RNA Amplification Research Articles

Benthic foraminiferal community structure and its response to environmental factors revealed using high-throughput sequencing in the Zhoushan Fishing Ground, East China Sea

Benthic foraminifera are excellent tools for monitoring marine environments and reconstructing paleoenvironments. This study investigated the structure and diversity of benthic foraminiferal communities in 20 superficial sediment samples obtained from the Zhoushan Fishing Ground (ZFG) using high-throughput sequencing based on small subunit ribosomal DNA and RNA amplification. The results revealed Rotaliida as the most dominant group, with spatial heterogeneity in foraminiferal distribution. Total benthic foraminiferal communities exhibited higher species richness and diversity compared to active communities. While heavy metal pollution in the ZFG was moderate, areas with elevated concentrations of heavy metals exhibited low diversity and richness in foraminiferal communities. Total foraminiferal community structure was primarily influenced by factors such as water depth and Hg, Pb, Cd, and Zn levels. Notably, Hg levels emerged as a critical factor impacting the structure and diversity of the active foraminiferal community. The dominant species, Operculina, exhibited tolerance toward heavy metal pollution.

HIV protease resistance mutations in patients receiving second-line antiretroviral therapy in Libreville, Gabon

IntroductionIn 2022, the WHO reported that 29.8 million people around the world were living with HIV (PLHIV) and receiving antiretroviral treatment (ART), including 25‌ 375 people in Gabon (54% of all those living with HIV in the country). The literature reports a frequency of therapeutic failure with first-line antiretrovirals (ARVs) of between 20% and 82%. Unfortunately, data relating to the failure of second-line ARVs are scarce in Gabon. This study aims to determine the profiles of HIV drug resistance mutations related to protease inhibitors in Gabon.MethodologyPlasma from 84 PLHIV receiving ARVs was collected from 2019 to 2021, followed by RNA extraction, amplification, and sequencing of the protease gene. ARV resistance profiles were generated using the Stanford interpretation algorithm version 8.9-1 (https://hivdb.stanford.edu) and statistical analyses were performed using EpiInfo software version 7.2.1.0 (CDC, USA).ResultsOf 84 HIV plasma samples collected from 45 men and 39 women, 342 mutations were detected. Of these, 43.3% (148/342) were associated with nucleoside reverse transcriptase inhibitors (NRTIs), 30.4% (104/342) with non-nucleoside reverse transcriptase inhibitors (NNRTIs), and 26.3% (90/342) with protease inhibitors (PIs). Most NRTI mutations were associated with thymidine analogues (TAMs) (50.7%; 75/148), including T215F/V (14.9%; 22/148), D67DN/E/G/N/T (10.1%; 15/148), M41L (9.5%; 14/148), and K70E/KN/S/R (9.5%; 14/148). Resistance mutations related to non-TAM NRTIs (33.1%; 49/148) were M184V (29.1%; 43/148), and L74I/V (8.1%; 12/148). NNRTI mutations were predominantly K103N/S (32.7%; 34/104), V108I (10.6%; 11/104), A98G (10.6%; 11/104), and P225H (9.6%; 10/104). Minor mutations associated with PIs (60.0%; 54/90) were predominantly K20I (15.6%; 14/90) and L10F/I/V (14.5%; 13/90). The major mutations associated with PIs (40.0%; 36/90) were M41L (12.2%; 11/90), I84V (6.7%; 06/90), and V82A (6.7%; 06/90). The four most prescribed therapeutic regimens were TDF + 3TC + LPV/r (20.3%; 17/84), ABC + DDI + LPV/r (17.9%; 15/84), TDF + FTC + LPV/r (11.9%; 10/84), and ABC + 3TC + LPV/r (11.9%; 10/84).ConclusionThis study revealed that HIV drug resistance mutations are common in Gabon. The major mutations associated with PIs were M41L, I84V, and V82A. There is a need for access to new NRTIs, NNRTIs, and PIs for a better therapeutic management of PLHIV in Gabon.

Quantitative detection of chikungunya, Zika, and dengue viruses by one-step real-time PCR in different cell substrates.

Chikungunya (CHIKV), Zika (ZIKV), and dengue viruses (DENV) are vector-borne pathogens that cause emerging and re-emerging epidemics throughout tropical and subtropical countries. The symptomatology is similar among these viruses and frequently co-circulates in the same areas, making the diagnosis arduous. Although there are different methods for detecting and quantifying pathogens, real-time reverse transcription-polymerase chain reaction (real-time RT-qPCR) has become a leading technique for detecting viruses. However, the currently developed assays frequently involve probes and high-cost reagents, limiting access in low-income countries. Therefore, this study aims to design and evaluate a quantitative one-step RT-qPCR assay to detect CHIKV, ZIKV, and DENV with high specificity, reproducibility, and low cost in multiple cell substrates. We established a DNA intercalating green dye-based RT-qPCR test that targets nsP1 of CHIKV, and NS5 gene of ZIKV, and DENV for the amplification reaction. The assay exhibited a high specificity confirmed by the melting curve analysis. No cross-reactivity was observed between the three viruses or unspecific amplification of host RNA. The sensitivity of the reaction was evaluated for each virus assay, getting a limit of detection of one RNA copy per virus. Standard curves were constructed, obtaining a reaction efficiency of ~ 100%, a correlation coefficient (R2) of ~ 0.97, and a slope of -3.3. The coefficient of variation (CV) ranged from 0.02 to 1.43. In addition, the method was optimized for viral quantification and tested in Vero, BHK-21, C6/36, LULO, and the Aedes cell lines. Thus, the DNA intercalating green dye-based RT-qPCR assay was a highly specific, sensitive, reproducible, and effective method for detecting and quantifying CHIKV, ZIKV, and DENV in different cell substrates that could also be applied in clinical samples.

Benchmarking a targeted 16S ribosomal RNA gene enrichment approach to reconstruct ancient microbial communities.

The taxonomic characterization of ancient microbiomes is a key step in the rapidly growing field of paleomicrobiology. While PCR amplification of the 16S ribosomal RNA (rRNA) gene is a widely used technique in modern microbiota studies, this method has systematic biases when applied to ancient microbial DNA. Shotgun metagenomic sequencing has proven to be the most effective method in reconstructing taxonomic profiles of ancient dental calculus samples. Nevertheless, shotgun sequencing approaches come with inherent limitations that could be addressed through hybridization enrichment capture. When employed together, shotgun sequencing and hybridization capture have the potential to enhance the characterization of ancient microbial communities. Here, we develop, test, and apply a hybridization enrichment capture technique to selectively target 16S rRNA gene fragments from the libraries of ancient dental calculus samples generated with shotgun techniques. We simulated data sets generated from hybridization enrichment capture, indicating that taxonomic identification of fragmented and damaged 16S rRNA gene sequences was feasible. Applying this enrichment approach to 15 previously published ancient calculus samples, we observed a 334-fold increase of ancient 16S rRNA gene fragments in the enriched samples when compared to unenriched libraries. Our results suggest that 16S hybridization capture is less prone to the effects of background contamination than 16S rRNA amplification, yielding a higher percentage of on-target recovery. While our enrichment technique detected low abundant and rare taxa within a given sample, these assignments may not achieve the same level of specificity as those achieved by unenriched methods.

Mycoplasmoides genitalium Macrolide Resistance Detection is Needed in University Settings.

Background: Mycoplasmoides genitalium remains a difficult sexually-transmitted infection (STI) to manage due to its potential for antimicrobial resistance and post-infection sequelae. University students are especially vulnerable, as this demographic has the highest rate of STI in the United States. As a result, investigating prevalence rates and therapeutic outcomes in this population is essential to minimize future impact of M. genitalium The purpose of this study was to investigate a university student population for M. genitalium distribution and treatment outcome.Design: Retrospective chart-review of university health clinic attendees, augmented by laboratory detection of M. genitalium following therapeutic intervention.Methods: A total of 1617 student encounters at a midwestern United States university health clinic over a 28-month interval from November 2017 through February 2020 were analyzed for M. genitalium and Chlamydia trachomatis positivity rates and prevalence. Detection of these sexually-transmitted pathogens occurred by commercial RNA amplification testing. Chart review was focused on participant outcomes following initial M. genitalium detection and therapeutic intervention.Results: C. trachomatis positivity and prevalence rates were 7.05% and 9.00%, respectively, while analogous rates for M. genitalium were 7.05% and 6.51%, respectively. An average of 1.83 positive results was generated from participants infected with M. genitalium at any time, with an average of 1.17 positive results for C. trachomatis (P < 0.0002). For students treated with azithromycin, 30.3% generated a negative M. genitalium result upon follow-up, with 1g daily and 2-day 500mg dosing regimens demonstrating less efficacy than a 4-day 250mg regimen or moxifloxacin.Conclusion: Data indicate a need for molecular M. genitalium macrolide resistance determination from primary specimens in the university setting.

Low-Cost Arduino Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) for Sensitive Nucleic Acid Detection.

This work presents a low-cost transcription loop-mediated isothermal amplification (RT-LAMP) instrument for nucleic acid detection, employing an Arduino Nano microcontroller. The cooling system includes customized printed circuit boards (PCBs) that serve as electrical resistors and incorporate fans. An aluminum block is designed to accommodate eight vials. The system also includes two PCB heaters-one for sample heating and the other for vial lid heating to prevent condensation. The color detection system comprises a TCS3200 color 8-sensor array coupled to one side of the aluminum heater body and a white 8-LED array coupled to the other side, controlled by two Multiplexer/Demultiplexer devices. LED light passes through the sample, reaching the color sensor and conveying color information crucial for detection. The top board is maintained at 110 ± 2 °C, while the bottom board is held at 65 ± 0.5 °C throughout the RT-LAMP assay. Validation tests successfully demonstrated the efficacy of the colorimetric RT-LAMP reactions using SARS-CoV-2 RNA amplification as a sample viability test, achieving 100% sensitivity and 97.3% specificity with 66 clinical samples. Our instrument offers a cost-effective (USD 100) solution with automated result interpretation and superior sensitivity compared to visual inspection. While the prototype was tested with SARS-CoV-2 RNA samples, its versatility extends to detecting other pathogens using alternative primers, showcasing its potential for broader applications in biosensing.

Real-Time On-Site Monitoring of Viruses in Wastewater Using Nanotrap® Particles and RICCA Technologies.

Wastewater-based epidemiology (WBE) is an effective and efficient tool for the early detection of infectious disease outbreaks in a community. However, currently available methods are laborious, costly, and time-consuming due to the low concentration of viruses and the presence of matrix chemicals in wastewater that may interfere with molecular analyses. In the present study, we designed a highly sensitive "Quick Poop (wastewater with fecal waste) Sensor" (termed, QPsor) using a joint approach of Nanotrap microbiome particles and RICCA (RNA Isothermal Co-Assisted and Coupled Amplification). Using QPsor, the WBE study showed a strong correlation with standard PEG concentrations and the qPCR technique. Using a closed format for a paper-based lateral flow assay, we were able to demonstrate the potential of our assay as a real-time, point-of-care test by detecting the heat-inactivated SARS-CoV-2 virus in wastewater at concentrations of 100 copies/mL and within one hour. As a proof-of-concept demonstration, we analyzed the presence of viral RNA of the SARS-CoV-2 virus and PMMoV in raw wastewater samples from wastewater treatment plants on-site and within 60 min. The results show that the QPsor method can be an effective tool for disease outbreak detection by combining an AI-enabled case detection model with real-time on-site viral RNA extraction and amplification, especially in the absence of intensive clinical laboratory facilities. The lab-free, lab-quality test capabilities of QPsor for viral prevalence and transmission in the community can contribute to the efficient management of pandemic situations.

Harnessing Catalytic RNA Circuits for Construction of Artificial Signaling Pathways in Mammalian Cells.

Engineering of genetic networks with artificial signaling pathways (ASPs) can reprogram cellular responses and phenotypes under different circumstances for a variety of diagnostic and therapeutic purposes. However, construction of ASPs between originally independent endogenous genes in mammalian cells is highly challenging. Here we report an amplifiable RNA circuit that can theoretically build regulatory connections between any endogenous genes in mammalian cells. We harness the system of catalytic hairpin assembly with combination of controllable CRISPR-Cas9 function to transduce the signals from distinct messenger RNA expression of trigger genes into manipulation of target genes. Through introduction of these RNA-based genetic circuits, mammalian cells are endowed with autonomous capabilities to sense the changes of RNA expression either induced by ligand stimuli or from various cell types and control the cellular responses and fates via apoptosis-related ASPs. Our design provides a generalized platform for construction of ASPs inside the genetic networks of mammalian cells based on differentiated RNA expression.

Harnessing Catalytic RNA Circuits for Construction of Artificial Signaling Pathways in Mammalian Cells

AbstractEngineering of genetic networks with artificial signaling pathways (ASPs) can reprogram cellular responses and phenotypes under different circumstances for a variety of diagnostic and therapeutic purposes. However, construction of ASPs between originally independent endogenous genes in mammalian cells is highly challenging. Here we report an amplifiable RNA circuit that can theoretically build regulatory connections between any endogenous genes in mammalian cells. We harness the system of catalytic hairpin assembly with combination of controllable CRISPR‐Cas9 function to transduce the signals from distinct messenger RNA expression of trigger genes into manipulation of target genes. Through introduction of these RNA‐based genetic circuits, mammalian cells are endowed with autonomous capabilities to sense the changes of RNA expression either induced by ligand stimuli or from various cell types and control the cellular responses and fates via apoptosis‐related ASPs. Our design provides a generalized platform for construction of ASPs inside the genetic networks of mammalian cells based on differentiated RNA expression.

HIV Immunocapture Reveals Particles Expressed in Semen Under Integrase Strand Transfer Inhibitor-Based Therapy Are Largely Myeloid Cell-Derived and Disparate.

As use of human immunodeficiency virus (HIV) integrase strand transfer inhibitors (INSTI) increases and formulations are being developed for maintenance therapies and chemoprophylaxis, assessing virus suppression under INSTI-based regimens in prevention-relevant biologic compartments, such as the male genital tract, is timely. We used cell-source marker virion immunocapture to examine amplification of particle RNA then assessed the phylogenetic relatedness of seminal and blood viral sequences from men with HIV who were prescribed INSTI-based regimens. Seminal plasma immunocaptures yielded amplifiable virion RNA from 13 of 24 (54%) men, and the sequences were primarily associated with markers indicative of macrophage and resident dendritic cell sources. Genetic distances were greatest (>2%) between seminal virions and circulating proviruses, pointing to ongoing low-level expression from tissue-resident cells. While the low levels in semen predict an improbable likelihood of transmission, viruses with large genetic distances are expressed under potent INSTI therapy and have implications for determining epidemiologic linkages if adherence is suboptimal.

Comparative Evaluation of Microbial Flora of Endodontic Origin in Teeth with Endo-Perio Lesions

ABSTRACT Background: Endodontic microbial flora plays a pivotal role in the development and persistence of periodontal endodontic lesions (PELs). Understanding the composition and prevalence of microbial species in PELs is essential for effective treatment strategies Materials and Methods: Microbial samples were collected from 50 teeth diagnosed with PELs. Sterile paper points were used to obtain samples from the root canals. Deoxyribonucleic acid (DNA) was extracted and subjected to polymerase chain reaction (PCR) amplification of the 16S ribosomal RNA (rRNA) gene to identify bacterial species. The obtained data were analyzed using statistical methods. Results: The microbial analysis revealed a diverse range of bacterial species in PELs. The most prevalent species were Porphyromonas gingivalis (32.5%), Treponema denticola (28.0%), and Fusobacterium nucleatum (22.5%). Streptococcus mutans (9.0%) and Actinomyces naeslundii (8.0%) were also frequently detected. Additionally, Prevotella intermedia (7.0%), Aggregatibacter actinomycetemcomitans (3.5%), and Enterococcus faecalis (2.5%) were present in lower frequencies Conclusion: The presence of a diverse microbial flora in teeth with PELs underscores the polymicrobial nature of these lesions. The predominance of periodontal pathogens such as Porphyromonas gingivalis, Treponema denticola, and Fusobacterium nucleatum suggests a strong association between periodontal and endodontic infections. A comprehensive understanding of the microbial profile in PELs is crucial for tailored therapeutic approaches targeting the specific pathogens involved.

The First Report on the Complete Sequence Characterization of Bluetongue Virus Serotype 3 in the Republic of Korea.

The bluetongue virus (BTV) is a significant animal pathogen with economic implications in the ruminant industry. Despite global reports on BTV detection and epidemiologic investigations, limited studies have focused on the virus in the ROK. In this study, BTV epidemiological research was conducted on blood samples from cattle and goat farms across nine regions during 2013-2014. The results showed that 3.33% of bovine blood samples (194/5824) and 0.19% of goat blood samples (2/1075) tested positive for BTV antibodies using ELISA. In Jeju-do, BTV RNA amplification occurred in 51 of 422 samples (12.1%) using real-time reverse transcription (RT-qPCR). The isolation of one sample revealed it as serotype 3, as indicated by the sequence of segments 2 (Seg-2) and 6 (Seg-6), associated with the eastern BTV topotype. However, based on Seg-1, -3, -4, -5, -7, -8, -9, and -10 analyses, the BTV-3/JJBB35 strain is more closely related to distinct BTV strains. These findings imply BTV circulation and that the Korean-isolated BTV might originate from Asian BTV strains due to multiple reassortment events. This study provides foundational data for ongoing BTV monitoring and disease-control policies in the ROK.

Vibration mixing for enhanced paper-based recombinase polymerase amplification.

Isothermal nucleic acid amplification tests (NAATs) are a vital tool for point-of-care (POC) diagnostics. These assays are well-suited for rapid, low-cost POC diagnostics for infectious diseases compared to traditional PCR tests conducted in central laboratories. There has been significant development of POC NAATs using paper-based diagnostic devices because they provide an affordable, user-friendly, and easy to store format; however, the difficulties in integrating separate liquid components, resuspending dried reagents, and achieving a low limit of detection hinder their use in commercial applications. Several studies report low assay efficiencies, poor detection output, and poorer limits of detection in porous membranes compared to traditional tube-based protocols. Recombinase polymerase amplification is a rapid, isothermal NAAT that is highly suited for POC applications, but requires viscous reaction conditions that has poor performance when amplifying in a porous paper membrane. In this work, we show that we can dramatically improve the performance of membrane-based recombinase polymerase amplification (RPA) of HIV-1 DNA and viral RNA by employing a coin cell-based vibration mixing platform. We achieve a limit of detection of 12 copies of DNA per reaction, nearly 50% reduction in time to threshold (from ∼10 minutes to ∼5 minutes), and an overall fluorescence output increase up to 16-fold when compared to unmixed experiments. This active mixing strategy enables reactions where the target and reaction cofactors are isolated from each other prior to the reaction. We also demonstrate amplification using a low-cost vibration motor for both temperature control and mixing, without the requirement of any additional heating components.

Brown rats (Rattus norvegicus) as potential reservoirs of Enterocytozoon bieneusi in Heilongjiang Province, China: high prevalence, genetic heterogeneity, and potential risk for zoonotic transmission.

Enterocytozoon bieneusi, an obligatory intracellular fungus, is prevalent among animals and humans. Due to their close interaction with humans and their extensive regional distribution, brown rats (Rattus norvegicus) are important pathogen reservoirs. To assess the zoonotic transmission potential of E. bieneusi, a molecular investigation was conducted on 817 R. norvegicus from four cities in Heilongjiang Province, China. A total of 817 R. norvegicus were collected from four cities in Heilongjiang Province, China. The genotyping of E. bieneusi was conducted through PCR amplification of the small subunit ribosomal RNA (SSU rRNA)'s internal transcribed spacer (ITS) segments. Phylogenetic and similarity analyses were used to examine zoonotic potential and genetic characteristics of the E. bieneusi-positive specimens. Among the 817 R. norvegicus, the total infection rate was 33.3% (272/817). Seventy-five genotypes were identified, including 14 known genotypes D (n = 167), A (n = 15), HLJ-CP1 (n = 12), WR8 (n = 6), EbpC (n = 2), BEB6 (n = 1), CS-4 (n = 1), CHPM1 (n = 1), Henan-II (n = 1), HNH-22 (n = 1), HNH-25 (n = 1), I (n = 1), JLD-XI (n = 1), SDD5 (n = 1), and 61 novel genotypes designated as SHWR1 (n = 10), SYSWR1 (n = 2), and SHWR2 to SHWR17, SYSWR2 to SYSWR36 and QTHWR1 to QTHWR8 (n = 1, each). Moreover, 10 samples exhibited mixed genotype infections, including D + A (n = 3), D + EbpC (n = 1), D + HLJ-CP1 (n = 1), D + SHWR1 (n = 1), D + SHWR16 (n = 1), D + SHWR17 (n = 1), SDD5 + WR8 (n = 1), and CS-4 + SYSWR36 (n = 1). Phylogenetic analysis grouped the genotypes into three main groups: group 1 (n = 67), group 2 (n = 5), and group 9 (n = 3). The high prevalence and genetic diversity of E. bieneusi in Heilongjiang Province's R. norvegicus imply that these animals spread the pathogen. The R. norvegicus that E. bieneusi carries can spread zoonotic disease, making it a serious hazard to the local human population. Therefore, it is imperative to raise awareness about the dangers posed by R. norvegicus and implement measures to reduce their population to prevent environmental contamination.

Rapid detection of avian leukemia virus using CRISPR/Cas13a based lateral flow dipstick.

Avian leukemia virus (ALV) is one of the main pathogens of poultry tumor diseases, and has caused significant economic losses to the poultry industry since its discovery. Therefore, establishing a rapid detection method is essential to effectively prevent and control the spread of ALV. In this study, specific CRISPR RNA (crRNA) and recombinase-aided amplification (RAA) primers with T7 promoter were designed based on the relatively conserved sequence of avian leukemia virus. When crRNA recognized the target sequence, Cas13a protein was activated to cut the reporting probes, and then the detection results were read by using lateral flow dipstick (LFD). The RAA-CRISPR/Cas13a-LFD reaction system was constructed. The RAA amplification time, Cas13a protein concentration, crRNA concentration and CRISPR reaction time were optimized to evaluate the specificity, sensitivity and reproducibility of the system. Finally, RAA-CRISPR/Cas13a-LFD method was compared with Polymerase chain reaction (PCR)-Agarose electrophoresis method and qPCR method in the detection of clinical samples, and the reliability of RAA-CRISPR/Cas13a-LFD method was evaluated. The results showed that the RAA-CRISPR/Cas13a-LFD method could effectively amplify the target gene at 37°C for 40 min, and the test results could be determined by LFD visual observation. The method had good specificity and no cross-reaction with Marek's disease virus (MDV), Fowl adenovirus (FAdV), Infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), Infectious laryngotracheitis virus (ILTV), and Infectious bronchitis virus (IBV). The minimum detection limit of the method was 100 copies/μL, and it had good repeatability and stability. The coincidence rate of clinical detection reached 97.69% and 99.23%. In summary, this study established a simple, efficient, accurate and visualized ALV detection method, which can be used for the prevention and rapid clinical diagnosis of avian leukosis (AL).

RNA Analysis Using Immunoassay Detection Format.

Oligonucleotide probe tagging and reverse transcriptase polymerase-chain reaction (RT-PCR) are the most widely used techniques currently used for detecting and analyzing RNA. RNA detection using labeled oligonucleotide probe-based approaches is suitable for point-of-care (POC) applications but lacks assay sensitivity, whereas RT-PCR requires complex instrumentation. As an alternative, immunoassay detection formats coupled with isothermal RNA amplification techniques have been proposed for handheld assay development. In this chapter, we describe a robust technique comprising of: (a) target RNA tagging with a complementary oligonucleotide probe labeled with a hapten moiety to form a DNA/RNA duplex hybrid; (b) complexing the DNA/RNA duplex with a pre-coated antibody (Ab) directed at the hapten moiety; (c) sandwich complex formation with an Ab that selectively recognizes the DNA/RNA structural motif; and (d) detection of the sandwich complex using a secondary Ab enzyme conjugate targeting the anti-DNA/RNA Ab followed by standard enzyme-linked immunosorbent assay (ELISA) visualization.

NASBA Coupled to Paper Microfluidics for RNA Detection.

The analysis of RNA sequences is crucial to obtain invaluable insights into disease prognosis. Reliable and rapid diagnostic solutions at the site of sample collection contribute toward optimal delivery of medical treatment. For this reason, the development of more sensitive and portable RNA detection techniques are expected to advance current point-of-care (POC) diagnostic capabilities. Advancements of POC diagnostic technologies will also contribute to counter the spread of emerging viruses. Reverse transcriptase polymerase chain reaction (RT-PCR) is the most commonly used technique to identify etiological organisms of infections. However, the need for thermocycler and fluorescent measurement renders RT-PCR less suitable for POC applications. Here, we provide a step-by-step protocol of Nucleic Acid Sequence-Based Amplification (NASBA), a robust isothermal RNA amplification technique, coupled with a portable paper microfluidics detection format.

Collagen Anchoring Protein-Nucleic Acid Chimeric Probe for In Situ In Vivo Mapping of a Tumor-Specific Protease.

In situ analysis of biomarkers in the tumor microenvironment (TME) is important to reveal their potential roles in tumor progression and early diagnosis of tumors but remains a challenge. In this work, a bottom-up modular assembly strategy was proposed for a multifunctional protein-nucleic chimeric probe (PNCP) for in situ mapping of cancer-specific proteases. PNCP, containing a collagen anchoring module and a target proteolysis-responsive isothermal amplification sensor module, can be anchored in the collagen-rich TME and respond to the target protease in situ and generate amplified signals through rolling cycle amplification of tandem fluorescent RNAs. Taking matrix metalloproteinase 2 (MMP-2), a tumor-associated protease, as the model, the feasibility of PNCP was demonstrated for the in situ detection of MMP-2 activity in 3D tumor spheroids. Moreover, in situ in vivo mapping of MMP-2 activity was also achieved in a metastatic solid tumor model with high sensitivity, providing a useful tool for evaluating tumor metastasis and distinguishing highly aggressive forms of tumors.

The HC-Pro cistron of Triticum mosaic virus is dispensable for systemic infection in wheat but is required for symptom phenotype and efficient genome amplification

Triticum mosaic virus (TriMV), the type species of the genus Poacevirus in the family Potyviridae, is an economically important wheat curl mite-transmitted wheat-infecting virus in the Great Plains region of the USA. In this study, the functional genomics of helper component-proteinase (HC-Pro) encoded by TriMV was examined using a reverse genetics approach. TriMV with complete deletion of HC-Pro cistron elicited systemic infection in wheat, indicating that HC-Pro cistron is dispensable for TriMV systemic infection. However, TriMV lacking HC-Pro caused delayed systemic infection with mild symptoms that resulted in little or no stunting of plants with a significant reduction in the accumulation of genomic RNA copies and coat protein (CP). Sequential deletion mutagenesis from the 5′ end of HC-Pro cistron in the TriMV genome revealed that deletions within amino acids 3 to 25, except for amino acids 3 and 4, elicited mild symptoms with reduced accumulation of genomic RNA and CP. Surprisingly, TriMV with deletion of amino acids 3 to 50 or 3 to 125 in HC-Pro elicited severe symptoms with a substantial increase in genomic RNA copies but a drastic reduction in CP accumulation. Additionally, TriMV with heterologous HC-Pro from other potyvirids produced symptom phenotype and genomic RNA accumulation similar to that of TriMV without HC-Pro, suggesting that HC-Pros of other potyvirids were not effective in complementing TriMV in wheat. Our data indicate that HC-Pro is expendable for replication of TriMV but is required for efficient viral genomic RNA amplification and symptom development. The availability of TriMV with various deletions in the HC-Pro cistron will facilitate the examination of the requirement of HC-Pro for wheat curl mite transmission.

Molecular Characterization of Colletotrichum gloeosporioides (Glomerella cingulata) Causing Mango Anthracnose Disease

Mango production in Tamil Nadu is known to be adversely affected by C. gloeosporioides in both field and post harvest stage of crop and drastically reduces market and edible value of fruit. To manage the disease it is essential to learn about the nature of pathogen and its way of interaction with host and its survival ability in environment. Hence a study has been carried out on Isolation of fungal pathogen was done using PDA medium in order to identify the disease's causative agent and its variation in morphology and colony characters. A total of twenty-four C. gloeosporioides isolates were obtained from various Tamil Nadu mango cultiavting areas of Tamil Nadu. The isolates were identified morphologically and molecularly. The microscopic features such as conidia shape and size were used to identify C. gloeosporioides. The CTAB technique was employed to extract the DNA from the isolates of C. gloeosporioides. A total of 24 isolates were subjected to ITS (Inter Transcribed Spacer) of 18s ribosomal RNA amplification; all of these isolates exhibited amplified products with a size range of 560 bp, indicating that they were fungal pathogen. The MkCgF and MkCgR PCR primer combination was unique to the ITS region of C. gloeosporioides species. It has been confirmed that all 24 of the C. gloeosporioides isolates are really C. gloeosporioides by amplifying the predicted size of 380 bp.