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 0.21 µM. 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 0.002 µM. 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.

Current Trends in RNA Virus Detection via Nucleic Acid Isothermal Amplification-Based Platforms.

Ribonucleic acid (RNA) viruses are one of the major classes of pathogens that cause human diseases. The conventional method to detect RNA viruses is real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), but it has some limitations. It is expensive and time-consuming, with infrastructure and trained personnel requirements. Its high throughput requires sophisticated automation and large-scale infrastructure. Isothermal amplification methods have been explored as an alternative to address these challenges. These methods are rapid, user-friendly, low-cost, can be performed in less specialized settings, and are highly accurate for detecting RNA viruses. Microfluidic technology provides an ideal platform for performing virus diagnostic tests, including sample preparation, immunoassays, and nucleic acid-based assays. Among these techniques, nucleic acid isothermal amplification methods have been widely integrated with microfluidic platforms for RNA virus detection owing to their simplicity, sensitivity, selectivity, and short analysis time. This review summarizes some common isothermal amplification methods for RNA viruses. It also describes commercialized devices and kits that use isothermal amplification techniques for SARS-CoV-2 detection. Furthermore, the most recent applications of isothermal amplification-based microfluidic platforms for RNA virus detection are discussed in this article.

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.

Prevalence of Plasmodium falciparum gametocytaemia in asymptomatic school children before and after treatment with dihydroartemisinin-piperaquine (DP).

Background: Asymptomatic Plasmodium carriers form the majority of malaria-infected individuals in most endemic areas. A proportion of these asymptomatically infected individuals carry gametocytes, the transmissible stages of malaria parasites, that sustain human to mosquito transmission. Few studies examine gametocytaemia in asymptomatic school children who may form an important reservoir for transmission. We assessed the prevalence of gametocytaemia before antimalarial treatment and monitored clearance of gametocytes after treatment in asymptomatic malaria children. Methods: A total of 274 primary school children were screened for P. falciparum parasitaemia by microscopy. One hundred and fifty-five (155) parasite positive children were treated under direct observation with dihydroartemisinin-piperaquine (DP). Gametocyte carriage was determined by microscopy seven days prior to treatment, day 0 before treatment, and on days 7, 14 and 21 post initiation of treatment. Results: The prevalence of microscopically-detectable gametocytes at screening (day -7) and enrolment (day 0) were 9% (25/274) and 13.6% (21/155) respectively. Following DP treatment, gametocyte carriage dropped to 4% (6/135), 3% (5/135) and 6% (10/151) on days 7, 14 and 21 respectively. Asexual parasites persisted in a minority of treated children, resulting in microscopically detectable parasites on days 7 (9%, 12/135), 14 (4%, 5/135) and 21 (7%, 10/151). Gametocyte carriage was inversely correlated with the age of the participants (p = 0.05) and asexual parasite density (p = 0.08). In a variate analysis, persistent gametocytaemia 7 or more days after treatment was significantly associated with post-treatment asexual parasitaemia at day 7 (P = 0.027) and presence of gametocytes on the day of treatment (P < 0.001). Conclusions: Though DP provides both excellent cure rates for clinical malaria and a long prophylactic half-life, our findings suggest that after treatment of asymptomatic infections, both asexual parasites and gametocytes may persist in a minority of individuals during the first 3 weeks after treatment. This indicates DP may be unsuitable for use in mass drug administration strategies towards malaria elimination in Africa.

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

Introduction: 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. Methods: 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. Results and discussion: 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.

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.

CRISPR-Cas13a cascade-based viral RNA assay for detecting SARS-CoV-2 and its mutations in clinical samples.

SARS-CoV-2 is one of the greatest threats to global human health. Point-of-care diagnostic tools for SARS-CoV-2 could facilitate rapid therapeutic intervention and mitigate transmission. In this work, we report CRISPR-Cas13a cascade-based viral RNA (Cas13C) assay for label-free and isothermal determination of SARS-CoV-2 and its mutations in clinical samples. Cas13a/crRNA was utilized to directly recognize the target of SARS-CoV-2 RNA, and the recognition events sequentially initiate the transcription amplification to produce light-up RNA aptamers for output fluorescence signal. The recognition of viral RNA via Cas13a-guide RNA ensures a high specificity to distinguish SARS-CoV-2 from MERS-CoV and SARS-CoV, as well as viral mutations. A post transcription amplification strategy was triggered after CRISPR-Cas13a recognition contributes to an amplification cascade that achieves high sensitivity for detecting SARS-CoV-2 RNA, with a limit of detection of 0.216 fM. In addition, the Cas13C assay could be able to discriminate single-nucleotide mutation, which was proven with N501Y in SARS-Cov-2 variant. This method was validated by a 100% agreement with RT-qPCR results from 12 clinical throat swab specimens. The Cas13C assay has the potential to be used as a routine nucleic acid test of SARS-CoV-2 virus in resource-limited regions.

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.

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens.

Rapid, sensitive, and timely diagnostics are essential for protecting plants from pathogens. Commonly, PCR techniques are used in laboratories for highly sensitive detection of DNA/RNA from viral, viroid, bacterial, and fungal pathogens of plants. However, using PCR-based methods for in-field diagnostics is a challenge and sometimes nearly impossible. With the advent of isothermal amplification methods, which provide amplification of nucleic acids at a certain temperature and do not require thermocyclic equipment, going beyond the laboratory has become a reality for molecular diagnostics. The amplification stage ceases to be limited by time and instruments. Challenges to solve involve finding suitable approaches for rapid and user-friendly plant preparation and detection of amplicons after amplification. Here, we summarize approaches for in-field diagnostics of phytopathogens based on different types of isothermal amplification and discuss their advantages and disadvantages. In this review, we consider a combination of isothermal amplification methods with extraction and detection methods compatible with in-field phytodiagnostics. Molecular diagnostics in out-of-lab conditions are of particular importance for protecting against viral, bacterial, and fungal phytopathogens in order to quickly prevent and control the spread of disease. We believe that the development of rapid, sensitive, and equipment-free nucleic acid detection methods is the future of phytodiagnostics, and its benefits are already visible.

A highly sensitive and rapid enzymatic method using a biochemical automated analyzer to detect inorganic pyrophosphate generated by nucleic acid sequence-based amplification.

BACKGROUND AND AIMS: Polymerase chain reaction-based techniques require expensive equipment for fluorescence detection of the products. However, the measurement of inorganic pyrophosphate (PPi) released during DNA synthesis can be used to quantify target genes without such equipment. Here, we devised a high-sensitivity enzymatic assay for detection of PPi. MATERIALS AND METHODS: In our assay method, PPi was converted to hypoxanthine by hypoxanthine phosphoribosyl transferase. Xanthine dehydrogenase converted the hypoxanthine to uric acid and yielded two molecules of NADH, which in turn reduced Fe3+ to Fe2+ (mediated by 1-methoxy-5-ethylphenazinium ethylsulfate). 2-Nitroso-5-(N-propyl-N-sulfopropylamino) phenol chelated the Fe2+, which resulted in an intensely colored product that could be measured using a biochemical automated analyzer. RESULTS: The assay was able to detect PPi within 10 min. It was linear between 0 and 10 µmol/L PPi, and intra-run and inter-run coefficients of variation were 1%-2%. Other validation tests with a biochemical automated analyzer were satisfactory. The assay could potentially be used to directly quantify samples after isothermal nucleic acid sequence-based amplification of a target gene. CONCLUSION: The method developed here for detection of PPi can be used to measure nucleic acid biomarkers in biological samples in clinical practice using a high-throughput biochemical automated analyzer.

Cascade of deoxyribozymes for the colorimetric analysis of drug resistance in Mycobacterium tuberculosis.

A visual cascade detection system has been applied to the detection and analysis of drug-resistance profile of Mycobacterium tuberculosis complex (MTC), a causative agent of tuberculosis. The cascade system utilizes highly selective split RNA-cleaving deoxyribozyme (sDz) sensors. When activated by a complementary nucleic acid, sDz releases the peroxidase-like deoxyribozyme apoenzyme, which, in complex with a hemin cofactor, catalyzes the color change of the sample's solution. The excellent selectivity of the cascade has allowed for the detection of point mutations in the sequences of the MTC rpoB, katG, and gyrA genes, which are responsible for resistance to rifampin, isoniazid, and fluoroquinolone, respectively. When combined with isothermal nucleic acid sequence based amplification (NASBA), the assay was able to detect amplicons of 16S rRNA and katG mRNA generated from 0.1 pg and 10 pg total RNA taken for NASBA, respectively, in less than 2 h, producing a signal detectable with the naked eye. The proposed assay may become a prototype for point-of-care diagnosis of drug resistant bacteria with visual signal output.

[Application of NASBA and RPA in detection of pathogenic bacteria].

Nucleic acid sequence-based amplification and recombinase polymerase amplification are the recently developed thermostatic amplification techniques based on PCR. This paper briefly summarizes the principle of reaction, design principle of primer and probe, advantage of these two techniques (simple, accurate, highly sensitive and rapid) and introduces the application of the techniques in the detection of pathogenic bacteria.

Evaluation of a novel micro/nanofluidic chip platform for the detection of influenza A and B virus in patients with influenza-like illness.

We introduced a novel micro/nanofluidic chip platform (MNCP), which is based on an isothermal nucleic acid amplification method. This study aimed to evaluate the MNCP method for influenza A and B viruses detecting and subtyping using throat swab samples from patients with influenza-like illness (ILI). A total of 266 throat swab samples from 266 non-repeated patients with ILI were tested for influenza A and B viruses using three methods, MNCP, a rapid influenza diagnostic test (RIDT), and real-time reverse transcription polymerase chain reaction (rRT-PCR). The results of MNCP were compared to those obtained by rRT-PCR and RIDT and the performance of MNCP was further evaluated. Compared with rRT-PCR results, the rates of sensitivity, specificity, overall concordance, and the kappa value of MNCP were 98.89%, 96.97%, 97.65%, and 0.95 for influenza A virus; 94.95%, 99.38%, 97.68%, and 0.95 for influenza B virus, respectively. Subtypes of influenza A viruses, e.g., A(H1N1)pdm09, A(H3N2), and A(not subtyped), and influenza B viruses could be distinguished in one MNCP assay within 1 h. Compared with rRT-PCR and MNCP, RIDT showed poor clinical sensitivity for influenza virus detection. This study showed MNCP is rapid, sensitive and versatile detecting system with potential for clinical application in pathogen diagnosis for patients with ILI.

Development of a Quantitative Real-time Nucleic Acid Sequence based Amplification (NASBA) Assay for Early Detection of Apple scar skin viroid.

An assay for detecting Apple scar skin viroid (ASSVd) was developed based on nucleic acid sequence based amplification (NASBA) in combination with real-time detection during the amplification process using molecular beacon. The ASSVd specific primers for amplification of the viroid RNA and molecular beacon for detecting the viroid were designed based on highly conserved regions of several ASSVd sequences including Korean isolate. The assay had a detection range of 1 × 104 to 1 × 1012 ASSVd RNA copies/µl with reproducibility and precision. Following the construction of standard curves based on time to positive (TTP) value for the serial dilutions ranging from 1 × 107 to 1 × 1012 copies of the recombinant plasmid, a standard regression line was constructed by plotting the TTP values versus the logarithm of the starting ASSVd RNA copy number of 10-fold dilutions each. Compared to the established RT-PCR methods, our method was more sensitive for detecting ASSVd. The real-time quantitative NASBA method will be fast, sensitive, and reliable for routine diagnosis and selection of viroid-free stock materials. Furthermore, real-time quantitative NASBA may be especially useful for detecting low levels in apple trees with early viroid-infection stage and for monitoring the influence on tree growth.

Nucleic acid amplification: Alternative methods of polymerase chain reaction.

Nucleic acid amplification is a valuable molecular tool not only in basic research but also in application oriented fields, such as clinical medicine development, infectious diseases diagnosis, gene cloning and industrial quality control. A comperehensive review of the literature on the principles, applications, challenges and prospects of different alternative methods of polymerase chain reaction (PCR) was performed. PCR was the first nucleic acid amplification method. With the advancement of research, a no of alternative nucleic acid amplification methods has been developed such as loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification. Most of the alternative methods are isothermal obviating the need for thermal cyclers. Though principles of most of the alternate methods are relatively complex than that of PCR, they offer better applicability and sensitivity in cases where PCR has limitations. Most of the alternate methods still have to prove themselves through extensive validation studies and are not available in commercial form; they pose the potentiality to be used as replacements of PCR. Continuous research is going on in different parts of the world to make these methods viable technically and economically.

Real-time nucleic acid sequence-based amplification to predict the clinical outcome of invasive aspergillosis.

Monitoring the response to therapy for invasive aspergillosis (IA) is essential for the management of patients with hematologic diseases. We evaluated the correlation between the outcome of real-time nucleic acid sequence-based amplification (RTi-NASBA) for Aspergillus 18S rRNA and the clinical outcome of IA. A total of 157 serum samples from 29 patients with IA were tested for RTi-NASBA. The treatment response and mortality were compared with the NASBA outcome (whether the NASBA value was converted to negative or not) at 12 weeks after the start of antifungal therapy. At 12 weeks, there was a moderate correlation between the treatment failure and persistently positive NASBA (κ = 0.482; P = 0.019). Deaths attributable to IA were more prevalent in patients without negative conversion of NASBA than in those with negative conversion (50% vs 5%; P = 0.013). Significant factors of treatment failure at 12 weeks were the status of hematologic disease (nonremission; P = 0.041) and the NASBA outcome (failure of negative conversion; P = 0.024). Survival was significantly better in patients with negative conversion of NASBA than those with persistently positive values (P = 0.036). This study suggests that the serial monitoring of RTi-NASBA could be useful for prediction of the clinical outcome in hematologic patients with IA.