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Under the new American Institute of Certified Public Accountants (AICPA)-National Association of State Boards of Accountancy (NASBA) Certified Public Accountant (CPA) Evolution Model Curriculum, students seeking to take the Tax Compliance and Planning (TCP) section of the uniform CPA exam will be evaluated heavily on Tax Compliance and Planning for Individuals and Personal Financial Planning. While some undergraduate and graduate finance curricula include personal financial planning, most accounting curricula do not. When modifying their accounting curricula to meet the new CPA exam specifications, universities must address how to cover and deliver new content areas. As many accounting instructors may have never focused on personal finance or financial planning, this may be a new and unfamiliar area for them. This paper aims to describe the proposed learning objectives of the course in Personal Financial Advisory Services (PFAS) recommended by the AICPA-NASBA CPA Evolution Model Curriculum. In addition, the paper provides current accounting instructors with the best pedagogy for delivering such courses based on prior research as well as provides accounting faculty with recommended resources to assist with curriculum development. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We herein describe rapid and accurate clinical testing for COVID-19 by nicking and extension chain reaction system-based amplification (NESBA), an ultrasensitive version of NASBA. The primers to identify SARS-CoV-2 viral RNA were designed to additionally contain the nicking recognition sequence at the 5'-end of conventional NASBA primers, which would enable nicking enzyme-aided exponential amplification of T7 RNA promoter-containing double-stranded DNA (T7DNA). As a consequence of this substantially enhanced amplification power, the NESBA technique was able to ultrasensitively detect SARS-CoV-2 genomic RNA (gRNA) down to 0.5 copies/μL (= 10 copies/reaction) for both envelope (E) and nucleocapsid (N) genes within 30 min under isothermal temperature (41 °C). When the NESBA was applied to test a large cohort of clinical samples (n = 98), the results fully agreed with those from qRT-PCR and showed the excellent accuracy by yielding 100% clinical sensitivity and specificity. By employing multiple molecular beacons with different fluorophore labels, the NESBA was further modulated to achieve multiplex molecular diagnostics, so that the E and N genes of SARS-CoV-2 gRNA were simultaneously assayed in one-pot. By offering the superior analytical performances over the current qRT-PCR, the isothermal NESBA technique could serve as very powerful platform technology to realize the point-of-care (POC) diagnosis for COVID-19.

Simple Summary: In this review, we examine the application of different molecular diagnostic techniques in the pig industry; qPCR, isothermal methods, and novel techniques such as CRISPR-Cas and microfluidics platforms are discussed in detail. The main viruses affecting the health of pigs were identified, including the African swine fever virus, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, and porcine circovirus. Furthermore, the main challenges for the implementation of large-scale molecular diagnostic tests in the swine industry are discussed, which include lower costs, ease of operation for any type of user, portability for use on farms, and rapid response times. Molecular diagnostic tests have evolved very rapidly in the field of human health, especially with the arrival of the recent pandemic caused by the SARS-CoV-2 virus. However, the animal sector is constantly neglected, even though accurate detection by molecular tools could represent economic advantages by preventing the spread of viruses. In this regard, the swine industry is of great interest. The main viruses that affect the swine industry are described in this review, including African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), and porcine circovirus (PCV), which have been effectively detected by different molecular tools in recent times. Here, we describe the rationale of molecular techniques such as multiplex PCR, isothermal methods (LAMP, NASBA, RPA, and PSR) and novel methods such as CRISPR-Cas and microfluidics platforms. Successful molecular diagnostic developments are presented by highlighting their most important findings. Finally, we describe the barriers that hinder the large-scale development of affordable, accessible, rapid, and easy-to-use molecular diagnostic tests. The evolution of diagnostic techniques is critical to prevent the spread of viruses and the development of viral reservoirs in the swine industry that impact the possible development of future pandemics and the world economy. [ABSTRACT FROM AUTHOR]

The inclusion of eight emerging topics on future Certified Public Accounting (CPA) exams challenges educators to prioritize these topics in the accounting curriculum. We analyze the emerging topics and discuss their importance and appropriate teaching locus in light of survey evidence collected from professionals. The results support the preeminence of data analytics above other emerging accounting topics and show the relative importance of other topics based on their statistically significant differences. The results are helpful as educators make decisions about where to teach particular topics as well as their relative emphasis. To augment the analysis, we also examine factors that influence professionals' judgement of the importance of data analytics to career success and find a positive relationship between years of experience and data analytics importance. [ABSTRACT FROM AUTHOR]

Many diagnostic tests are currently used to diagnose chronic toxoplasmosis, yet the biggest challenge remains the rapid diagnosis of active infection, either primary or secondary. The late appearance of antibodies or their inability to emerge in case of defective immunity represents obstacles with serological testing. Molecular diagnosis relied on the detection of DNA are available, however, detecting DNA does not mean the presence of viable infectious organisms. Therefore, the switch to RNA detection technologies is necessary. In this initial study, a trial was done for early detecting genomic materials of viable circulating Toxoplasma gondii in the blood of 24 infected mice at variable doses, ranging from 10/ml to 108/ml Toxoplasma gondii RH strain tachyzoites, applying Real-time Nucleic acid sequence-based amplification technique (NASBA) and primer sets targeting B1 rRNA gene. The standard curve of real-time NASBA was created using serially diluted specific RNA samples that all generated signals for absolute genomic quantitation and crossing points ranging between 11.07 and 31.32. Fluorescence signals were created from all samples isolated from the 24 infected mice with different infection doses with quantitative genomic equivalent ranging from 1.3 × 10 to 9.6 × 1010 with crossing points ranging between 12.1 and 37.43, while no signals were detected from all negative control samples. In conclusion, NASBA is a relatively rapid primer-dependent molecular technique that can be performed in a single tube at a constant temperature for continuous amplification of a huge amount of nucleic acid. The technique can be used in medical diagnostics as an alternative to PCR, being quicker and sensitive to detect viable Toxoplasma circulating parasites. [ABSTRACT FROM AUTHOR]

Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by the Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables the optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for the fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20-200 aM sensitivity. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.

Nucleic acid tests (NATs) are considered as gold standard in molecular diagnosis. To meet the demand for onsite, point-of-care, specific and sensitive, trace and genotype detection of pathogens and pathogenic variants, various types of NATs have been developed since the discovery of PCR. As alternatives to traditional NATs (e.g., PCR), isothermal nucleic acid amplification techniques (INAATs) such as LAMP, RPA, SDA, HDR, NASBA, and HCA were invented gradually. PCR and most of these techniques highly depend on efficient and optimal primer and probe design to deliver accurate and specific results. This chapter starts with a discussion of traditional NATs and INAATs in concert with the description of computational tools available to aid the process of primer/probe design for NATs and INAATs. Besides briefly covering nanoparticles-assisted NATs, a more comprehensive presentation is given on the role CRISPR-based technologies have played in molecular diagnosis. Here we provide examples of a few groundbreaking CRISPR assays that have been developed to counter epidemics and pandemics and outline CRISPR biology, highlighting the role of CRISPR guide RNA and its design in any successful CRISPR-based application. In this respect, we tabularize computational tools that are available to aid the design of guide RNAs in CRISPR-based applications. In the second part of our chapter, we discuss machine learning (ML)- and deep learning (DL)-based computational approaches that facilitate the design of efficient primer and probe for NATs/INAATs and guide RNAs for CRISPR-based applications. Given the role of microRNA (miRNAs) as potential future biomarkers of disease diagnosis, we have also discussed ML/DL-based computational approaches for miRNA-target predictions. Our chapter presents the evolution of nucleic acid-based diagnosis techniques from PCR and INAATs to more advanced CRISPR/Cas-based methodologies in concert with the evolution of deep learning (DL)- and machine learning (ml)-based computational tools in the most relevant application domains., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Molecular diagnostic tests have evolved very rapidly in the field of human health, especially with the arrival of the recent pandemic caused by the SARS-CoV-2 virus. However, the animal sector is constantly neglected, even though accurate detection by molecular tools could represent economic advantages by preventing the spread of viruses. In this regard, the swine industry is of great interest. The main viruses that affect the swine industry are described in this review, including African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), and porcine circovirus (PCV), which have been effectively detected by different molecular tools in recent times. Here, we describe the rationale of molecular techniques such as multiplex PCR, isothermal methods (LAMP, NASBA, RPA, and PSR) and novel methods such as CRISPR-Cas and microfluidics platforms. Successful molecular diagnostic developments are presented by highlighting their most important findings. Finally, we describe the barriers that hinder the large-scale development of affordable, accessible, rapid, and easy-to-use molecular diagnostic tests. The evolution of diagnostic techniques is critical to prevent the spread of viruses and the development of viral reservoirs in the swine industry that impact the possible development of future pandemics and the world economy.

Abstract Background Cryptococcosis 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. Methods We 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. Results The 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. Conclusions NASBA 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.

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. • Universal Electrochemical detection of HIV group M, subtype B, and HIV group O. • A nucleic acid sequence-based amplification protocol to amplified and tagged different HIV types. • A modular electrochemical biosensor that uses a universal set of probes coupled with an isothermal amplification procedure. [ABSTRACT FROM AUTHOR]

Introduction. Rotavirus A is the most common pathogen causing diarrhoea in children less than 5 years, leading to severe complications such as dehydration, electrolyte imbalances, acidosis, myocarditis, convulsions, pneumonia, and other life-threatening conditions. Gap statement. There is an urgent need for a rapid and efficient nucleic acid detection strategy to enable early diagnosis and treatment, preventing rotavirus transmission and associated complications. Aim. This article aimed to develop a nuclear acid sequence-based amplification (NASBA)-Cas12a system for detecting rotavirus A using fluorescence intensity or lateral flow strips. Methodology. The NASBA technology was combined with the clustered regularly interspaced short palindromic repeats-Cas12a system to establish a NASBA-Cas12a system for detecting rotavirus A. Results. The NASBA-Cas12a system could detect rotavirus A at 37 ℃ within 70 min and had no cross-reactivity with other viruses, achieving a limit of detection of 1.2 copies μl -1 . This system demonstrated a sensitivity of 100%, specificity of 90%, positive predictive value of 97.22% and negative predictive value of 100%. The kappa value was 0.933, indicating that the NASBA-Cas12a system was highly consistent with reverse transcription-PCR. Conclusion. The NASBA-Cas12a system exhibited high sensitivity and specificity for detecting rotavirus A, showing great potential for clinical application.

Background: Cryptococcosis 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.Methods: We 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.Results: The 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.Conclusions: NASBA 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. [ABSTRACT FROM AUTHOR]

Background: Fungal infections have increased during the last years due to the AIDS epidemic and immunosuppressive therapies. The available diagnostic methods, such as culture, histopathology and serology, have several drawbacks regarding sensitivity, specificity and time‐consuming, while molecular methods are still expensive and dependent on many devices. In order to overcome these challenges, isothermal nucleic acid amplification techniques (INAT) arose as promising diagnostic methods for infectious diseases. Objective: This review aimed to present and discuss the main contributions of the isothermal nucleic acid amplification techniques applied in medical mycology. Methods: Papers containing terms for each INAT (NASBA, RCA, LAMP, CPA, SDA, HAD or PSR) and the terms 'mycoses' or 'disease, fungal' were obtained from National Center for Biotechnology Information database until August 2019. Results: NASBA, RCA, LAMP and PSR are the INAT reported in the literature for detection and identification of pathogenic fungi. Despite the need of a previous conventional PCR, the RCA technique might also be used for genotyping or cryptic species differentiation, which may be important for the treatment of certain mycoses; nevertheless, LAMP is the most used INAT for pathogen detection. Conclusion: Among all INATs herein reviewed, LAMP seems to be the most appropriate method for fungal detection, since it is affordable, sensitive, specific, user‐friendly, rapid, robust, equipment‐free and deliverable to end‐users, fulfilling all ASSURED criteria of the World Health Organization for an ideal diagnostic method. [ABSTRACT FROM AUTHOR]

• This review article describes the amplification chemistries that are commonly used in the clinical virology laboratory. • The article includes different techniques for target, signal, and probe amplification chemistries. • Major features and limitations of each chemistry are described. • Commercial applications of these chemistries for viral pathogens that infect humans are included. Molecular diagnostic methods have evolved and matured considerably over the last several decades and are constantly being evaluated and adopted by clinical laboratories for the identification of infectious pathogens. Advancement in other technologies such as fluorescence, electronics, instrumentation, automation, and sensors have made the overall diagnostic process more accurate, sensitive, and rapid. Nucleic acid based detection procedures, which rely on the fundamental principles of DNA replication have emerged as a popular and standard diagnostic method, and several commercial assays are currently available based on different nucleic acid amplification techniques. This review focuses on the major amplification chemistries that are used for developing commercial assays and discusses their application in the clinical virology laboratory. [ABSTRACT FROM AUTHOR]

Highlights • A three-stage colorimetric test for the naked-eye detection of Zika virus. • Isothermal amplification combined with a label-free split deoxyribozyme probe. • Differentiation of Zika virus from closely related dengue and West Nile viruses. • Total test time of 2 h without expensive instrumentation. • Detects clinically relevant concentrations of viral RNA. Abstract A colorimetric nucleic acid based test for label-free pathogen detection has been developed and used for the detection of the Zika virus. The test relies on nucleic acid sequence-based amplification (NASBA) of a viral RNA followed by interrogation of the amplicon by a cascade of deoxyribozymes constituting a visual split deoxyribozyme (vsDz) probe. The probe consists of a split phosphodiesterase deoxyribozyme, which forms its catalytic core upon binding to a specific amplicon fragment. The catalytically active complex recognizes and cleaves an inhibited peroxidase-like deoxyribozyme (PDz), thereby activating it. Active PDz catalyzes hydrogen peroxide-mediated oxidation of a colorless substrate into a colored product, thereby generating a visible signal. Viral RNA (106 copies/mL or higher) triggers intense color within 2 h. The test selectively differentiates between Zika and closely related dengue and West Nile viruses. The reported technology combines isothermal amplification and visual detection and therefore represents a basis for the future development of a cost-efficient and instrument-free method for point-of-care nucleic acid analysis. [ABSTRACT FROM AUTHOR]

Dengue is a global health problem, caused by the dengue virus (DENV), which belongs to the Flaviviridae family of viruses. The transmission of DENV occurs through vectors, Ae. aegypti and Ae. Albopictus mosquitoes, to the human host, classifying it as a vector-borne disease. The disease incidence is increasing at an alarming rate and needs to be tackled to reduce the morbidity and mortality caused by the disease. Environmental and clinical surveillance, detection of the virus, and diagnostics are critical tools to address this issue. In this comprehensive review, we explore various diagnostic techniques and the associated challenges within the context of dengue. While we briefly touch upon dengue's epidemiology, serotypes, and pathogenesis, our primary emphasis remains on diagnostics. We delve into the intricacies of these diagnostic methods, considering both the challenges they entail and the potential they hold in terms of accuracy and accessibility. It's important to note that the review does not extensively cover clinical aspects or regional variations of the disease. [ABSTRACT FROM AUTHOR]

Jembrana disease is an infectious disease in Bali cattle cause by a member of lentivirus calledjembrana disease virus (JDV). It causes an acute and severe disease syndrome with short incubationperiod. As the disease has spread to several areas in Indonesia, a simple and rapid detection method isrequired. The objective of this study to apply rapid diagnostic method for JDVTabanan 1995 strain basedon Nucleic Acid Sequence Based Amplification (NASBA) methods targeting env-tm gene. The steps of thisresearch consisted of viral RNA isolation from organ and blood of cattle experimentaly infected withJDVTabanan 1995 strain . RNA amplification was conducted by NASBA using waterbath. The NASBAproducts were then separated on 2 % agarose gel. Using this technique JDV positive result was obtainedfrom organ samples such as spleen, liver, lung, prefemoralis lymph node, prescapularis lymph node andblood generating a RNA fragment of 207 bp. In this study, diagnosis method for env tm of JDV Tabanan1995 strain can be conducted by isothermal amplification NASBA.

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., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

AbstrakTujuan yang ingin dicapai dalam penulisan gagasan tertulis ini adalah menjelaskan kajianbiologi molekuler, imunologi dan aspek genetik pada infeksi HIV-1 serta memaparkandiagnosa yang efektif untuk mengetahui infeksi HIV-1 yang dapat diterapkan. Pengumpulandata dan informasi didapatkan melalui buku dan jurnal-jurnal ilmiah hasil penelitian. Datadan informasi yang diverifikasikan lebih lanjut terbatas pada bukti yang menunjukkanjenis-jenis diagnosa HIV-1 dan membuat jenis diagnosa yang lebih efektif. Setelah semuadata yang dibutuhkan terkumpul, dilakukan pengelolaan data dengan menyusun secarasistematis dan logis. Tes Double-detect Protein kemungkinan memiliki keefektifan lebihtinggi dari tes yang mendeteksi antigen p24 ataupun tes yang mendeteksi antibodi.Diagnosa dini pada infeksi HIV merupakan diagnosa yang dapat membantu pendeteksianHIV pada fase awal infeksi hingga sebelum masuknya fase serokonversi. Pada saat inilahtes Double-Detect Protein dapat dilakukan. Namun, perlu dilakukan tes NASBA sebagai followup test.AbstractThe objectives of this writing were to explain the topic of molecular biology,immunology, and the genetic aspect of HIV infection type I. And also to give out a moreeffective diagnose of HIV type I that can be applied. The data and information werecollected from various books and scientific journals resulted from research. Data andinformation was verified further limited to the evidence that shows the types of diagnosesof HIV-1 and created a more effective type of diagnoses. Once all the required datacollected, data management was done by arranging a systematic and logical manner. TheProtein Double-Detect test had the possibility of having a higher effectiveness compared to p24antigen test or antibody detection tests. Early diagnosis of HIV infection is a diagnosis thatcan help the detection of HIV in the early phase of infection prior to the entry phase ofseroconversion. At this time Double-Detect Proteins Test can be done. However, NASBA testsneeded as a follow-up test.

SUMMARY: A perennial challenge in the accounting profession is how to aggregate and share instances of practitioner misconduct among numerous relevant parties. At present, both the American Institute of Certified Public Accountants (AICPA) and National Association of State Boards of Accountancy (NASBA) offer solutions for centralized collection of misconduct, but both likely experience issues with incomplete reporting from key constituents. I propose a novel use of blockchain technology to address this issue, such that all key parties in the accounting profession leverage an accountancy blockchain to aggregate and share instances of practitioner misconduct across the country on a nearly real-time basis. Such a network creates an immutable record of misconduct and allows key constituents in the accounting profession to work together and share information as peers without the risk of one party taking control of the ledger. I close by discussing blockchain-specific roadblocks to realizing this proposed model. [ABSTRACT FROM AUTHOR]

This paper presents a novel portable sample filtration/concentration system, designed for use on samples of microorganisms with very low cell concentrations and large volumes, such as water-borne parasites, pathogens associated with faecal matter, or toxic phytoplankton. The example application used for demonstration was the in-field collection and concentration of microalgae from seawater samples. This type of organism is responsible for Harmful Algal Blooms (HABs), an example of which is commonly referred to as “red tides”, which are typically the result of rapid proliferation and high biomass accumulation of harmful microalgal species in the water column or at the sea surface. For instance, Karenia brevis red tides are the cause of aquatic organism mortality and persistent blooms may cause widespread die-offs of populations of other organisms including vertebrates. In order to respond to, and adequately manage HABs, monitoring of toxic microalgae is required and large-volume sample concentrators would be a useful tool for in situ monitoring of HABs. The filtering system presented in this work enables consistent sample collection and concentration from 1 L to 1 mL in five minutes, allowing for subsequent benchtop sample extraction and analysis using molecular methods such as NASBA and IC-NASBA. The microalga Tetraselmis suecica was successfully detected at concentrations ranging from 2 × 10 5  cells/L to 20 cells/L. Karenia brevis was also detected and quantified at concentrations between 10 cells/L and 10 6  cells/L. Further analysis showed that the filter system, which concentrates cells from very large volumes with consequently more reliable sampling, produced samples that were more consistent than the independent non-filtered samples (benchtop controls), with a logarithmic dependency on increasing cell numbers. This filtering system provides simple, rapid, and consistent sample collection and concentration for further analysis, and could be applied to a wide range of different samples and target organisms in situations lacking laboratories. [ABSTRACT FROM AUTHOR]

Malaria is the most threatening disease protozoal and a major health problem worldwide especially in developing countries. WHO recommended that for every case of suspected malaria, diagnostic test must be done to confirm the disease. A more advanced Diagnostic Techniques were developed to overcome the problem of conventional microscopy. These techniques are known as Molecular diagnostic techniques, and they detect specific sequence in DNA, RNA and proteins to provide clinical information for human pathogens including malaria parasites. There are several techniques involve in molecular diagnostics, some are however discussed in this review. They include Polymerase Chain Reaction (PCR), Loop-mediated isothermal amplification (LAMP), Flow Cytometric technique (FCM), Nucleic acid based sequence amplification (NASBA), and Luminex xMax technology. Among these techniques; LAMP technique is the best techniques that can be deployed in the field settings (clinical and rural settings) because of its simplicity, reliability, stability, detection method as well as point-of- care and confirmatory ability. On the other hand, PCR-based technique is more suitable for research purposes because it can be used to identify drug resistance, follow-up therapeutic response, and detect asymptomatic malaria carriers who may be targeted for treatment. Hence, molecular diagnostic techniques are most innovative science and technical implementations that can be used to diagnose malaria infection and to overcome the limitations.

Abstract Background and Objective: Culture, microscopic method is a gold standard method for identification of Lishmania parasite. The use of Molecular methods such as RT- PCR compared to microscopic methods has a higher sensitivity and specificity however, it is not widely used due to its expensive equipment and the time requested. The use of nucleic acid sequence based amplification (NASBA) method is highly valuable for diagnosis of live parasite because there is no need for to use Thermo cycler. We aimed to assess sensitivity and specificity of NASBA for molecular detection of cutaneous Leishmaniasis. Material and Methods: First, the RNA was extracted from 28 skin biopsies suspected cutaneous Leishmaniasis. Then, by means of specific primers designed for 18srRNA region, this region was amplified using NASBA isothemal amplification. To increase the sensitivity, the product was electroforesed in TBE (IX) buffer, using Syber Gold Flourecent probes. Using specific primers, RT- PCR was conducted on the samples too. Result: For diagnosis of Leishmania parasites, NASBA and RT-PCR had the sensitivity of 81% and 51%, respectively, and specificity of 100%. Conclusion: NASBA isothermal method with high sensitivity and specificity can be applied for identification of cutaneous leishmaniasis. Keywords: Cutaneous Leishmanisis, NASBA, 18S rRNA

Dengue is a rapidly spreading mosquito-borne viral disease. Early diagnosis is important for clinical screening, medical management, and disease surveillance. The objective of this study was to develop a colorimetric lateral flow biosensor (LFB) for the visual detection of dengue-1 RNA using dextrin-capped gold nanoparticle (AuNP) as label. The detection was based on nucleic acid sandwich-type hybridization among AuNP-labeled DNA reporter probe, dengue-1 target RNA, and dengue-1 specific DNA capture probe immobilized on the nitrocellulose membrane. Positive test generated a red test line on the LFB strip, which enabled visual detection. The optimized biosensor has a cut-off value of 0.01 µM using synthetic dengue-1 target. Proof-of-concept application of the biosensor detected dengue-1 virus in pooled human sera with a cut-off value of 1.2 × 104 pfu/mL. The extracted viral RNA, when coupled with nucleic acid sequence-based amplification (NASBA), was detected on the LFB in 20 min. This study first demonstrates the applicability of dextrin-capped AuNP as label for lateral flow assay. The biosensor being developed provides a promising diagnostic platform for early detection of dengue infection in high-risk resource-limited areas.

Now and again, the rapid proliferation of certain species of phytoplankton can give rise to Harmful Algal Blooms, which pose a serious threat to marine life and human health. Current methods of monitoring phytoplankton are limited by poor specificity or by the requirement to return samples to a highly resourced, centralised lab. The Lab Card is a small, microfluidic cassette which, when used in tandem with a portable Lab Card Reader can be used to sensitively and specifically quantify harmful algae in the field, from nucleic acid extracts using RNA amplification; a sensitive and specific method for the enumeration of potentially any species based on their unique genetic signatures. This study reports the culmination of work to develop a Lab Card-based genetic assay to quantify the harmful algae Karenia brevis using mRNA amplification by the Nucleic Acid Sequence Based Amplification (NASBA) method. K. brevis cells were quantified by amplification of the rbcL gene transcript in nucleic acid extracts of K. brevis cell samples. A novel enzyme dehydration and preservation method was combined with a pre-existing reagent Gelification method to prepare fully preserved Lab Cards with a shelf-life of at least six weeks prior to use. Using an internal control (IC), the Lab Card-based rbcL NASBA was demonstrated for the quantification of K. brevis from cell extracts containing between 50 and 5000 cells. This is the first demonstration of quantitation of K. brevis using IC-NASBA on a Lab Card. [ABSTRACT FROM AUTHOR]

[Display omitted] • The NASBA-LFD for detecting MrNV-chin was developed by amlification at 41 °C in a single tube within 90 min followed by hybridization. • The NASBA-LFD was specific for MrNV. • Sensitivity of NASBA-LFD was about 104 times higher in comparison to PCR-based detection. • The NASBA-LFD would be a simple, rapid and reliable method for detection of WTD. 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. [ABSTRACT FROM AUTHOR]

Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development.

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. [Display omitted] • SNP and viability of ARB in water were simultaneously detected by AlleRNA. • AlleRNA enables the detection of as low as 0.5 % SNP in mRNAs. • A LOD of 80 CFU of viable ARB was achieved by AlleRNA. • AlleRNA was successfully applied for the estimation of disinfectant performance. [ABSTRACT FROM AUTHOR]

A critical challenge for the successful application of antifungal therapies for invasive aspergillosis (IA) is a lack of reliable biomarkers to assess early treatment response. Patients with proven or probable IA were prospectively enrolled, and serial blood samples were collected at 8 specified time points during 12-week antifungal therapy. Total nucleic acid was extracted from 2.5 ml blood and tested for Aspergillus-specific RNA by a pan- Aspergillus real-time nucleic acid sequence-based amplification (NASBA) assay. Serum 1, 3-β-D-glucan (BG) and galactomannan (GM) were measured in parallel. Clinical outcome was evaluated at 6 and 12 weeks. Overall, 48/328 (14.6%) blood samples from 29/46 (63%) patients had positive NASBA detection at baseline and/or some point during the study. Positive NASBA results during the first 4 and 6 weeks of treatment are significantly associated with the 12-week outcome. Blood RNA load change during weeks 4-6 may be informative to predict outcome at 12 weeks. While independent of serum GM, the kinetic change of circulating Aspergillus RNA appears to be well correlated with that of BG on some patient individuals. Monitoring blood Aspergillus RNA during the first 4-6 weeks of antifungal treatment may help assess therapeutic response. Combination of circulating Aspergillus RNA and BG may be a useful adjunct to assess response. [ABSTRACT FROM AUTHOR]

The proof of concept of a nanoparticle displacement assay that enables the use of large diameter nanopores for the detection of targets of smaller molecular dimensions is presented. We hypothesized that an inherent signal amplification should arise from the selective displacement of nanoparticles preloaded in a nanopore by a much smaller molecular target. The method is demonstrated using peptide nucleic acid (PNA)-functionalized gold nanopore arrays in which short DNA-modified gold nanoparticles are anchored by weak interaction. Complementary microRNAs are detected via the resistance change caused by competitive displacement of nanoparticles from the PNA-functionalized nanopores. [ABSTRACT FROM AUTHOR]

Sample preparation has recently been an issue in the detection of food poisoning pathogens, particularly viruses such as norovirus (NoV), in food because of the complexity of foods and raw fresh materials. Here, we demonstrate a total analytical microfluidic chip module to automatically perform a series of essential processes (cell concentration, lysis (RNA extraction), nucleic acid amplification, and detection) for the fast but sensitive detection of norovirus in oysters. The murine NoV spiked oyster was stomached using a standard method. The supernatant was first loaded into a shape switchable sample preparation chamber consisting of charge switchable micro-beads. Murine NoV, which was adsorbed on microbeads by electrostatic physisorption, was lysed using bead beating. The extracted RNA was transferred to the detection chamber to be amplified using Nucleic Acid Sequence Based Amplification (NASBA). The optimal surface functionality, size, and number of microbeads were achieved for the virus concentration and the stable RNA extraction in the shape-switchable micro-channel. As a result, murine NoV in a single oyster was successfully detected within 4 h by the microfluidic chip developed here, and could be directly applied to the large volume environmental sample as well as the food sample. [ABSTRACT FROM AUTHOR]

In countries where malaria transmission has decreased substantially, thanks to the scale-up of control interventions, malaria elimination may be feasible. Nevertheless, this goal requires new strategies such as the active detection and treatment of infected individuals. As the detection threshold for the currently used diagnostic methods is 100 parasites/μL, most low-density, asymptomatic infections able to maintain transmission cannot be detected. Identifying them by molecular methods such as PCR is a possible option but the field deployment of these tests is problematic. Isothermal amplification of nucleic acids (at a constant temperature) offers the opportunity of addressing some of the challenges related to the field deployment of molecular diagnostic methods. One of the novel isothermal amplification methods for which a substantial amount of work has been done is the loop-mediated isothermal amplification (LAMP) assay. The present review describes LAMP and several other isothermal nucleic acid amplification methods, such as thermophilic helicase-dependent amplification, strand displacement amplification, recombinase polymerase amplification and nucleic acid sequence-based amplification, and explores their potential use as high-throughput, field-based molecular tests for malaria diagnosis. [ABSTRACT FROM PUBLISHER]

The incidence of foodborne diseases has increased over the years and resulted in major public health problem globally. Foodborne pathogens can be found in various foods and it is important to detect foodborne pathogens to provide safe food supply and to prevent foodborne diseases. The conventional methods used to detect foodborne pathogen are time consuming and laborious. Hence, a variety of methods have been developed for rapid detection of foodborne pathogens as it is required in many food analyses. Rapid detection methods can be categorized into nucleic acid-based, biosensor-based and immunological-based methods. This review emphasizes on the principles and application of recent rapid methods for the detection of foodborne bacterial pathogens. Detection methods included are simple polymerase chain reaction (PCR), multiplex PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP) and oligonucleotide DNA microarray which classified as nucleic acid-based methods; optical, electrochemical and mass-based biosensors which classified as biosensor-based methods; enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay which classified as immunological-based methods. In general, rapid detection methods are generally time-efficient, sensitive, specific and labor-saving. The developments of rapid detection methods are vital in prevention and treatment of foodborne diseases. [ABSTRACT FROM AUTHOR]