutations (530, 568, and 659 aa) were done at the RT connection domain and RNase H active site, respectively. High expression levels of rRT-L and rRT-K were obtained in E. coli BL21(DE3) and BL21(shuffle) strains, 0.5 mM IPTG concentration at 37 °C, and 8 hours’ post-induction condition. Then, recombinant enzymes were purified and verified by Ni-NTA resin and western blotting. Insilico analysis (IUpred 3.0) showed that the directed mutation in the RNase H domain caused the formation of disorder regions or instability in the RNase H domain of rRT-K compared to rRT-L. The modified RT-PCR and the RT-LAMP reactions proved the RNase H inactivity of rRT-K. In addition, increasing of thermostability of rRT-K compared to rRT-L and commercial RT was evaluated by the RT-PCR and RT-LAMP reactions. The results showed that rRT-K could successfully tolerate 60 ºC in the two methods. This study revealed that the directed mutations and the versatile sequence optimization can promise to produce thermostable commercial enzymes to decrease non-specific one-step RT-PCR and RT-LAMP products.

B-026 Transforming PCR protocols in pandemic response with automated non-contact liquid dispensing

Abstract Background During the COVID-19 pandemic, effective Polymerase Chain Reaction (PCR) protocols were crucial for detecting SARS-CoV-2 in clinical samples, playing a key role in rapid infection control and easing the strain on healthcare services. However, with the critical need for fast and accurate pathogen surveillance, the manual execution of PCR workflows presented numerous challenges, including limited throughput, risk of human error and variability, and risk to human health. Several automated liquid handling systems from a range of vendors were considered to prepare extraction plates for this process. Ultimately, RFL decided on dragonfly® discovery, a non-contact positive displacement dispenser. Methods The analytical sensitivity for the UltraDx SARS-CoV-2 N1/N2/RP assay was evaluated by the RFL development team using RNA extracted from a panel of virus inputs from a Qnostics Analytical Q Panel, Product Number: SCV2AQP. Samples were extracted using a modified RNA extraction procedure for Thermo MagMax reagents and consumables with dragonfly discovery. dragonfly discovery was utilized to distribute a combination of inactivation buffer and beads into a 96-well kingfisher flex plate. This process was accomplished using a peristaltic pump and a 6-way Auto Feed Reservoir (AFR) to ensure a constant buffer flow. Simultaneously, the AFR ensured that the beads were kept in constant suspension, resulting in precise dispensing across all wells. The EPCR assay, UltraDx SARS-CoV-2 N1/N2/RP assay (LGC, Biosearch Technologies), utilized two CDC-characterized amplicon primer sets alongside two additional probes using fluorescence-based (FAM dye) detection. The input RNA extracted from clinical samples was combined with RT-PCR reaction mixes, before the reaction arrays were amplified. The reactions were then measured by a single scan on a multi-channel fluorescence reader. Results The UltraDx SARS-CoV-2 N1/N2/RP assay (when performed using the manufacturer method and in the absence of dragonfly discovery) has an LoD of 80 copies per reaction for the N1 assay, and 20 copies per reaction for the N2 assay, across 20 replicates for each copy number tested. Using the same/equivalent reagents and control materials, inclusion of dragonfly discovery showed an LoD of 70 copies/mL (0.9 copies/reaction) sensitivity when compared to the LoD suggested by the manufacture. Conclusions Integrating dragonfly discovery into the UltraDx SARS-CoV-2 N1/N2/RP assay led to an 80-fold reduction in the limit of detection for the N1 assay compared to the reagent manufacturer's recommendation. Some of this improvement can be attributed to dragonfly discovery's non-contact positive displacement dispensing, coupled with the AFR pump mechanism that ensures a continuous suspension of magnetic beads, enhancing accuracy and precision. dragonfly discovery's versatility supports a robust business-as-usual (BAU) process and is easily scalable for pandemic-level testing. Its user-friendly software interface requires zero computer programming knowledge, providing end-users with the flexibility to run multiple protocols on the same instrument, making it ideal for BAU assay processing. The instrument played a pivotal role in the UltraDx SARS-CoV-2 N1/N2/RP assay conducted by UKHSA during the pandemic, and SPT Labtech will continue to support UKHSA in their mission to protect communities from the impact of health threats.

Detection of Hepatitis E Virus (HEV) in Pork Sold in Saint-Louis, the North of Senegal.

The hepatitis E virus (HEV) is a zoonotic pathogen with various hosts, including pigs, which act as reservoirs. In industrialized countries, sporadic cases caused by genotype 3, contracted by ingesting contaminated uncooked or undercooked meat, have been reported. However, in developing countries, HEV infection is mainly dominated by genotype 2 and often associated with poor hygiene conditions and drinking water supplies. HEV infection and its circulation in domestic fauna in West Africa are poorly documented. This study aimed to assess the presence of HEV in pork sold in Saint-Louis, Senegal. Meat products (250 g samples, n = 74) were purchased in August 2022 from three locations. Then, 2 g/sample was minced to extract total nucleic acids using the Purelink™ Viral DNA/RNA kit. RT-PCR reactions were performed using the One-Taq™ One-Step RT-PCR kit targeting the HEV ORF2 genomic region. The products obtained were visualized on a 1% agarose gel. Of a total of 74 samples, divided into pork meat (n = 65) and pork liver (n = 9), 5.4% (n = 4) tested positive for HEV. In both cases, two samples were positive, representing a rate of 3.1% and 22.2% for meat and pork liver, respectively. All new viral sequences were obtained from a monophyletic group within HEV genotype 3. This study is the first to report the presence of HEV in pork sold in Senegal and the results reveal a potential circulation of HEV in the pig population. The high proportion of contamination in the pork liver samples highlights a major risk associated with their consumption.

RLP system: A single-tube two-step approach with dual amplification cascades for rapid identification of EGFR T790M

BackgroundThe detection of cancer gene mutations in biofluids plays a pivotal role in revolutionizing disease diagnosis. The presence of a large background of wild-type sequences poses a challenge to liquid biopsy of tumor mutation genes. Suppressing the detection of wild-type sequences can reduce their interference, however, due to the minimal difference between mutant and wild-type sequences (such as single nucleotide variants differing by only one nucleotide), how to suppress the detection of wild-type sequences to the greatest extent without compromising the sensitivity of mutant sequence detection remains to be explored. SignificanceThe RLP system addresses the incompatibility between RPA and RT-PCR reactions through a physical separation strategy. Besides, due to the remarkable flexibility of locked nucleic acid probes, the RLP system emerges as a potent tool for detecting mutations across diverse genes. It excels in sensitivity and speed, tolerates plasma matrix, and is cost-effective. This bodes well for advancing the field of precision medicine. ResultsThe recombinase-assisted locked nucleic acid (LNA) probe-mediated dual amplification biosensing platform (namely RLP), which combines recombinase polymerase amplification (RPA) and LNA clamp PCR method in one tube, enabling highly sensitive and selective detection of EGFR T790M mutation under the help of well-designed LNA probes. This technique can quantify DNA targets with a limit of detection (LoD) at the single copy level and identify point mutation with mutant allelic fractions as low as 0.007 % in 45 min. Moreover, RLP has the potential for the direct detection of plasma samples without the need for nucleic acid extraction and the cost of a single test is less than 1USD. Furthermore, the RLP system is a cascading dual amplification reaction conducted in a single tube, which eliminates the risk of cross-contamination associated with opening multiple tubes and ensures the reliability of the results.

Evaluation of Direct Detection Protocols for Poliovirus from Stool Samples of Acute Flaccid Paralysis Patients.

Polio surveillance in the Global Polio Eradication Initiative has been conducted with virus isolation from stool samples of acute flaccid paralysis (AFP) cases. Under the current biorisk management/regulations, challenges arise in the timelines of the report, sensitivity of the test and containment of poliovirus (PV) isolates. In the present study, we evaluated protocols of previously reported direct detection (DD) methods targeting the VP1 or VP4-VP2 regions of the PV genome in terms of sensitivity and sequencability. An optimized protocol targeting the entire-capsid region for the VP1 sequencing showed a high sensitivity (limit of detection = 82 copies of PV genome) with a simpler and faster reaction than reported ones (i.e., with the addition of all the primers at the start of the reaction, the RT-PCR reaction finishes within 2.5 h). The DD methods targeting the VP1 region detected PV in 60 to 80% of PV-positive stool samples from AFP cases; however, minor populations of PV strains in the samples with virus mixtures were missed by the methods. Sequencability of the DD methods was primarily determined by the efficiency of the PCRs for both Sanger and nanopore sequencing. The DD method targeting the VP4-VP2 region showed higher sensitivity than that targeting the VP1 region (limit of detection = 25 copies of PV genome) and successfully detected PV from all the stool samples examined. These results suggest that DD methods are effective for the detection of PV and that further improvement of the sensitivity is essential to serve as an alternative to the current polio surveillance algorithm.

Charge Engineering of the Nucleic Acid Binding Cleft of a Thermostable HIV-1 Reverse Transcriptase Reveals Key Interactions and a Novel Mechanism of RNase H Inactivation

Coupled with PCR, reverse transcriptases (RTs) have been widely used for RNA detection and gene expression analysis. Increased thermostability and nucleic acid binding affinity are desirable RT properties to improve yields and sensitivity of these applications. The effects of amino acid substitutions in the RT RNase H domain were tested in an engineered HIV-1 group O RT, containing mutations K358R/A359G/S360A and devoid of RNase H activity due to the presence of E478Q (O3MQ RT). Twenty mutant RTs with Lys or Arg at positions interacting with the template-primer (i.e., at positions 473–477, 499–502 and 505) were obtained and characterized. Most of them produced significant amounts of cDNA at 37, 50 and 65 °C, as determined in RT-PCR reactions. However, a big loss of activity was observed with mutants A477K/R, S499K/R, V502K/R and Y505K/R, particularly at 65 °C. Binding affinity experiments confirmed that residues 477, 502 and 505 were less tolerant to mutations. Amino acid substitutions Q500K and Q500R produced a slight increase of cDNA synthesis efficiency at 50 and 65 °C, without altering the KD for model DNA/DNA and RNA/DNA heteroduplexes. Interestingly, molecular dynamics simulations predicted that those mutations inactivate the RNase H activity by altering the geometry of the catalytic site. Proof of this unexpected effect was obtained after introducing Q500K or Q500R in the wild-type HIV-1BH10 RT and mutant K358R/A359G/S360A RT. Our results reveal a novel mechanism of RNase H inactivation that preserves RT DNA binding and polymerization efficiency without substituting RNase H active site residues.

Rapid and Sensitive Human-Specific DNA Quantitation Using a Microfluidic Amplification Module at the Point-of-Care.

A rapid microfluidic human-specific DNA quantitation assay module was developed for chip-based amplification of the human TH01 and Alu loci in the presence of PicoGreen. The method makes use of the thermal cycler and 488 nm Solid State laser-based optical train that are components of the fully-integrated, sample-in to results out, ANDE Rapid Nucleic Acid Analysis system. The assay was effective in quantitating human DNA from a variety of sample types, including blood, buccal, and forensic touch samples mixed with varying amounts of non-human DNA. The 28-cycle TH01 and 10-cycle Alu reactions were completed in 18 minutes and 7 minutes, respectively. The observed limit of detection (LOD) of the assay is approximately 0.3 ng, and the flexibility of assay design allows an LOD of as little as 0.005 femtograms.Clinical Relevance-We have developed a fully-integrated, sample-in to results-out, Rapid Nucleic Acid Analysis system that characterizes nucleic acid fragments (whether generated by PCR, rt-PCR, sequencing, or SNP reactions) by electrophoresis in plastic microfluidic channels. Here we describe the development, characterization, and validation of the microfluidic quantitation module. The quantitation module is the first that can be incorporated into integrated microfluidic workflows for the analysis of highly-multiplexed clinical diagnostic assays interrogating hundreds of genomic targets in a single sample. In particular, the use of a microfluidic quantitation module allows reaction volumes, thermal cycling conditions, and electrophoretic injection protocols to be determined based on nucleic acid content during and throughout fully-automated processing-dramatically enhancing the power of the fully-automated diagnostic system.

Development, Validation, and Application of Reverse Transcription Real-Time and Droplet Digital PCR Assays for the Detection of the Potyviruses Watermelon Mosaic Virus and Zucchini Yellow Mosaic Virus in Cucurbits.

Among the cucurbit-infecting viruses, watermelon mosaic virus (WMV) and zucchini yellow mosaic virus (ZYMV) (Potyvirus: Potyviridae) are responsible for severe symptoms on cucumber, melon, watermelon, and zucchini cultivations worldwide. In this study, reverse transcription real-time PCR (real-time RT-PCR) and droplet-digital PCR (RT-ddPCR) assays targeting the coat protein (CP) genes of WMV and ZYMV were developed and validated according to the international standards of plant pest diagnosis (EPPO PM 7/98 (5)). First, the diagnostic performance of WMV-CP and ZYMV-CP real-time RT-PCRs was evaluated, and the assays displayed an analytical sensitivity of 10-5 and 10-3, respectively. The tests also showed an optimal repeatability, reproducibility and analytical specificity, and were reliable for the virus detection in naturally infected samples and across a wide range of cucurbit hosts. Based on these results, the real-time RT-PCR reactions were adapted to set up RT-ddPCR assays. These were the first RT-ddPCR assays aiming at the detection and quantification of WMV and ZYMV and showed a high sensitivity, being able to detect until 9 and 8 copies/µL of WMV or ZYMV, respectively. The RT-ddPCRs allowed the direct estimation of the virus concentrations and opened to a broad range of applications in disease management, such as the evaluation of partial resistance in breeding processes, identification of antagonistic/synergistic events, and studies on the implementation of natural compounds in the integrated management strategies.

A simplified viral RNA extraction method based on magnetic nanoparticles for fast and high-throughput detection of SARS-CoV-2.

The ongoing outbreak of the novel coronavirus disease 2019 (COVID-19) draws worldwide concerns due to its long incubation period and strong infectivity. Although RT-PCR-based methods are being widely applied for clinical diagnosis, timely and accurate diagnosis towards COVID-19 causing virus, the SARS-CoV-2, is still limited due to labor-intensive and time-consuming operations. Herein, we report a new viral RNA extraction method based on poly-(amino ester) with carboxyl group (PC)-coated magnetic nanoparticles (pcMNPs) for the sensitive detection of SARS-CoV-2. This method combines the lysis and binding steps into one step, and refines multiple washing steps into one step, giving a turnaround time of less than 9min. Furthermore, the extracted pcMNP-RNA complexes can be directly introduced into subsequent RT-PCR reactions without elution. This simplified viral RNA method could be well adapted in fast manual and automated high-throughput nucleic acids extraction protocols suitable for different scenarios. A high sensitivity down to 100 copies/mL and a linear correlation between 100 and 106 copies/mL of SARS-CoV-2 pseudovirus particles are achieved in both protocols. Benefitting from the simplicity and excellent performances, this new method can dramatically improve the efficiency and reduce operational requirements for the early clinical diagnosis and large-scale SARS-CoV-2 nucleic acid screening.

One assay to test them all: Multiplex assays for expansion of respiratory virus surveillance.

Molecular multiplex assays (MPAs) for simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza and respiratory syncytial virus (RSV) in a single RT-PCR reaction reduce time and increase efficiency to identify multiple pathogens with overlapping clinical presentation but different treatments or public health implications. Clinical performance of XpertXpress® SARS-CoV-2/Flu/RSV (Cepheid, GX), TaqPath™ COVID-19, FluA/B, RSV Combo kit (Thermo Fisher Scientific, TP), and PowerChek™ SARS-CoV-2/Influenza A&B/RSV Multiplex RT-PCR kit II (KogeneBiotech, PC) was compared to individual Standards of Care (SoC). Thirteen isolates of SARS-CoV-2, human seasonal influenza, and avian influenza served to assess limit of detection (LoD). Then, positive and negative residual nasopharyngeal specimens, collected under public health surveillance and pandemic response served for evaluation. Subsequently, comparison of effectiveness was assessed. The three MPAs confidently detect all lineages of SARS-CoV-2 and influenza viruses. MPA-LoDs vary from 1 to 2 Log10 differences from SoC depending on assay and strain. Clinical evaluation resulted in overall agreement between 97 and 100%, demonstrating a high accuracy to detect all targets. Existing differences in costs, testing burden and implementation constraints influence the choice in primary or community settings. TP, PC and GX, reliably detect SARS-CoV-2, influenza and RSV simultaneously, with reduced time-to-results and simplified workflows. MPAs have the potential to enhance diagnostics, surveillance system, and epidemic response to drive policy on prevention and control of viral respiratory infections.

Alleviating Cell Lysate-Induced Inhibition to Enable RT-PCR from Single Cells in Picoliter-Volume Double Emulsion Droplets.

Microfluidic droplet assays enable single-cell polymerase chain reaction (PCR) and sequencing analyses at unprecedented scales, with most methods encapsulating cells within nanoliter-sized single emulsion droplets (water-in-oil). Encapsulating cells within picoliter double emulsion (DE) (water-in-oil-in-water) allows sorting droplets with commercially available fluorescence-activated cell sorter (FACS) machines, making it possible to isolate single cells based on phenotypes of interest for downstream analyses. However, sorting DE droplets with standard cytometers requires small droplets that can pass FACS nozzles. This poses challenges for molecular biology, as prior reports suggest that reverse transcription (RT) and PCR amplification cannot proceed efficiently at volumes below 1 nL due to cell lysate-induced inhibition. To overcome this limitation, we used a plate-based RT-PCR assay designed to mimic reactions in picoliter droplets to systematically quantify and ameliorate the inhibition. We find that RT-PCR is blocked by lysate-induced cleavage of nucleic acid probes and primers, which can be efficiently alleviated through heat lysis. We further show that the magnitude of inhibition depends on the cell type, but that RT-PCR can proceed in low-picoscale reaction volumes for most mouse and human cell lines tested. Finally, we demonstrate one-step RT-PCR from single cells in 20 pL DE droplets with fluorescence quantifiable via FACS. These results open up new avenues for improving picoscale droplet RT-PCR reactions and expanding microfluidic droplet-based single-cell analysis technologies.

Single-shot multi-channel plasmonic real-time polymerase chain reaction for multi-target point-of-care testing.

Plasmonic nucleic acid amplification tests demand high-throughput and multi-target detection of infectious diseases as well as short turnaround time and small size for point-of-care molecular diagnostics. Here, we report a multi-channel plasmonic real-time reverse-transcription polymerase chain reaction (mpRT-qPCR) assay for ultrafast and on-chip multi-target detection. The mpRT-qPCR system features two pairs of plasmonic thermocyclers for rapid nanostructure-driven amplification and microlens array fluorescence microscopes for in situ multi-color fluorescence quantification. Each channel shows a physical dimension of 32 mm, 75 mm, and 25 mm in width, length, and thickness. The ultrathin microscopes simultaneously capture four different fluorescence images from two PCR chambers of a single cartridge at a single shot exposure per PCR cycle of four different excitation light sources. The experimental results demonstrate a single assay result of high-throughput amplification and multi-target quantification for RNA-dependent RNA polymerase, nucleocapsid, and human ribonuclease P genes in SARS-CoV-2 RNA detection. The mpRT-PCR increases the number of tests four times over the single RT-PCR and exhibits a short detection time of 15 min for the four RT-PCR reactions. This point-of-care molecular diagnostic platform can reduce false negative results in clinical applications of virus detection and decentralize healthcare facilities with limited infrastructure.

Identification of viral RNA sequences in vampire bats (Desmodus rotundus) from central Mexico

Bats are important reservoirs of viral entities that cause diverse economical and health problems in several sectors. The purpose of this study was to determine the prevalence of the rabies virus in vampire bats. We also tested for 3 other zoonotic viruses in the collected tissue. We captured 45 vampire bats (Desmodus rotundus), which were measured and sexed under standard parameters. We followed an RT-PCR reaction to amplify the viral products of four virus types: chikungunya (CHIKV), dengue (DENV), rabies (RABV) and Zika virus (ZIKV) from cerebral tissue. We obtained an amplicon of 100 bp for ZIKV in 2 samples, representing a prevalence of 4.4%. An amplicon of 581 bp for RABV was observed in 9 individuals, which is equivalent to 20% of our sample. We obtained 4 different haplotypes for RABV and a single haplotype for ZIKV. We did not record the presence of DENV and CHIKV. We corroborated the presence of the family Rhabdoviridae in the vampire bat and recorded the presence of ZIKV for the first time in this bat species.

Multi-omics analysis reveals the pathogenesis of db/db mice diabetic kidney disease and the treatment mechanisms of multi-bioactive compounds combination from Salvia miltiorrhiza.

Diabetic kidney disease (DKD) is a common diabetic complication. Salvia miltiorrhiza has significant therapeutic effects on diabetes complications, although the mechanism remains unclear. Here, biochemical indicators and pathological changes were used to screen out the optimal Salvia miltiorrhiza multi-bioactive compounds combination. Metabolomics, transcriptomics and proteomics were used to explore the pathogenesis of DKD. RT-PCR and parallel reaction monitoring targeted quantitative proteome analysis were utilized to investigate treatment mechanisms of the optimal Salvia miltiorrhiza multi-bioactive compounds combination. The db/db mice showed biochemical abnormalities and renal lesions. The possible metabolic pathways were steroid hormone biosynthesis and sphingolipid metabolism. The 727 differential genes found in transcriptomics were associated with biochemical indicators via gene network to finally screen 11 differential genes, which were mainly key genes of TGF-β/Smad and PI3K/Akt/FoxO signaling pathways. Salvia miltiorrhiza multi-bioactive compounds combination could significantly regulate the Egr1, Pik3r3 and Col1a1 genes. 11 differentially expressed proteins involved in the two pathways were selected, of which 9 were significantly altered in db/db mice compared to db/m mice. Salvia miltiorrhiza multi-bioactive compounds combination could callback Q9DBM2, S4R1W1, Q91Y97, P47738, A8DUK4, and A2ARV4. In summary, Salvia miltiorrhiza multi-bioactive compounds combination may ameliorate kidney injury in diabetes through regulation of TGF-β/Smad and PI3K/Akt/FoxO signaling pathways.

Development and diagnostic validation of a one-step multiplex RT-PCR assay as a rapid method to detect and identify Nervous Necrosis Virus (NNV) and its variants circulating in the Mediterranean.

Nervous Necrosis Virus (NNV) represents one of the most threatening pathogens for Mediterranean aquaculture. Several NNV strains are currently co-circulating in the Mediterranean Basin with a high prevalence of the RGNNV genotype and the RGNNV/SJNNV reassortant strain and a more limited diffusion of the SJNNV genotype and the SJNNV/RGNNV reassortant. In the present study, a one-step multiplex RT-PCR (mRT-PCR) assay was developed as an easy, cost-effective and rapid diagnostic technique to detect RGNNV and the reassortant RGNNV/SJNNV strain and to distinguish them from SJNNV and the reassortant SJNNV/RGNNV strain in a single RT-PCR reaction. A unique amplification profile was obtained for each genotype/reassortant enabling their rapid identification from cell culture lysates or directly from brain tissues of suspected fish. The method’s detection limit varied between 102.3 and 103.4 TCID50 ml-1 depending on viral strains. No cross-reacitivty with viruses and bacteria frequently associated with gilthead seabream, European seabass and marine environment was observed. The mRT-PCR was shown to be an accurate, rapid and affordable method to support traditional diagnostic techniques in the diagnosis of VNN, being able to reduce considerably the time to identify the viral genotype or the involvement of reassortant strains.

The Matrix Effect in the RT-PCR Detection of SARS-CoV-2 Using Saliva without RNA Extraction.

The present work focuses on the detection of SARS-CoV-2 in saliva, contributing to understanding the inhibition effect of the matrix and its influence on the results. Detection of viral genes ORF1ab, N, and E was performed by RT-PCR using saliva directly in the reaction without RNA extraction. Different amounts of saliva were spiked with increasing amounts of viral RNA from COVID-19 patients and subjected to RT-PCR detection. In parallel, 64 saliva samples from confirmed COVID-19 patients were used in two different amounts directly in the RT-PCR reaction and their results compared. The presence of saliva in the RT-PCR always causes a positive shift of the Ct values, but a very high between-person variability of its magnitude was obtained, with increases ranging from 0.93 to 11.36. Viral targets are also affected differently depending on the initial number of viral particles. Due to inhibitors present in saliva, the duplication of sample volume causes only 48 to 61% of the expected Ct value decrease depending on the viral target gene. The use of saliva has advantages, but also limitations, due to potential inhibitors present in the matrix. However, the choice of the target and the right amount of sample may significantly influence the results.

High throughput SARS-CoV-2 variant analysis using molecular barcodes coupled with next generation sequencing.

The identification of SARS-CoV-2 variants across the globe and their implications on the outspread of the pandemic, infection potential and resistance to vaccination, requires modification of the current diagnostic methods to map out viral mutations rapidly and reliably. Here, we demonstrate that integrating DNA barcoding technology, sample pooling and Next Generation Sequencing (NGS) provide an applicable solution for large-population viral screening combined with specific variant analysis. Our solution allows high throughput testing by barcoding each sample, followed by pooling of test samples using a multi-step procedure. First, patient-specific barcodes are added to the primers used in a one-step RT-PCR reaction, amplifying three different viral genes and one human housekeeping gene (as internal control). Then, samples are pooled, purified and finally, the generated sequences are read using an Illumina NGS system to identify the positive samples with a sensitivity of 82.5% and a specificity of 97.3%. Using this solution, we were able to identify six known and one unknown SARS-CoV-2 variants in a screen of 960 samples out of which 258 (27%) were positive for the virus. Thus, our diagnostic solution integrates the benefits of large population and epidemiological screening together with sensitive and specific identification of positive samples including variant analysis at a single nucleotide resolution.

Development of a Singleplex Real-Time Reverse Transcriptase PCR Assay for Pan-Dengue Virus Detection and Quantification.

Dengue virus (DENV) infection is a significant global health problem. There are no specific therapeutics or widely available vaccines. Early diagnosis is critical for patient management. Viral RNA detection by multiplex RT-PCR using multiple pairs of primers/probes allowing the simultaneous detection of all four DENV serotypes is commonly used. However, increasing the number of primers in the RT-PCR reaction reduces the sensitivity of detection due to the increased possibility of primer dimer formation. Here, a one tube, singleplex real-time RT-PCR specific to DENV 3′-UTR was developed for the detection and quantification of pan-DENV with no cross reactivity to other flaviviruses. The sensitivity of DENV detection was as high as 96.9% in clinical specimens collected at the first day of hospitalization. Our assay provided equivalent PCR efficiency and RNA quantification among each DENV serotype. The assay’s performance was comparable with previously established real-time RT-PCR targeting coding sequences. Using both assays on the same specimens, our results indicate the presence of defective virus particles in the circulation of patients infected with all serotypes. Dual regions targeting RT-PCR enhanced the sensitivity of viral genome detection especially during the late acute phase when viremia rapidly decline and an incomplete viral genome was clinically evident.