RNA Amplification Research Articles

Contrasting patterns of Asaia association with Plasmodium falciparum between field-collected Anopheles gambiae and Anopheles coluzzii from Cameroon.

The widespread prevalence of Asaia in mosquitoes makes it a potential candidate for paratrangenic control in Anopheles. To better understand whether this bacterium could be used for malaria control, we quantified Asaia in An. gambiae s.l populations in malaria endemic regions examining co-infection with Plasmodium falciparum. Adult Anopheles mosquitoes were collected across two different eco-geographical localities in Cameroon, during both the dry and wet seasons. DNA was extracted from whole individual mosquitoes, and real time-qPCR amplification of the 16S ribosomal RNA was used to quantify Asaia in both An. gambiae and An. coluzzii samples. We also detected and quantified P. falciparum infection in the same mosquitoes. The density of Asaia was successfully quantified in a total of 864 field mosquitoes, comprising of 439 An. gambiae from Bankeng and 424 An. coluzii collected from Gounougou. Interestingly, a higher prevalence of Asaia in An. gambiae (88.3%) compared to An. coluzzii (80.9%) was observed. Moreover, the density of Asaia in both species was significantly affected by seasonal changes in the two localities. Furthermore, a significant difference between the infection densities of Asaia and the Plasmodium infection status in the two species was recorded. However, no correlation was observed between the number of Asaia and P. falciparum infections. This study provides evidence that naturally occurring Asaia infection is not correlated to P. falciparum development within An. gambiae and An. coluzzii. Nevertheless, further studies incorporating experimental infections are required to better investigate the correlation between Anopheles mosquitoes, Asaia, and Plasmodium.IMPORTANCEThe symbiont Asaia has emerged as a promising candidate for paratransgenic control of malaria, but further analysis of its biology and genetics across Africa is necessary. In this study, we investigated and quantified the influence of Asaia in naturally infected An. gambiae s.l. populations with the malaria parasite Plasmodium falciparum. Genomic DNA was extracted from whole individual mosquitoes collected from two localities, and Asaia was quantified using real-time qPCR by amplification of the 16S ribosomal RNA gene. We also detected and quantified Plasmodium falciparum infection in the same mosquitoes and established the correlation between Asaia and Plasmodium coinfection. This study provides evidence that naturally occurring Asaia infection is not correlated with P. falciparum development within An. gambiae and An. coluzzii mosquitoes.

Simple, direct amplification of RNA-containing paper discs for diagnosing the hepatitis C virus.

Nucleic acid extraction (NAE) is crucial for molecular diagnostics but presents challenges in point-of-care testing (POCT) and decentralized settings. We developed a streamlined, paper-based NAE method for hepatitis C virus (HCV) RNA amplification, suitable for integration into POCT and lab-on-a-chip systems. This method uses Fusion 5 paper discs, completing extraction in under 30 min without centrifugation. The nucleic acids on the disc can be directly used or eluted for amplification. We validated this method's compatibility with reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR (RQ-PCR), and loop-mediated isothermal amplification (LAMP), demonstrating versatility across amplification platforms. Clinical evaluation (n = 60) showed 100% sensitivity and specificity with a low detection limit of ~101 IU/mL. Results matched those from standard HCV RQ-PCR, confirming accuracy. Additionally, incorporating polyethylene glycol (PEG) improves extraction efficiency, eliminating the need for ethanol treatment and washing/drying steps. This modification enhances performance and suitability for field applications. Our paper-based HCV amplification is affordable and user-friendly, making it valuable for decentralized HCV detection and supporting global health initiatives.

Highly sensitive and specific electrochemical biosensor for direct detection of hepatitis C virus RNA in clinical samples using DNA strand displacement

Hepatitis C virus (HCV) is a common blood-borne infection that can lead to long-term illnesses such as hepatocellular cancer and liver cirrhosis. Early diagnosis is crucial for effective management, as no vaccine is available for preventing HCV infection. However, the high cost and complexity of current molecular diagnostic tools hinder efforts to achieve early diagnosis and prevent transmission, particularly in resource-limited settings. We developed a novel electrochemical biosensor for point-of-care testing (POCT) of HCV RNA. The sensor utilizes a strand displacement method, where the target RNA displaces a gold nanoparticle-labeled reporter probe (AuRP) from a pre-hybridized duplex with a magnetic nanoparticle (MNP)-labeled capture probe. The amount of displaced AuRP, detected using differential pulse anodic stripping voltammetry (DPASV), is directly proportional to the target RNA concentration. The biosensor exhibited excellent analytical performance, with a detection limit of 4 fM for synthetic targets and 43 ng/µL for RT-PCR products. Importantly, it successfully detected HCV RNA directly in clinical plasma samples without the need for RNA extraction or amplification. The sensor was used to analyze 30 RNA samples from HCV-positive patients, 20 cDNA samples from viral RNA, 30 HCV-positive plasma samples, and 22 HCV-negative plasma samples. The sensor results show good concordance with the RT-PCR results, demonstrating the sensor’s potential for detecting HCV in clinical samples.

LAMP-CRISPR/Cas12a-based impedimetric biosensor powered by Fe3O4@Au-(S-polyA-S)-Au for detection of SARS-CoV-2.

Alow-cost, lab-made polytetrafluoroethylene micro-cell, equipped with three electrodes, wasd evelopedfor the impedimetric detection of SARS-CoV-2. The gold working electrode was modified with a double-ended thiolated poly-adenine probe, which was conjugated with magnetic Fe₃O₄@Au nanoparticles (Fe3O4@Au-(S-polyA-S)-Au). After the loop-mediated isothermal amplification (LAMP) of viral RNA, the single-guide RNA (sgRNA), specifically bound to the SARS-CoV-2 target sequence, activates Cas12a. Cas12a then cleaved the immobilized probe. As a result, the magnetic Fe3O4@Au nanoparticles were released and adsorbed onto the gold electrode surface, using an external magnet. This process increased the physical surface area of the gold electrode, facilitating redox ion ([FeIII/II(CN)6]3-/4-) electron transfer. The decrease in the charge transfer resistance was utilized for SARS-CoV-2 detection. Our LAMP-CRISPR/Cas12a-based impedimetric biosensor, powered by Fe3O4@Au-(S-polyA-S)-Au, demonstrated impressive capabilities, including a remarkable detection limit of 0.8 aM (0.48 copies/µL) and a linear range of 0.01 to 36.06 fM.

Enhancing the Accuracy of Peripheral Pulmonary Tuberculosis Diagnosis: A Comparative Evaluation of CapitalBioTM Mycobacterium Nucleic Acid Detection Test and Mycobacterium tuberculosis Isothermal RNA Amplification Assay Using Endobronchial Ultrasonography with Guide Sheath.

The diagnosis of tuberculosis located in the peripheral zone remains challenging and requires ultrasound bronchoscopy-guided maneuvers. We assessed the precision of CapitalBioTM Mycobacterium nucleic acid detection test (CapitalBio MTB test) and Mycobacterium tuberculosis isothermal RNA amplification test (MTB-RNA) using endobronchial ultrasonography with a guide sheath (EBUS-GS) for peripheral pulmonary tuberculosis (PTB) and compared it with those of acid-fast bacilli (AFB) smear and MTB culture tests. This retrospective analysis included 287 patients suspected of peripheral pulmonary tuberculosis who underwent EBUS-GS examinations, medical examination results of AFB smears, MTB culture, CapitalBio MTB test, and MTB-RNA were analyzed. We evaluated the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the receiver operating characteristic curve (AUC), and its diagnostic accuracy for peripheral PTB was evaluated in comparison with the final clinical diagnosis. The overall sensitivity, specificity, PPV, NPV, and AUC of CapitalBio MTB test were 44.83%, 100.00%, 100.00%, 54.07%, and 0.72, respectively; those of MTB-RNA were 22.99%, 100.00%, 100.00%, 45.75%, and 0.61, respectively, and those for parallel test (CapitalBio MTB test or MTB-RNA) were 46.55%, 100.00%, 100.00%, 54.85%, and 0.73, respectively. These values for AFB smear were 9.2%, 97.35%, 84.21%, 41.04%, and 0.53, respectively, and those of MTB culture were 31.03%, 100.00%, 100.00%, 48.50%, and 0.69, respectively. The CapitalBio MTB test showed the best diagnostic performance compared with AFB smear, MTB culture, and MTB-RNA assays and was similar to the parallel test (CapitalBio MTB test or MTB-RNA). The CapitalBio MTB test combined with EBUS-GS had satisfactory diagnostic accuracy for diagnosing peripheral PTB.

Occurrence rate and species and subtypes of Cryptosporidium spp. in pet dogs in Yunnan Province, China

BackgroundCryptosporidium spp. is a ubiquitous, globally distributed intestinal protozoan infecting humans and at least 260 animal hosts. Due to close human contact with pet dogs and identification of zoonotic Cryptosporidium species and subtypes in these animals, dog health is not only a veterinarian issue but also a public health issue. This study aimed to understand occurrence and genetic characterization at both genotype and subtype levels in pet dogs in Yunnan Province, China.ResultsA total of 589 fresh fecal specimens were collected from adult pet dogs in the rural areas of eight cities/autonomous prefectures of Yunnan Province, China. 16 fecal specimens were positive for Cryptosporidium spp. by polymerase chain reaction (PCR) amplification and sequence analysis of the small subunit ribosomal RNA (SSU rRNA) gene, with an average occurrence rate of 2.7% (16/589) being observed. Three zoonotic Cryptosporidium species were identified: C. parvum (n = 7), C. suis (n = 5) and C. canis (n = 4). At the 60-kDa glycoprotein (gp60) locus, only three C. parvum and two C. canis specimens were successfully amplified and sequenced, with subtype IIaA17G2R1 (n = 3) and subtypes XXa4 (n = 1) and XXa5 (n = 1) being identified, respectively.ConclusionsThe present finding of three zoonotic Cryptosporidium species in dogs implied that dogs infected with Cryptosporidium spp. may pose a threat to human health. C. suis was identified in dogs in this study for the first time, expanding the host range of this species. Identification of C. parvum subtype IIaA17G2R1 and C. canis subtypes XXa4 and XXa5 will be helpful to explore the source attribution of infection/contamination and assess the transmission dynamics of C. parvum and C. canis in the investigated areas in the future.

A multiplexed real‐time PCR assay for simultaneous quantification of human immunodeficiency virus and Hepatitis B virus for low‐and‐middle‐ income countries

Due to shared routes of transmission, including sexual contact and vertical transmission, HIV-HBV co-infection is common, particularly in sub-Saharan Africa. Measurement of viral load (VL), for both HIV and HBV, plays a critical role for determining their infectious phase and monitoring response to antiviral therapy. Implementation of viral load testing in clinical settings is a significant challenge in resource-limited countries, notably because of cost and availability issues. We designed HIV and HBV primers for conserved regions of the HIV and HBV genomes that were specifically adapted to viral strains circulating in West Africa that are HIV-1 subtype CRF02AG and HBV genotype E. We first validated two monoplex qPCR assays for individual quantification and, then developed a multiplex qPCR for simultaneous quantification of both viruses. HIV RNA and HBV DNA amplification was performed in a single tube using a one-step reverse transcription-PCR reaction with primers and probes targeting both viruses. Performance characteristics such as the quantification range, sensitivity, and specificity of this multiplex qPCR assay were compared to reference qPCR tests for both HIV and HBV viral load quantification. The multiplex assay was validated using clinical samples from co- or mono-infected patients and gave comparable viral load quantification to the HIV and HBV reference test respectively. The multiplex qPCR demonstrated an overall sensitivity of 71.25 % [68.16–74.3] for HBV and 82 % [78.09–85.90] for HIV and an overall specificity of 100 % [94.95–100] for both viruses. Although the overall sensitivities of the HIV and HBV assays were lower than the commercial comparator assays, the sensitivity in the clinical decision range of >1000 copies/mL for HIV was 80 % [71.26–88.73] and >1000 IU/mL for HBV was 100 % [95.51–100] which indicates the test results can be used to guide treatment decisions. This in-house developed multiplex qPCR assay represents a useful diagnostic tool as it can be performed on affordable "open" real-time PCR platforms currently used for HIV or SARS-Cov-2 infection surveillance in Mali.

A multi-epitope self-amplifying mRNA SARS-CoV-2 vaccine design using a reverse vaccinology approach

Background and purpose: Massive vaccine distribution is a crucial step to prevent the spread of SARS-CoV2 as the causative agent of COVID-19. This research aimed to design the multi-epitope self-amplifying mRNA (saRNA) vaccine from the spike and nucleocapsid proteins of SARS-CoV2. Experimental approach: Commonly distributed constructions class I and II alleles of the Indonesian population were used to determine peptide sequences that trigger this population’s high specificity T-cell response. The best vaccine candidate was selected through the analysis of tertiary structure validation and molecular docking of each candidate with TLR-4, TLR-8, HLA-A*24:02, and HLA-DRB1*04:05. The selected multi-epitope vaccine combined with the gene encoding the replication machinery that allows the RNA amplification in the host cell. Findings/Results: Seven B-cell and four T-cell epitopes from the protein target were highly antigenic and conserved, non-allergen, non-toxic, and hydrophilic. Tertiary structure validation then determined the best multi-epitope construction with 269 AA in length containing hBD-2 adjuvant and PADRE. Most residues are predicted to be accessible by solvent and show high population coverage (99,26%). Molecular docking analysis demonstrated a stable and strong binding affinity with immune receptors. A recombinant plasmid as the template for mRNA production was constructed by inserting the multi-epitope DNA and non-structural polyprotein 1-4 gene of VEEV, which encodes the RNA replication complex to the cloning site of pcDNA3.1(+). Conclusion and implication: In silico, design of self-amplifying mRNA could be a potential COVID-19 vaccine candidate since its ability to be amplified in the host cell can efficiently reduce the intake doses.

New Approaches to Plant Pathogen Detection and Disease Diagnosis.

Detecting plant pathogens and diagnosing diseases are critical components of successful pest management. These key areas have undergone significant advancements driven by breakthroughs in molecular biology and remote sensing technologies within the realm of precision agriculture. Notably, nucleic acid amplification techniques, with recent emphasis on sequencing procedures, particularly next-generation sequencing, have enabled improved DNA or RNA amplification detection protocols that now enable previously unthinkable strategies aimed at dissecting plant microbiota, including the disease-causing components. Simultaneously, the domain of remote sensing has seen the emergence of cutting-edge imaging sensor technologies and the integration of powerful computational tools, such as machine learning. These innovations enable spectral analysis of foliar symptoms and specific pathogen-induced alterations, making imaging spectroscopy and thermal imaging fundamental tools for large-scale disease surveillance and monitoring. These technologies contribute significantly to understanding the temporal and spatial dynamics of plant diseases.

An Integrated Micro-Heating System for On-Chip Isothermal Amplification of African Swine Fever Virus Genes.

The loop-mediated isothermal amplification (LAMP) is widely used in the laboratory to facilitate rapid DNA or RNA detection with a streamlined operational process, whose properties are greatly dependent on the uniformity and rise rate of temperature in the reaction chambers and the design of the primers. This paper introduces a planar micro-heater equipped with an embedded micro-temperature sensor to realize temperature tunability at a low energy cost. Moreover, a control system, based on the Wheatstone bridge and proportional, integral, and derivative (PID) control, is designed to measure and adjust the temperature of the micro-heater. The maximum temperature rise rate of the designed micro-heater is ≈8°Cs-1, and it only takes ≈60s to reach the target temperature. Furthermore, a designed plasmid, containing the B646L gene of African Swine Fever Virus (ASFV), and a set of specific primers, are used to combine with the designed micro-heating system to implement the LAMP reaction. Finally, the lateral flow assay is used to interpret the amplification results visually. This method can achieve highly sensitive and efficient detection of ASFV within 40min. The sensitivity of this on-chip gene detection method is 8.4 copies per reaction, holding great potential for applications in DNA and RNA amplification.

CRISPR/Cas13a analysis based on NASBA amplification for norovirus detection

Human norovirus (HuNoV) is a leading cause of foodborne diseases worldwide, making rapid and accurate detection crucial for prevention and control. In recent years, the CRISPR/Cas13a system, known for its single-base resolution in RNA recognition and unique collateral cleavage activity, is particularly suitable for sensitive and rapid RNA detection. However, isothermal amplification-based CRISPR/Cas13 assays often require an external transcription step, complicating the detection process. In our study, an efficient diagnostic technique based on the NASBA/Cas13a system was established to identify conserved regions at the ORF1-ORF2 junction of norovirus. The RNA amplification techniques [Nucleic Acid Sequence-Based Amplification (NASBA)] integrates reverse transcription and transcription steps, enabling sensitive, accurate, and rapid enrichment of low-abundance RNA. Furthermore, the CRISPR/Cas13a system provides secondary precise recognition of the amplified products, generating a fluorescence signal through its activated accessory collateral cleavage activity. We optimized the reaction kinetics parameters of Cas13a and achieved a detection limit as low as 51pM. The conditions for the cascade reaction involving CRISPR analysis and RNA amplification were optimized. Finally, we validated the reliability and accuracy of the NASBA/Cas13a method by detecting norovirus in shellfish, achieving results comparable to qRT-PCR in a shorter time and detecting viral loads as low as 10 copies/μL.

Target-specific requirements for RNA interference can arise through restricted RNA amplification despite the lack of specialized pathways

Since double-stranded RNA (dsRNA) is effective for silencing a wide variety of genes, all genes are typically considered equivalent targets for such RNA interference (RNAi). Yet, loss of some regulators of RNAi in the nematode Caenorhabditis elegans can selectively impair the silencing of some genes. Here, we show that such selective requirements can be explained by an intersecting network of regulators acting on genes with differences in their RNA metabolism. In this network, the Maelstrom domain-containing protein RDE-10, the intrinsically disordered protein MUT-16, and the Argonaute protein NRDE-3 work together so that any two are required for silencing one somatic gene, but each is singly required for silencing another somatic gene, where only the requirement for NRDE-3 can be overcome by enhanced dsRNA processing. Quantitative models and their exploratory simulations led us to find that (1) changing cis-regulatory elements of the target gene can reduce the dependence on NRDE-3, (2) animals can recover from silencing in non-dividing cells, and (3) cleavage and tailing of mRNAs with UG dinucleotides, which makes them templates for amplifying small RNAs, are enriched within ‘pUG zones’ matching the dsRNA. Similar crosstalk between pathways and restricted amplification could result in apparently selective silencing by endogenous RNAs.

Molecular Detection and Phylogenetic Relationships of Honey Bee-Associated Viruses in Bee Products.

In the last few years, the isolation and amplification of DNA or RNA from the environment (eDNA/eRNA) has proven to be an alternative and non-invasive approach for molecular identification of pathogens and pests in beekeeping. We have recently demonstrated that bee pollen and bee bread represent suitable biological material for the molecular identification of viral RNA. In the present study, we extracted total RNA from different bee products (pollen, n = 25; bee bread, n = 17; and royal jelly, n = 15). All the samples were tested for the presence of six of the most common honey bee-associated viruses-Deformed wing virus (DWV), Acute bee paralysis virus (ABPV), Chronic bee paralysis virus (CBPV), Sacbrood virus (SBV), Kashmir bee virus (KBV), and Black queen cell virus (BQCV)-using a reverse transcription polymerase chain reaction (RT-PCR). We successfully detected six records of DWV (10.5%, 6/57), four of ABPV (7.0%, 4/57), three of Israeli acute paralysis virus (IAPV) (5.3%, 3/57), and two of BQCV (3.5%, 2/57). Using ABPV primers, we also successfully detected the presence of IAPV. The obtained viral sequences were analyzed for phylogenetic relationships with the highly similar sequences (megablast) available in the GenBank database. The Bulgarian DWV isolates revealed a high homology level with strains from Syria and Turkey. Moreover, we successfully detected a DWV strain B for the first time in Bulgaria. In contrast to DWV, the ABPV isolates formed a separate clade in the phylogenetic tree. BQCV was closely grouped with Russian isolates, while Bulgarian IAPV formed its own clade and included a strain from China. In conclusion, the present study demonstrated that eRNA can be successfully used for molecular detection of honey bee-associated viruses in bee products. The method can assist the monitoring of the health status of honey bee colonies at the local, regional, and even national levels.

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.

Circulating B Cell-Derived Small RNA Delivered by Extracellular Vesicles: A Dialogue Mechanism for Long-Range Targeted Renal Mitochondrial Injury in Obesity.

The intricate processes that govern the interactions between peripatetic immune cells and distal renal injury in obesity are not fully understood. Employing transcriptomic analysis of circulating extracellular vesicles (EVs), a marked amplification of small RNA (miR-3960) is discerned within CD3-CD19+ B cells. This RNA is found to be preferentially augmented in kidney tissues, contrasting with its subdued expression in other organs. By synthesizing dual-luciferase reporter assay with co-immunoprecipitation analysis, it is pinpointed that miR-3960 specifically targets the nuclear gene TRMT5, a pivotal actor in the methylation of mitochondrial tRNA. This liaison instigates aberrations in the post-transcriptional modifications of mitochondrial tRNA, engendering deficiencies within the electron respiratory chain, primarily attributable to the diminution of the mitochondrial bioenergetic compound (NDUFA7) complex I. Such perturbations lead to a compromised mitochondrial respiratory capacity in renal tubular cells, thereby exacerbating tubular injury. In contrast, EV blockade or miR-3960 depletion markedly alleviates renal tubular injury in obesity. This investigation unveils a hitherto unexplored pathway by which obesity-induced circulating immune cells remotely manipulate mitochondrial metabolism in target organs. The strategic targeting of obese EVs or infiltrative immune cells and their specifically secreted RNAs emerges as a promising therapeutic avenue to forestall obesity-related renal afflictions.

An alternative method for SARS-CoV-2 detection with use modified fluorescent in situ hybridization

The real-time reverse-transcriptase polymerase-chain-reaction (rRT-PCR) tests are the gold standard in detecting SARS-CoV-2 virus infection. However, despite high sensitivity and specificity, they have limitations that in some cases may result in false negative results. Therefore, it is reasonable to search for additional tools that could support microbiological diagnosis of SARS-CoV-2. The aim of the study was to develop a highly specific molecular test capable of detecting and visualizing SARS-CoV-2 infection. A universal probe and a set of 18 specific oligonucleotides with a FLAP sequence attached to them on both sides were designed to visualize SARS-CoV-2 virus infection based on the fluorescence in situ hybridization method (FISH). FISH conditions using the developed kit were standardized on the Vero CCL-81 cell line infected by SARS-CoV-2 virus. The method was tested on 290 nasopharyngeal swabs (collected in a doublet) from patients with clinical symptoms of SARS-CoV-2. Each one swab from the doublet was subjected to RNA isolation and amplification by rRT-PCR. From the second swab, a microscopic preparation was performed for FISH. The use of the rRT-PCR allowed obtaining 200 positive and 90 negative results, while our FISH method allowed for 220 positive results and 70 negative results. The differences obtained using both methods were statistically significant (p = 0.008). The obtained results support the use of FISH as an additional method in microbiological diagnostics of SARS-CoV-2.

New Haplotypes of Blastocystis sp. Identified in Faeces from Various Animal Groups in Algeria.

Blastocystis sp. is a single-celled, anaerobic, parasitic protozoan commonly found in the intestinal tract of animals and humans globally. Genetic analysis has revealed significant diversity within its species, leading to the identification of at least 40 subtypes (ST1-ST40). This study aimed to identify and differentiate Blastocystis in faeces samples from various animal hosts in Algeria. A total of 403 fecal samples, collected from both domestic and zoo animals, were subjected to PCR amplification and sequencing of Blastocystis-specific small subunit ribosomal RNA (SSU-RNA) gene. The overall prevalence of Blastocystis in animals was found to be 38.9%. Through comprehensive phylogenetic and phylogeographic analyses, we identified four distinct subtypes (ST1 in both domestic and zoo animals, and ST3, ST4, and ST5 exclusively in zoo animals), encompassing nine different haplotypes, including five that appear original to Algeria. This study represents the first epidemiological molecular investigation of Blastocystis sp. in animals in Algeria.

Target-specific requirements for RNA interference can arise through restricted RNA amplification despite the lack of specialized pathways.

Since double-stranded RNA (dsRNA) is effective for silencing a wide variety of genes, all genes are typically considered equivalent targets for such RNA interference (RNAi). Yet, loss of some regulators of RNAi in the nematode C. elegans can selectively impair the silencing of some genes. Here we show that such selective requirements can be explained by an intersecting network of regulators acting on genes with differences in their RNA metabolism. In this network, the Maelstrom domain-containing protein RDE-10, the intrinsically disordered protein MUT-16, and the Argonaute protein NRDE-3 work together so that any two are required for silencing one somatic gene, but each is singly required for silencing another somatic gene, where only the requirement for NRDE-3 can be overcome by enhanced dsRNA processing. Quantitative models and their exploratory simulations led us to find that (1) changing cis-regulatory elements of the target gene can reduce the dependence on NRDE-3, (2) animals can recover from silencing in non-dividing cells and (3) cleavage and tailing of mRNAs with UG dinucleotides, which makes them templates for amplifying small RNAs, is enriched within 'pUG zones' matching the dsRNA. Similar crosstalk between pathways and restricted amplification could result in apparently selective silencing by endogenous RNAs.

LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue

Whole transcriptome analysis (WTA) using RNA extracted from Formalin Fixed Paraffin Embedded (FFPE) tissue is an invaluable tool to understand the molecular pathology of disease. RNA extracted from FFPE tissue is either degraded and/or in very low quantities hampering gene expression analysis. Earlier studies described protocols applied for cellular RNA using poly-A primer-based linear amplification. The current study describes a method, LINCATRA (LINear amplifiCAtion of RNA for whole TRAnscriptome analysis). It employs random nonamer primer based method which can amplify short, fragmented RNA with high fidelity from as low as 5 ng to obtain enough material for WTA. The two-cycle method significantly amplified RNA at ∼1000 folds (p < 0.0001) improving the mean read lengths (p < 0.05) in WTA. Overall, increased mean read length positively correlated with on-target reads (Pearson's r = 0.71, p < 0.0001) in both amplified and unamplified RNA-seq analysis. Gene expression analysis compared between unamplified and amplified group displayed substantial overlap of the differentially expressed genes (DEGs) (log2 fold change cut-off < −2 and >2, p < 0.05) identified between lung cancer and asthma cohorts validating the method developed. This method is applicable in clinical molecular pathology field for both diagnostics and elucidation of disease mechanisms.

Contact lenses as novel tear fluid sampling vehicles for total RNA isolation, precipitation, and amplification.

The tear fluid is a readily accessible, potential source for biomarkers of disease and could be used to monitor the ocular response to contact lens (CL) wear or ophthalmic pathologies treated by therapeutic CLs. However, the tear fluid remains largely unexplored as a biomarker source for RNA-based molecular analyses. Using a rabbit model, this study sought to determine whether RNA could be collected from commercial CLs and whether the duration of CL wear would impact RNA recovery. The results were referenced to standardized strips of filtered paper (e.g., Shirmer Strips) placed in the inferior fornix. By performing total RNA isolation, precipitation, and amplification with commercial kits and RT-PCR methods, CLs were found to have no significant differences in RNA concentration and purity compared to Schirmer Strips. The study also identified genes that could be used to normalize RNA levels between tear samples. Of the potential control genes or housekeeping genes, GAPDH was the most stable. This study, which to our knowledge has never been done before, provides a methodology for the detection of RNA and gene expression changes from tear fluid that could be used to monitor or study eye diseases.