Nucleic Acid Sequence-based Amplification Research Articles

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.

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.

Detection of micro-RNA by a combination of nucleic acid sequence-based amplification and a novel chemiluminescent pyrophosphate assay.

Micro-RNA has attracted much attention as a biomarker for disease progression and malignancy. A compact, simple, rapid, and highly sensitive method is required to perform simple genetic analyses, such as point-of-care testing (POCT), at the clinic or bedside. Nucleic acid sequence-based amplification (NASBA) is a specific amplification method for a single-stranded RNA fragment that is useful for the highly sensitive detection of miRNAs. In this work, we developed a novel miRNA analytical system for POCT by combining the NASBA and chemiluminescence methods. Because the NASBA reaction is conducted at a constant temperature (41°C) and detection by chemiluminescence reaction does not require a light source, these methods could be combined to amplify 100 ng/assay miRNA. This combined miRNA detection method could be useful for the future development of compact POCT systems.

Isothermal RNA Amplification for the Detection of Viable Pathogenic Bacteria to Estimate the Salmonella Virulence for Causing Enteritis.

Viable foodborne pathogens can cause intestinal infection and food poisoning. Herein, we reported an RNA assay allowing for sensitive (close to 1 CFU and 1% viable bacteria detectable) and rapid (within 2.5 h) detection of viable pathogenic bacteria by coupling isothermal RNA amplification (nucleic acid sequence-based amplification, NASBA) with a CRISPR/Cas13a system. NASBA allowed direct amplification of 16S rRNA extracted from viable S. enterica (RNAs degrade rapidly in dead bacteria), and the specificity of amplification was ensured using Cas13a/crRNA to recognize the amplicons. We used the CRISPR/Cas13-based NASBA assay (termed cNASBA assay) to investigate the in vivo colonization and intestinal infection of S. enterica in mice. We found that S. enterica was mainly colonized at the cecum, colon, and rectum, and the severity of enteritis caused by S. enterica was determined by the number of viable S. enterica rather than the total count of S. enterica. The cNASBA assay can quantify viable S. enterica and thus can improve the accuracy of virulence estimation compared to qPCR. It shows promise as a reliable tool for monitoring pathogen contamination and biosafety control.

A duplex real-time NASBA assay targeting a serotype-specific gene for rapid detection of viable Salmonella Paratyphi C in retail foods of animal origin.

Salmonella enterica serovar Paratyphi C is highly adapted to humans and can cause a typhoid-like disease with high mortality rates. In this study, three serovar-specific genes were identified by comparative genomics for Salmonella Paratyphi C, SPC_0871, SPC_0872, and SPC_0908. Based on the SPC_0908 and xcd genes for testing Salmonella spp., we developed a duplex real-time nucleic acid sequence-based amplification (real-time NASBA) with a molecular beacon approach for the simultaneous detection of viable cells of Salmonella spp. and serotype Paratyphi C. The test selectively and consistently detected 53 Salmonella spp. (representing 31 serotypes) and 18 non-Salmonella strains. Additionally, the method showed high resistance to interference from natural background flora in pork and chicken samples. The sensitivity of the established approach was determined to be 4.89 cfu/25 g in artificially contaminated pork and chicken samples after pre-enrichment. We propose this NASBA-based protocol as a potential detection method for Salmonella spp. and serotype Paratyphi C in foods of animal origin.

IVT cell-free biosensors for tetracycline and macrolide detection based on allosteric transcription factors (aTFs).

In recent years, the issue of food safety has received a lot of attention. The Food and Drug Administration (FDA) prescribes the antibiotic's maximum residue limit (MRL) in food production. The standard detection methods of antibiotics are liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and high-performance liquid chromatography (HPLC), with complex operations and precision instruments. In this study, allosteric transcription factor (aTF)-based in vitro transcription (IVT) cell-free biosensors were developed for tetracyclines and macrolides with nucleic acid sequence-based amplification (NASBA). Characterization of binding and dissociation processes between aTF and DNA was carried out by BIAcore assay and electrophoretic mobility shift assay (EMSA). BIAcore was innovatively used to directly observe the real-time process of binding and dissociation of aTF with DNA. The biosensors produce more fluorescence RNA when target antibiotics are added to the three-way junction dimeric Broccoli (3WJdB). Four tetracyclines and two macrolides were quantified in the 0.5-15 μM range, while erythromycin and clarithromycin were detected over a range of 0.1-15 μM. NASBA, commonly used for viral detection, was used to amplify 3WJdB RNA generated by IVT, which greatly increased the LOD for tetracyclines and macrolides to 0.01 μM. The use of biosensors in milk samples demonstrated their on-site detection performance. Overall, our proposed biosensors are simple, rapid, selective, and sensitive, with the potential for field application.

Loop-Mediated Isothermal Amplification (LAMP) as a Promising Point-of-Care Diagnostic Strategy in Avian Virus Research.

Simple SummaryMany of the existing screening methods of avian viruses depend on clinical symptoms and pathological gross examinations that still necessitate confirmatory microscopic testing. Confirmation of a virus is often conducted at centralized laboratories that are well-equipped with instruments for virus isolation, hemagglutinin inhibition, virus neutralization, ELISA, PCR and qPCR. These assays are known for their great accuracy and sensitivity, and hence are set as standard practices. Nevertheless, limitations arise due to the time, cost and on-site applicability. As the technology progresses, molecular diagnostics should be more accessible to isolated areas and even practicable for use by non-skilled personnel such as farmers and private breeders. One of the point-of-care diagnostic strategies to consider for such matters is loop-mediated isothermal amplification (LAMP).Over the years, development of molecular diagnostics has evolved significantly in the detection of pathogens within humans and their surroundings. Researchers have discovered new species and strains of viruses, while mitigating the viral infections that occur, owing to the accessibility of nucleic acid screening methods such as polymerase chain reaction (PCR), quantitative (real-time) polymerase chain reaction (qPCR) and reverse-transcription qPCR (RT-qPCR). While such molecular detection methods are widely utilized as the benchmark, the invention of isothermal amplifications has also emerged as a reliable tool to improvise on-field diagnosis without dependence on thermocyclers. Among the established isothermal amplification technologies are loop-mediated isothermal amplification (LAMP), recombinant polymerase amplification (RPA), strand displacement activity (SDA), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA) and rolling circle amplification (RCA). This review highlights the past research on and future prospects of LAMP, its principles and applications as a promising point-of-care diagnostic method against avian viruses.

HPV RNA and DNA testing in Polish women screened for cervical cancer – A single oncological center study

ObjectivesTo evaluate the incidence of HPV infection, and the frequency of the various genotypes, using mRNA and DNA testing; to assess their relationship with the cervical lesions and women’s age in the Polish patients. Study designA group of 1840 women, most of whom had abnormal cytology, from the Franciszek Łukaszczyk Oncology Centre in Bydgoszcz, Poland were screened for presence of at least one of 13 high risk HPV. Following that, 545 HPV DNA positive women were tested for HPV infection using HPV mRNA with the Nucleic Acid Sequence-Based Amplification Assay (NASBA) method. ResultsIn our study group, 70.1% had DNA HPV positive results. Only 4% of the women had normal cytology. Among 545 HPV DNA positive patients, 36.3% had HPV mRNA positive tests. Moreover, 48% of the HPV mRNA positive patients were infected with HPV 16, followed by 18 (12.6%), 31 (10.1%), 33 (8.6%%), 45 (4.5%), and 16.2% of HPV mRNA positive women were infected with more than one HPV genotype. Furthermore, we found that in women under 30, HPV DNA positivity was higher than HPV mRNA positivity, supporting the hypothesis that younger women's infections are mostly temporary. ConclusionsThe differences in HPV prevalence and genotype distribution observed in our study may have an impact on the efficacy of HPV vaccinations for cervical cancer and the development of screening programs, which should be examined further in future studies.

Application and evaluation of nucleic acid sequence-based amplification, PCR and cryptococcal antigen test for diagnosis of cryptococcosis

BackgroundCryptococcosis is a major opportunistic invasive mycosis in immunocompromised patients, but it is also increasingly seen in immunocompetent patients. In the early stages of cryptococcosis, limitations of the detection method may hinder the diagnosis. A molecular diagnostic technique based on nucleic acid sequence-based amplification (NASBA) method was developed to fulfil the need for efficient diagnosis of cryptococcosis.MethodsWe compared the diagnostic performance of NASBA, PCR and cryptococcal antigen (CrAg) test (colloidal gold method) in clinical samples from 25 cryptococcosis patients (including 8 cryptococcal meningoencephalitis and 17 pulmonary cryptococcosis) who were categorized as proven cases (n = 10) and probable cases (n = 15) according to the revised EORTC/MSG definitions. 10 patients with non-Cryptococcus infection and 30 healthy individuals were categorized as control group.ResultsThe lowest detection limit of NASBA was 10 CFU/mL, and RNA of non-target bacteria or fungi was not amplified. The sensitivity of NASBA, PCR and colloidal gold method was 92.00% (95% CI 72.50–98.60%), 64.00% (95% CI 42.62–81.29%), 100.00% (95% CI 83.42–100.00%), and the specificity was 95.00% (95% CI 81.79–99.13%), 80.00% (95% CI 63.86–90.39%) and 82.50% (95% CI 66.64–92.11%) respectively. The highest specificity (97.50%), accuracy (95.38%) and k value (0.90) were achieved when both NASBA and colloidal gold results were positive.ConclusionsNASBA is a new alternative detection method for cryptococcosis which is both accurate and rapid without expensive equipment and specialised personnel. It may be used as a tool for confirming current infection as well as monitoring the effectiveness of antifungal treatment. The use of NASBA to detect Cryptococcus RNA in blood samples is of great significance for the diagnosis of pulmonary cryptococcosis. The combination of NASBA and colloidal gold can improve the diagnostic accuracy of cryptococcosis.