Nucleic Acid Sequence-based Amplification Research Articles

Detection of Potato Pathogen Clavibacter sepedonicus by CRISPR/Cas13a Analysis of NASBA Amplicons.

The ring rot of potato caused by the bacterial pathogen Clavibacter sepedonicus is a quarantine disease posing a threat to the potato industry worldwide. The sensitive and selective detection of C. sepedonicus is of a high importance for its effective control. Here, the detection system is reported to determine viable bacteria of C. sepedonicus in potato tubers, based on the coupling of CRISPR/Cas13a nuclease with NASBA (Nucleic Acid Sequence Based Amplification)-the method of isothermal amplification of RNA. Detection can be conducted using both instrumental and non-instrumental (visual inspection of test tubes under blue light) modes. When NASBA and Cas13a analyses were carried out in separate test tubes, the limit of detection (LOD) for the system was 1000 copies of purified target 16S rRNA per NASBA reaction or about 24 colony-forming units (CFUs) of C. sepedonicus per 1 g of tuber tissue. The testing can also be conducted in the "one-pot" format (a single test tube), though with lower sensitivity: LOD was 10,000 copies of target RNA or about 100 CFU per 1 g of tuber tissue for both instrumental and visual detection modes. The overall time of NASBA/Cas13a analysis did not exceed 2 h. The developed NASBA/Cas13a detection system has the potential to be employed as a routine test of C. sepedonicus, especially for on-site testing.

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.

A cell-free fluorescence biosensor based on allosteric transcription factor NalC for detection of pentachlorophenol.

Pentachlorophenol (PCP) was once used as a pesticide, germicide, and preservative due to its stable properties and resistance to degradation. This study aimed to design a biosensor for the quantitative and prompt detection of capable of PCP. A cell-free fluorescence biosensor was developed while employing NalC, an allosteric Transcription Factor responsive to PCP and In Vitro Transcription. By adding a DNA template and PCP and employing Electrophoretic Mobility Shift Assay while monitoring the dynamic fluorescence changes in RNA, this study offers evidence of NalC's potential applicability in sensor systems developed for the specific detection of PCP. The biosensor showed the capability for the quantitative detection of PCP, with a Limit of Detection (LOD) of 0.21μM. Following the addition of Nucleic Acid Sequence-Based Amplification, the fluorescence intensity of RNA revealed an excellent linear relationship with the concentration of PCP, showing a correlation coefficient (R2) of 0.9595. The final LOD was determined to be 0.002μM. This study has successfully translated the determination of PCP into a fluorescent RNA output, thereby presenting a novel approach for detecting PCP within environmental settings.

Innovative approaches to microbial identification enhancing accuracy and speed in infectious disease diagnostics

The rapid and accurate identification of microbial pathogens is crucial for effective management of infectious diseases. Traditional methods, though reliable, are often time-consuming. Recent advancements in molecular diagnostics, mass spectrometry, and next-generation sequencing (NGS) have revolutionized the field by enhancing both the speed and accuracy of microbial identification. Polymerase chain reaction (PCR) and its derivatives, such as real-time PCR, enable rapid amplification of genetic material, significantly reducing diagnostic times. Techniques like nucleic acid sequence-based amplification (NASBA) and loop-mediated isothermal amplification (LAMP) further extend these capabilities to resource-limited settings by operating at constant temperatures. Mass spectrometry, particularly matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), provides rapid and accurate microbial identification by analyzing protein profiles. This method has high throughput, is cost-effective, and offers the ability to detect antimicrobial resistance, significantly impacting clinical decision-making and patient management. NGS technologies offer comprehensive pathogen detection by sequencing entire microbial genomes. This approach is invaluable in diagnosing complex infections, tracking outbreaks, and identifying antibiotic resistance genes, though its high cost and complexity limit widespread use. The integration of these innovative technologies into clinical practice enhances the ability to diagnose and manage infectious diseases effectively. Molecular diagnostics, mass spectrometry, and NGS each bring unique strengths, from rapid and precise pathogen detection to comprehensive genetic analysis and resistance profiling. These advancements contribute to improved patient outcomes and more effective public health strategies. The continued development and implementation of these technologies are essential to address the evolving challenges in microbial diagnostics, paving the way for more precise and personalized approaches in infectious disease management.

Molecular Diagnosis of Human Cutaneous Leishmaniasis and Identification of the Causative Leishmania Species in Iran: A Narrative Review

Cutaneous leishmaniasis (CL) is a common form of leishmaniasis in underdeveloped countries. Although CL tends to be self-limiting, it can cause significant scars and may progress to more severe manifestations. Additionally, Leishmania species vary in susceptibility to the available treatments. The selection of treatment and clinical outcome of CL depend on the accurate determination of the Leishmania species. This mini-review aims to provide an overview of the molecular diagnosis techniques such as PCR-based assays, nucleic acid sequence-based amplification, and loop-mediated isothermal amplification utilized in the identification of Leishmania species in Iran.

Sequence-independent assay for viral load quantification of HIV subtypes

A sequence-independent assay for the detection and quantification of the Human Immunodeficiency Virus (HIV) has been successfully developed for HIV group M, subtype B, and HIV group O. This assay comprises a modified nucleic acid sequence-based amplification (NASBA) protocol coupled with an electrochemical biosensor (E-biosensor). Our modified NASBA reaction involves the addition of a 22-nucleotide tag at the 5′ end of each amplicon. Consequently, all HIV sequences that are amplified with this protocol, would be detected with our E-biosensor transducer regardless of the sequence between the primer binding regions. An E-biosensor employing a four-way junction (4WJ) system has been meticulously designed to detect the 22-nucleotide tag. This development leads to the creation of a standardized E-Biosensor capable of potentially detecting any HIV subtype.

Microsphere-Enhanced Fluorescence-Lightened Solid-Phase Hybridization Assay: The Strategy to Highly Selective Detection of Micro Ribonucleic Acids.

The detection of low-abundance microribonucleic acid (miRNA) frequently adopted nucleic acid sequence-based amplification detection, which was found to have poor selectivity for the nonspecific amplification of template-dependent ligation in enzyme-mediated cascade reactions. Here, a highly selective detection of miRNAs was developed that combined microsphere-enhanced fluorescence (MSEF) and solid-phase base-paired hybridization. The target miRNA could be accurately and quantitatively identified through the solid-phase hybridization assay on the surface of an optical microsphere, while the detected fluorescence signal could be physically amplified by MSEF. Hereinto, the optical microsphere acted as the fluorescence amplifier and whose surface supplied the space to carry out base-paired hybridization to recognize the target miRNA via the immobilized capture DNA sequence. The detected fluorescence signal of the single-base mismatched miRNA-21 sequence was just around 12% of that of the target miRNA-21 sequence in the measurement of model miRNA-21, while the limit of detection of miRNA-21 could be 1.0 fM. The developed detection of miRNA on an optical microsphere was demonstrated to be an excellent physically amplified method to selectively and sensitively detect the target miRNA and magnificently avoid the nonspecific amplification and false-positive results, which is expected to have wide applications in pathematology, pharmacology, clinic diagnosis, and on-site screening fields as well.

A Single Electrochemical Biosensor Designed to Detect Any Virus.

Viruses are the primary cause of many infectious diseases in both humans and animals. Various testing methods require an amplification step of the viral RNA sample before detection, with quantitative reverse transcription polymerase chain reaction (RT-qPCR) being one of the most widely used along with lesser-known methods like Nucleic Acid Sequence-Based Amplification (NASBA). NASBA offers several advantages, such as isothermal amplification and high selectivity for specific sequences, making it an attractive option for low-income facilities. In this research, we employed a single electrochemical biosensor (E-Biosensor) designed for potentially detecting any virus by modifying the NASBA protocol. In this modified protocol, a reverse primer is designed with an additional 22-nucleotide sequence (tag region) at the 5'-end, which is added to the NASBA process. This tag region becomes part of the final amplicon generated by NASBA. It can hybridize with a single specific E-Biosensor probe set, enabling subsequent virus detection. Using this approach, we successfully detected three different viruses with a single E-Biosensor design, demonstrating the platform's potential for virus detection.

Disposable impedance sensors based on novel hybrid MoS2 nanosheets and microparticles to detect Escherichia Coli DNA.

The rapid and accurate detection of pathogenic bacteria is essential for food safety and public health. Conventional detection techniques, such as nucleic acid sequence-based amplification and polymerase chain reaction, are time-consuming and require specialized equipment and trained personnel. Here, we present quick, disposable impedance sensors based on the novel hybrid MoS2 nanomaterial for detecting Escherichia coli DNA. Our results indicate that the proposed sensors operate linearly between 10- 20 and 10-15 M concentrations, achieving an impressive detection limit of 10-20 M with the highest sensitivity observed at a 0.325 nM probe concentration sensor. Furthermore, the electrochemical impedance spectroscopy biosensors exhibited potential selectivity for Escherichia coli DNA over Bacillus subtilis and Vibrio proteolyticus DNA sequences. The findings offer a promising avenue for efficient and precise DNA detection, with potential implications for broader biotechnology and medical diagnostics applications.

Human Papillomavirus (HPV16 and HPV18) Infection; Pathogenesis, Vaccination, and Detection by Loop-Mediated Isothermal Amplification with Lateral Flow Dipstick Tests: A Review

Context: Human papillomavirus (HPV) infections contribute to the cause of 15% - 20% of all human cancers. This review aims to examine and provide updated information on various aspects, with a particular focus on topics that are of interest to dermatologists. Evidence Acquisition: Using published studies, the pathogenicity of HPV was investigated. Subsequently, the existing vaccines were explored, followed by a review of the loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) method. Results: For HPV detection, the polymerase chain reaction (PCR), self-sustained sequence replication (3SR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA) were used. These methods can have good detection but still have problems. In comparison with nested PCR, the detection of HPV16 and HPV18 using LAMP-LFD has higher sensitivity. Conclusions: Loop-mediated isothermal amplification-lateral flow dipstick is a simple and rapid method for the specific and sensitive detection of HPV. Thus, along with the previous HPV16 and HPV18 diagnostic tools, LAMP-LFD might be useful in field studies or local hospitals.

Characterization of viral infections in children with influenza-like-illness during December 2018-January 2019.

Respiratory viral infection (RVI) is of very concern after the outbreak of COVID-19, especially in pediatric departments. Learning pathogen spectrum of RVI in children previous the epidemic of COVID-19 could provide another perspective for understanding RVI under current situation and help to prepare for the post COVID-19 infection control. A nucleic acid sequence-based amplification (NASBA) assay, with 19 pairs of primers targeting various respiratory viruses, was used for multi-pathogen screening of viral infections in children presenting influenza-like illness (ILI) symptoms. Children with ILI at the outpatient department of Beijing Tsinghua Changgung Hospital during the influenza epidemic from 12/2018 to 01/2019 were included. Throat swabs were obtained for both the influenza rapid diagnostic test (IRDT) based on the colloidal gold immunochromatographic assay and the NASBA assay, targeting various respiratory viruses with an integrated chip technology. Of 519 patients, 430 (82.9%) were positive in the NASBA assay. The predominant viral pathogens were influenza A H1N1 pdm1/2009 (pH1N1) (48.4%) and influenza A (H3N2) (18.1%), followed by human metapneumovirus (hMPV) (8.8%) and respiratory syncytial virus (RSV) (6.1%). Of the 320 cases identified with influenza A by NASBA, only 128 (40.0%) were positive in the IRDT. The IRDT missed pH1N1 significantly more frequently than A (H3N2) (P<0.01). Influenza A pH1N1 and A (H3N2) were the major pathogens in <6 years and 6-15 years old individuals respectively (P<0.05). In summary, influenza viruses were the major pathogens in children with ILI during the 2018-2019 winter influenza epidemic, while hMPV and RSV were non-negligible. The coexistence of multiple pathogen leading to respiratory infections is the normalcy in winter ILI cases.

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.

Rapid and effective detection of Macrobrachium rosenbergii nodavirus using a combination of nucleic acid sequence-based amplification test and immunochromatographic strip

Nucleic acid sequence-based amplification (NASBA) provides a fast and convenient approach for nucleic acid amplification under isothermal conditions, and its combination with an immunoassay-based lateral flow dipstick (LFD) could produce a higher detection efficiency for M. rosenbergii nodavirus isolated from China (MrNV-chin). In this study, two specific primers and a labelled probe of the capsid protein gene of MrNV-chin were constructed. The process of this assay mainly included a single-step amplification at a temperature of 41 ℃ for 90 min, and hybridization with an FITC-labeled probe for 5 min, with the hybridization been required for visual identification during LFD assay. The test results indicated that, the NASBA-LFD assay showed sensitivity for 1.0 fg M. rosenbergii total RNA with MrNV-chin infection, which was 104 times that of the present RT-PCR approach for the detection of MrNV. In addition, no products were created for shrimps with infection of other kinds of either DNA or RNA virus, which indicated that the NASBA-LFD was specific for MrNV. Therefore, the combination of NASBA and LFD is a new alternative detection method for MrNV which is rapid, accurate, sensitive and specific without expensive equipment and specialised personnel. Early detection of this infectious disease among aquatic organisms will help implement efficient therapeutic strategy to prevent its spread, enhance animal health and limit loss of aquatic breeds in the event of an outbreak.

Bacterial drug-resistance and viability phenotyping upon disinfectant exposure revealed by single-nucleotide resolved-allele specific isothermal RNA amplification

Disinfectant abuse poses a risk of bacterial evolution against stresses, especially during the coronavirus disease 2019 (COVID-19) pandemic. However, bacterial phenotypes, such as drug resistance and viability, are hard to access quickly. Here, we reported an allele specific isothermal RNA amplification (termed AlleRNA) assay, using an isothermal RNA amplification technique, i.e., nucleic acid sequence-based amplification (NASBA), integrated the amplification refractory mutation system (ARMS), involving the use of sequence-specific primers to allow the amplification of the targets with complete complementary sequences. AlleRNA assay enables rapid and simultaneous detection of the single nucleotide polymorphism (SNP) (a detection limit, a LOD of 0.5 % SNP) and the viability (a LOD of 80 CFU) of the quinolone resistant Salmonella enterica. With the use of AlleRNA assay, we found that the quinolone resistant S. enterica exhibited higher survival ability during exposure toquaternary ammonium salt, 75 % ethanol and peracetic acid, which might be attributed to the upregulation of stress response-associated genescompared with the susceptible counterparts. Additionally, the AlleRNA assay indicated the potential risk in a high-frequency occurrence of viable but nonculturable (VBNC) quinolone resistant S. enterica induced by disinfectants due to the depression of ATP biosynthesis. The excessive usage of disinfectants during the COVID-19 pandemic should be carefully evaluated due to the latent threat to ecological and human health.

Detection of Multiplex NASBA RNA Products Using Colorimetric Split G Quadruplex Probes.

Structural RNA is a challenging target for recognition by hybridization probes. This chapter addresses the recognition problem of RNA amplicons in samples obtained by multiplex nucleic acid sequence-based amplification (NASBA). The method describes the design of G-quadruplex binary (split) DNA peroxidase sensors that produces colorimetric signal upon recognition of NASBA amplicons.

Molecular Methods for Identification and Quantification of Foodborne Pathogens

Foodborne pathogens that enter the human food chain are a significant threat worldwide to human health. Timely and cost-effective detection of them became challenging for many countries that want to improve their detection and control of foodborne illness. We summarize simple, rapid, specific, and highly effective molecular technology that is used to detect and identify foodborne pathogens, including polymerase chain reaction, isothermal amplification, loop-mediated isothermal amplification, nucleic acid sequence-based amplification, as well as gene chip and gene probe technology. The principles of their operation, the research supporting their application, and the advantages and disadvantages of each technology are summarized.

Rapid Detection of Viable Toxoplasma gondii by Nucleic Acid Sequence-Based Amplification (NASBA), Using Primer Sets Targeting B1 rRNA Gene

Rapid Detection of Viable Toxoplasma gondii by Nucleic Acid Sequence-Based Amplification (NASBA), Using Primer Sets Targeting B1 rRNA Gene

Trigger impact of Chlamydia trachomatis on various types of seronegative spondyloarthritis

Introduction. To date, clinical manifestations, laboratory changes and the results of instrumental examinations of the joints and spine in axial seronegative spondyloarthritis (aSpA) caused by Chlamydia trachomatis have not been fully elucidated. Purpose of the study: evaluation of clinical manifestations features, diagnosis, and evolution of seronegative spondyloarthritis associated with Chlamydia trachomatis infection. Objectives of the study: To compare clinical manifestations, changes in laboratory parameters, results of instrumental examinations of the joints and spine in seronegative spondyloarthritis, the axial, peripheral, and mixed form. To appreciate the significance of Chlamydia trachomatis in the development of SpA by detecting the presence of DNA using the polymerase chain reaction (PCR) in real time and by isothermal amplification of NASBA-PCR (Nucleic acid sequence-based amplification). Material and methods. During 2015-2021 were examined 138 patients with SpA hospitalized in the Republican Clinical Hospital „Timofei Moşneaga”. Chlamydia trachomatis in the form of monoinfection was detected in 87 (6.3%) patients, of which 52 (59.8%) were women and 35 (40.2%) men. Results. The results of the diagnosis of Chlamydia trachomatis infection in patients with arthritis: Chlamydia trachomatis infection was detected in 78 (89.7%) out of 87 patients examined by PCR, and in 9 (10.3%) cases by ELISA. The peripheral form begins mainly with inflammation of the joints of the lower extremities (81.8%), of which the knee joints are mainly involved in the pathological process (63.6%). SpA axial form associated with Chlamydia trachomatis infection occurs mainly in young men (85.7%). In more than half of the patients, large joints participate in the pathological process (58.8%), especially the lower extremities. The axial variant of the SpA can begin with an injury in any part of the spine. Discussions. SpA associated with Chlamydia trachomatis infection has more expressed clinical, laboratory and instrumental manifestations both in the onset and in the advanced stage of the disease.

Nanomolecular Magnetic Probe for Detection of Nucleic Acid Sequence-Based Amplification of Covid-19 RNA with the Naked Eye

Nucleic acid amplification of Covid-19 RNA is the main subject for molecular detection of SARS-COV-2. However, the employment of target amplification methods such as PCR needs a converting step for Covid-19 RNA to DNA template to be amplified. In addition, Covid-19 RNA isolation needs some RNA extraction kits that their providing could increase the time and costs for the molecular detection of the virus. In this study, we introduced a magnetic nanoprobe that could be used to capture and amplify Covid-19 RNA through an isothermal amplification process, so-called nucleic acid sequence-based amplification, without needing to perform a separate step for the viral RNA converting to DNA template. By using engineered sequences appropriate to the target nucleic acid attached to the magnetic nanoparticles, identifying the target RNA from the virus could be possible by clumping the particles that could be seen with naked eyes. According to the isothermal amplification of the viral RNA via nucleic acid sequence-based amplification assisted with the magnetic nanoprobe, the nanomolecular method eliminated the need for special pieces of equipment and the time for detection of Covid-19 in specimens.

An improved nucleic acid sequence-based amplification method mediated by T4 gene 32 protein.

The uptake of Nucleic Acid Sequence-Based Amplification (NASBA) for point of care testing may be hindered by a complexity in the workflow due the requirement of a thermal denaturation step to initiate the cyclic isothermal amplification before the addition of the amplification enzymes. Despite reports of successful enhancement of other DNA and RNA amplification methods using DNA and RNA binding proteins, this has not been reported for NASBA. Here, three single-stranded binding proteins, RecA, Extreme Thermostable Single-stranded binding protein (ET SSB) and T4 gene gp32 protein (gp32), were incorporated in NASBA protocol and used for single pot, one-step NASBA at 41 °C. Indeed, all SSBs showed significantly improved amplifications compared with the 2-step process, but only gp32 showed no non-specific aberrant amplification, and slightly improved the time-to-positivity in comparison with the conventional NASBA. For synthetic HIV-1 RNA, gp32 was found to improve the time-to-positivity (ttp) by average of 13.6% of one-step NASBA and 6.7% of conventional NASBA for the detection of HIV-1 RNA, showing its potential for simplifying the workflow as desirable for point of care applications of NASBA.