Helicase-dependent Amplification Research Articles

Construction of a glycosylation-mediated fluorescent biosensor for label-free measurement of site-specific 5-hydroxymethylcytosine in cancer cells with zero background signal

Background5-hydroxymethylcytosine (5hmC) as an epigenetic modification can regulate gene expression, and its abnormal level is related with various tumor invasiveness and poor prognosis. Nevertheless, the current methods for 5hmC assay usually involve expensive instruments/antibodies, radioactive risk, high background, laborious bisulfite treatment procedures, and non-specific/long amplification time. ResultsWe develop a glycosylation-mediated fluorescent biosensor based on helicase-dependent amplification (HDA) for label-free detection of site-specific 5hmC in cancer cells with zero background signal. The glycosylated 5hmC-DNA (5ghmC) catalyzed by β-glucosyltransferase (β-GT) can be cleaved by AbaSI restriction endonuclease to generate two dsDNA fragments with sticky ends. The resultant dsDNA fragments are complementary to the biotinylated probes and ligated by DNA ligases, followed by being captured by magnetic beads. After magnetic separation, the eluted ligation products act as the templates to initiate HDA reaction, generating abundant double-stranded DNA (dsDNA) products within 20 min. The dsDNA products are measured in a label-free manner with SYBR Green I as an indicator. This biosensor can measure 5hmC with a detection limit of 2.75 fM and a wide linear range from 1 × 10−14 to 1 × 10−8 M, and it can discriminate as low as 0.001% 5hmC level in complex mixture. Moreover, this biosensor can measure site-specific 5hmC in cancer cells, and distinguish tumor cells from normal cells. SignificanceThis biosensor can achieve a zero-background signal without the need of either 5hmC specific antibody or bisulfite treatment, and it holds potential applications in biological research and disease diagnosis.

Detection of monkeypox virus using helicase dependent amplification and recombinase polymerase amplification combined with lateral flow test

The monkeypox virus (MPXV) is a zoonotic DNA virus that belongs to the poxvirus family. Conventional laboratory methods for detecting MPXV are complex and expensive, making them unsuitable for detecting the virus in regions with limited resources. In this study, we using the Helicase dependent amplification (HDA) method and the Recombinase polymerase amplification (RPA) technique in combination with the lateral flow test (LFT), together with a self-designed qPCR technique for the detection of the MPXV specific conserved fragment F3L, to compare the sensitivity and specificity of the three assays. By analyzing the sensitivity detection results using Probit, it can be seen that the limit of detection (LOD) of the HDA-LFT detection target is 9.86 copies/µL (95% confidence interval, CI 7.52 copies/µL lower bound), the RPA-LFT detection target is 6.97 copies/µL (95% CI 3.90 copies/µL lower bound), and the qPCR detection target is 479.24 copies/mL (95% CI 273.81 copies/mL lower bound). The specificity test results showed that the specificity of the three methods mentioned above was higher than 90% in detecting pseudoviruses of the same genus of MPXV. The simple, highly sensitive, and specific MPXV assay developed in this study is anticipated to provide a solid foundation for future applications in the early screening, diagnosis, and evaluation of the efficacy of MPXV. This is the first time the HDA-LFT assay has been utilized to detect MPXV infection.

Detection of Extended-spectrum β-lactamase-producing Escherichia coli isolates by isothermal amplification and association of their virulence genes and phylogroups with extraintestinal infection

Extraintestinal pathogenic Escherichia coli (ExPEC) producing extended-spectrum β-lactamases (ESBL) cause serious human infections due to their virulence and multidrug resistance (MDR) profiles. We characterized 144 ExPEC strains (collected from a tertiary cancer institute) in terms of antimicrobial susceptibility spectrum, ESBL variants, virulence factors (VF) patterns, and Clermont’s phylogroup classification. The developed multiplex recombinase polymerase amplification and thermophilic helicase-dependent amplification (tHDA) assays for blaCTX-M, blaOXA, blaSHV, and blaTEM detection, respectively, were validated using PCR-sequencing results. All ESBL-ExPEC isolates carried blaCTX-M genes with following prevalence frequency of variants: blaCTX-M-15 (50.5%) > blaCTX-M-55 (17.9%) > blaCTX-M-27 (16.8%) > blaCTX-M-14 (14.7%). The multiplex recombinase polymerase amplification assay had 100% sensitivity, and specificity for blaCTX-M, blaOXA, blaSHV, while tHDA had 86.89% sensitivity, and 100% specificity for blaTEM. The VF genes showed the following prevalence frequency: traT (67.4%) > ompT (52.6%) > iutA (50.5%) > fimH (47.4%) > iha (33.7%) > hlyA (26.3%) > papC (12.6%) > cvaC (3.2%), in ESBL-ExPEC isolates which belonged to phylogroups A (28.4%), B2 (28.4%), and F (22.1%). The distribution of traT, ompT, and hlyA and phylogroup B2 were significantly different (P < 0.05) between ESBL-ExPEC and non-ESBL-ExPEC isolates. Thus, these equipment-free isothermal resistance gene amplification assays contribute to effective treatment and control of virulent ExPEC, especially antimicrobial resistance strains.

Implementation of helicase-dependent amplification with SYBR Green I for prompt naked-eye detection of bacterial contaminants in platelet products

Platelet transfusions may lead to more significant risks of infection and septic transfusion reactions that can be fatal to the recipient. Platelet products should be screened to limit or detect bacterial contamination before application to patients to minimise any adverse reactions. This study aimed to develop a helicase-dependent amplification (HDA) technique targeting a universal highly conserved bacterial gene, 16S rRNA, in combination with naked-eye detection using SYBR Green I (HDA/SYBR Green I) to detect bacterial contamination in platelet products. Thirty positive samples were obtained from spiked platelet products by five transfusion-relevant bacterial strains and were screened for bacterial contamination by HDA/SYBR Green I. HDA/SYBR Green I showed an enhanced yield of bacterial contaminant detection when performed with medium to late shelf life, Day 2 of storage or later platelet products (98.67% sensitivity and 100% specificity compared to the BACT/ALERT culture system). The limit of detection of HDA/SYBR Green I was 1 ng, and there was no cross-reaction with other organisms that could likely contaminate platelet products. The developed HDA/SYBR Green I assay is rapid and simplistic and only requires an easy-to-find heat box, available in general blood bank laboratories, for the amplification step. This technique is suitable for further development as an alternative method to detect bacterial contamination in platelet products in the near future.

Thermophilic helicase-dependent amplification-based CRISPR/Cas12a system: Detection of stx2 in Escherichia coli O157:H7 by controlling primer dimers

Backgrounds: To overcome the limitation of polymerase chain reaction (PCR), isothermal amplification methods such as thermophilic helicase-dependent amplification (tHDA) have been developed. However, formation of primer dimer due to the single amplification temperature are major problems of tHDA. When cross-dimerization of forward and reverse primer occurred, false-positive results can be found on the lateral flow assay (LFA) which is one of the major detection methods widely used as a point of care diagnosis. Therefore, specific method of detecting only the target amplicon is required. ResultsIn this study, a tHDA-based CRISPR/Cas12a system was developed to detect low levels of Escherichia coli O157:H7 in fresh salad mix without the false-positive results produced by primer dimers. For the comparison of the effect in eliminating false-positive results by CRISPR/Cas12a system, LFA was also evaluated. The tHDA-based CRISPR/Cas12a system detected as low as 101 CFU/mL E. coli O157:H7 in bacterial pure culture. In LFA false-positive results were produced due to the primer dimer, whereas the primer dimer produced by tHDA was not detected in the CRISPR/Cas12a system. These results indicated that the CRISPR/Cas12a system eliminated the formation of primer dimer. In fresh salad mix, the tHDA-based CRISPR/Cas12a system combined with the filter concentration method detected 103 CFU/g E. coli O157:H7. ConclusionThis study was the first to amplify stx2 of E. coli O157:H7 with tHDA as an isothermal amplification method and detected the amplicon without false-positive results by combining tHDA with CRISPR/Cas12a. Therefore, this study showed great potential for detecting low levels of E. coli O157:H7 present in fresh salad mix.

Detection of SARS-CoV-2 Using Reverse Transcription Helicase Dependent Amplification and Reverse Transcription Loop-Mediated Amplification Combined with Lateral Flow Assay

Rapid and accurate detection and identification of pathogens in clinical samples is essential for all infection diseases. However, in the case of epidemics, it plays a key role not only in the implementation of effective therapy but also in limiting the spread of the epidemic. In this study, we present the application of two nucleic acid isothermal amplification methods—reverse transcription helicase dependent amplification (RT-HDA) and reverse transcription loop-mediated amplification (RT-LAMP)—combined with lateral flow assay as the tools for the rapid detection of SARS-CoV-2, the etiological agent of COVID-19, which caused the ongoing global pandemic. In order to optimize the RT-had, the LOD was 3 genome copies per reaction for amplification conducted for 10–20 min, whereas for RT-LAMP, the LOD was 30–300 genome copies per reaction for a reaction conducted for 40 min. No false-positive results were detected for RT-HDA conducted for 10 to 90 min, but false-positive results occurred when RT-LAMP was conducted for longer than 40 min. We concluded that RT-HDA combined with LFA is more sensitive than RT-LAMP, and it is a good alternative for the development of point-of-care tests for SARS-CoV-2 detection as this method is simple, inexpensive, practical, and does not require qualified personnel to perform the test and interpret its results.

Colorimetric detection of norovirus by helicase-dependent amplification method based on specific primers integrated with HRPzyme.

Noroviruses (NoVs) are the most common causes of epidemic gastroenteritis, responsible for at least 50% of all gastroenteritis outbreaks worldwide and significant causes of foodborne illness. In the USA, approximately 21 million illnesses attributable to NoVs have annually occurred. Therefore, there is a great demand to develop a rapid, low-cost, and accurate detection method for NoVs. This study first reported colorimetric helicase-dependent amplification (HDA) methods based on specific primers integrated with HRPzyme for the rapid and sensitive detection of NoV GI and GII. The colorimetric HDA methods exhibited a detection limit of 10 copies mL-1 of each NoV GI and GII and were confirmed to be specific to each NoV GI and GII. The period required to complete the HDA method was 2h, including a step of RNA extraction and cDNA synthesis without expensive instruments such as a thermal cycler and detector. The cutoff value of the method for the oyster artificially inoculated with a known amount of NoV was all 102 copies g-1 for NoV GI and GII. Therefore, the HDA method developed in this study can be useful tool for the on-site detection of NoVs in food samples.

An isothermal amplification-coupled dipstick for the rapid detection of COVID-19.

Early detection of coronavirus disease 2019 (COVID-19) is critical for both initiating appropriate treatment and preventing the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent. A simple and rapid diagnostic test that can be performed without any expensive equipment would be valuable for clinicians working in a low-resource setting. Here, we report a point-of-care detection technique for COVID-19 that combines the power of isothermal amplification (reverse transcription helicase-dependent amplification, RT-HDA) and dipstick technologies. The limit of detection of this diagnostic test is six copies of SARS-CoV-2 µl-1 in clinical specimens. Of the 22 clinical specimens tested, RT-HDA-coupled dipstick correctly identified all positive and negative specimens. The RT-HDA can be performed over a heating block and the results can be interpreted visually with the dipstick technology without any specialized equipment. Furthermore, the RT-HDA-coupled dipstick could be performed in a short turnaround time of ~2 h. Thus, the RT-HDA-coupled dipstick could serve as a point-of-care diagnostic test for COVID-19 in a low-resource environment.

Comparative analysis of methods for isothermal amplification of nucleic acids

In this review, methods for isothermal amplification of nucleic acids are considered and analyzed, in particular, loop isothermal amplification of DNA and RNA (LAMP/RT-LAMP), helicase-dependent amplification (HDA) and recombinase polymerase amplification (RPA). The advantages and disadvantages of each of the techniques are described. The possibility of their application in the molecular diagnostics of infectious diseases is evaluated. A brief review of the literature on the use of LAMP, HDA, RPA in the diagnostics of viral, bacterial infections and diseases of protozoal etiology was conducted. It has been shown that the LAMP method has a number of advantages over other diagnostic methods: high efficiency, specificity, simplicity, turnaround time and minimum requirements for instrument equipment. As a result, it is concluded that loop isothermal amplification is a promising method for detecting the DNA/RNA of various pathogens. The data on the introduction of the LAMP method in the diagnostics of particularly dangerous bacterial and viral infections, including for the detection of RNA of a new coronavirus infection (SARS-CoV-2) in clinical samples, are presented.

Tunable Duplex Semiquantitative Detection of Nucleic Acids with a Visual Lateral Flow Immunoassay Readout.

Quantitative nucleic acid amplification testing (NAAT) is a key enabling technology for infectious disease management, especially in instances where viral load informs therapeutic decisions. Inadequate access to quantitative NAATs remains a challenge to the successful deployment of antiretroviral therapy (ART) regimens for patients with chronic hepatitis B virus (CHB) in low resourced settings (LRS). Current field-deployable NAATs are generally qualitative (yes/no) rather than quantitative in nature, making them ill-suited for viral load monitoring programs for CHB patients. Here, we report the development of a proof-of-concept molecular diagnostic test, the semiquantitative ligation and amplification (SQLA) assay, which achieves semiquantitative detection of input target DNA at two independently tunable detection thresholds with a simple visual readout. The SQLA assay utilizes a duplex competitive thermophilic helicase-dependent amplification (tHDA) chemistry and can be performed in under 1 h.

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.

Strategies for Engineering Affordable Technologies for Point-of-Care Diagnostics of Infectious Diseases.

Disease prevalence is highest in low-resource settings (LRS) due to the lack of funds, infrastructure, and personnel required to carry out laboratory-based molecular tests. In high-resource settings, gold-standard molecular tests for diseases consist of nucleic acid amplification tests (NAATs) due to their excellent sensitivity and specificity. These tests require the extraction, amplification, and detection of nucleic acids from clinical samples. In high-resource settings, all three of these steps require highly specialized, costly, and onerous equipment that cannot be used in LRS. Nucleic acid extraction involves multiple centrifugation steps. Amplification consists of the polymerase chain reaction (PCR), which requires thermal cyclers. The detection of amplified DNA is typically done with specialized thermal cyclers that are capable of fluorescence detection. Traditional methods used to extract, amplify, and detect nucleic acids cannot be used outside of a laboratory in LRS. Thus, there is a need for affordable point-of-care devices to ease the high burden of disease in LRS.The past decade of work on paper-based fluidic devices has resulted in the invention of many paper-based biosensors for disease detection as well as isothermal amplification techniques that replace PCR. However, a challenge still remains in detecting pathogenic biomarkers from complex human samples without specialized laboratory equipment. Our research has focused on the development of affordable technologies to extract and detect nucleic acids in clinical samples with minimal equipment. Here we describe methods for the paper-based extraction, amplification, and detection of nucleic acids. This Account provides an overview of our latest technologies developed to detect an array of diseases in low-resource settings. We focus on detecting nucleic acids of H1N1, human papillomavirus (HPV), Neisseria gonorrheae (NG), Chlamydia trachomatis (CT), Trichomonas vaginalis (TV), and malaria from a variety of clinical sample types. H1N1 RNA was extracted from nasopharyngeal swabs; HPV, NG, and CT DNA were extracted from either cervical, urethral, or vaginal swabs; TV DNA was extracted from urine; and malaria DNA was extracted from whole blood. Different sample types necessitate different nucleic extraction protocols; we provide guidelines for assay design based on the clinical sample type used. We compare the pros and cons of different isothermal amplification techniques, namely, helicase-dependent amplification (HDA), loop-mediated isothermal amplification (LAMP), and a novel isothermal amplification technique that we developed: isothermal-identical multirepeat sequences (iso-IMRS). Finally, we compare various detection mechanisms, including lateral-flow and electrochemical readouts. Electrochemical readouts frequently employ gold electrodes due to strong gold-thiol coupling. However, the high cost of gold precludes their use in LRS. We discuss our development of novel gold leaf electrodes that can be made without specialized equipment for a fraction of the cost of commercially available gold electrodes.

Detection and disinfection of COVID-19 virus in wastewater.

The coronavirus disease 2019, COVID-19, caused by the severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, appears as a major pandemic having adverse impact on public health and economic activities. Since viral replication in human enterocytes results in its faecal shedding, wastewater surveillance is an ideal, non-invasive, cost-effective and an early warning epidemiological approach to detect the genetic material of SARS-CoV-2. Here, we review techniques for the detection of SARS-CoV-2 in municipal wastewater, and disinfectants used to control viral spread. For detection, concentration of ribonucleic acid involves ultrafiltration, ultracentrifugation and polyethylene glycol precipitation. Identification is done by reverse transcriptase amplification, nucleic acid sequence-based amplification, helicase dependent amplification, loop-mediated isothermal amplification, recombinase polymerase amplification, high throughput screening and biosensor assays. Disinfectants include ultraviolet radiations, ozone, chlorine dioxide, hypochlorites and hydrogen peroxide. Wastewater surveillance data indicates viral presence within longer detection window, and provides transmission dynamics earlier than classical methods. This is particularly relevant for pre-symptomatic and asymptomatic COVID-19 cases.

Biosensors Based on Isothermal DNA Amplification for Bacterial Detection in Food Safety and Environmental Monitoring.

The easy and rapid spread of bacterial contamination and the risk it poses to human health makes evident the need for analytical methods alternative to conventional time-consuming laboratory-based techniques for bacterial detection. To tackle this demand, biosensors based on isothermal DNA amplification methods have emerged, which avoid the need for thermal cycling, thus facilitating their integration into small and low-cost devices for in situ monitoring. This review focuses on the breakthroughs made on biosensors based on isothermal amplification methods for the detection of bacteria in the field of food safety and environmental monitoring. Optical and electrochemical biosensors based on loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), helicase dependent amplification (HDA), strand displacement amplification (SDA), and isothermal strand displacement polymerisation (ISDPR) are described, and an overview of their current advantages and limitations is provided. Although further efforts are required to harness the potential of these emerging analytical techniques, the coalescence of the different isothermal amplification techniques with the wide variety of biosensing detection strategies provides multiple possibilities for the efficient detection of bacteria far beyond the laboratory bench.

LAMP-based foldable microdevice platform for the rapid detection of Magnaporthe oryzae and Sarocladium oryzae in rice seed

Rice blast (caused by Magnaporthe oryzae) and sheath rot diseases (caused by Sarocladium oryzae) are the most predominant seed-borne pathogens of rice. The detection of both pathogens in rice seed is essential to avoid production losses. In the present study, a microdevice platform was designed, which works on the principles of loop-mediated isothermal amplification (LAMP) to detect M. oryzae and S. oryzae in rice seeds. Initially, a LAMP, polymerase chain reaction (PCR), quantitative PCR (qPCR), and helicase dependent amplification (HDA) assays were developed with primers, specifically targeting M. oryzae and S. oryzae genome. The LAMP assay was highly efficient and could detect the presence of M. oryzae and S. oryzae genome at a concentration down to 100 fg within 20 min at 60 °C. Further, the sensitivity of the LAMP, HDA, PCR, and qPCR assays were compared wherein; the LAMP assay was highly sensitive up to 100 fg of template DNA. Using the optimized LAMP assay conditions, a portable foldable microdevice platform was developed to detect M. oryzae and S. oryzae in rice seeds. The foldable microdevice assay was similar to that of conventional LAMP assay with respect to its sensitivity (up to 100 fg), rapidity (30 min), and specificity. This platform could serve as a prototype for developing on-field diagnostic kits to be used at the point of care centers for the rapid diagnosis of M. oryzae and S. oryzae in rice seeds. This is the first study to report a LAMP-based foldable microdevice platform to detect any plant pathogens.

An isothermal amplification-based point-of-care diagnostic platform for the detection of Mycobacterium tuberculosis: A proof-of-concept study.

The timely diagnosis of active tuberculosis disease (TB) is crucial to interrupt the transmission and combat the spread of Mycobacterium tuberculosis (Mtb), the causative agent for TB. Here, we demonstrate the development of a specimen-direct rapid diagnostic method for TB which consists of an isothermal amplification device, Tiny Isothermal Nucleic acid quantification sYstem (TINY), coupled with helicase-dependent amplification (HDA). HDA, an isothermal amplification technique is established over TINY using pUCIDT-AMP vector carrying IS6110, the target DNA sequence for Mtb. The limit of detection of this technique for detecting the IS6110 within a threshold time of 50 min is 2.5 × 105 copies of IS6110. HDA in TINY for TB detection was evaluated using three IS6110-positive Mtb strains – H37Rv, CDC 1551, and Erdman wild-type and one IS6110-negative Mycobacterium avium. For spiked oral swabs, HDA in TINY detects IS6110 without any non-specificity in relatively short turnaround time (<1.5 h), highlighting its potential utility as a specimen-direct point-of-care diagnostic for TB. TINY does not require an uninterrupted power supply and its lightweight and small footprint offers portability and easier operation in clinical settings with poor infrastructure. Overall, HDA in TINY could serve as an efficient rapid, and portable platform for the qualitative detection of TB at the point-of-care.

A visualized nucleic acid test of 2019-nCoV based on helicase isothermal amplification

Objective: To develop a novel method for detection of genomic RNA of 2019-nCoV with reverse-transcription thermophilic helicase-dependent amplification (tHDA) and lateral flow dipsticks. Methods: This study included 143 PCR-negative nucleic acid specimens and 20 PCR- positive nucleic acid specimens collected from patients from January to April 2020 at Institute of Clinical Laboratory, Jinling Hospital. 5 pairs of primers were designed for conserved sequence of both gene N and E of 2019-nCoV, and the amplicons were analyzed by gel electrophoresis to screen out the most efficient primers. High level (5×105 copies/ml) and low level (5×102 copies/ml) templates were amplified, and lateral flow dipsticks (LFD) were used to detect amplification products. The amplification time and visualization time were optimized to visualize the results, and the optimal tHDA-LFD reaction system was established. Simulated specimens with low, medium, and high concentrations were detected for 15 times, and precision was assessed. The limit of detection was evaluated using tenfold serial dilutions of 2019-nCoV in contrived samples. To evaluate the cross-reactivity, six samples of respiratory viruses, including influenza A virus, influenza B virus, human adenovirus, human respiratory syncytial virus, human parainfluenza virus and coronavirus 229E were tested using this assay. The tHDA-LFD assay was performed to detect 2019-nCoV in 163 clinical specimens stored in our laboratory. The clinical diagnostic efficacy was determined. Results: The established RT-tHDA one-step method combined with LFD was adopted, which could be conducted within 60 min and was reproducible with good precision, showing 100% positive concordance rate. The assay showed no cross reaction with other six human respiratory pathogens. The limit of detection of the RT-tHDA assay was 5×102 copies/ml for both N and E genes detected by LFD. The diagnostic efficacy evaluation showed that the sensitivity of the method was 95.00%(19/20), and the specificity was 100.00%(143/143). The positive predicted value of the method was 100.00% (19/19)and the negative predicted value was 99.31%(143/144). Compared with real-time RT-qPCR assay, it showed Kappa value of 0.971 (P<0.0001). Conclusion: The proposed tHDA-LFD assay is a rapid and visualized method to detect 2019-nCoV. © 2021 Chinese Medical Association

5-Hydroxymethylcytosine Glucosylation-Triggered Helicase-Dependent Amplification-Based Fluorescent Biosensor for Sensitive Detection of β-Glucosyltransferase with Zero Background Signal

β-glucosyltransferase (β-GT) catalyzes the glucosylation of 5-hydroxymethylcytosine (5-hmC) to enable the survival of bacteriophage and parasite in host cells, and it is a critical tool enzyme for 5-hmC assay. However, few methods are available for β-glucosyltransferase assay, and they usually have the drawbacks of radioactive contamination, high background, laborious procedures, and unsatisfactory sensitivity. Herein, we develop a new fluorescent biosensor with zero background signal for sensitive detection of β-GT activity based on 5-hmC glucosylation-triggered helicase-dependent amplification (HDA). The detection probe we designed may act as both a probe for β-GT recognition and a template for HDA amplification. The β-GT-catalyzed 5-hmC glucosylation can protect the detection probes from both the cleavage by MfeI restrictive enzyme and the digestion by exonucleases I and III. The remaining detection probes can subsequently act as the templates for exponential HDA amplification to generate numerous double-stranded DNA products, which can be easily detected by SYBR Green I in a label-free manner. The zero background can be achieved by efficient elimination of primer-dimer nonspecific amplification and complete digestion of nonglucosylated detection probes. This biosensor exhibits high sensitivity and good specificity, and it can be further used to analyze β-GT kinetic parameters and screen the inhibitors, providing a powerful platform for deeper understanding of β-GT biological functions and promoting β-GT-related epigenetic studies. Furthermore, this biosensor can be extended to detect various DNA-modifying enzymes by simply replacing the recognition sequence and restriction enzyme.

Developments in integrating nucleic acid isothermal amplification and detection systems for point-of-care diagnostics

Early disease detection through point-of-care (POC) testing is vital for quickly treating patients and preventing the spread of harmful pathogens. Disease diagnosis is generally accomplished using quantitative polymerase chain reaction (qPCR) to amplify nucleic acids in patient samples, permitting detection even at low target concentrations. However, qPCR requires expensive equipment, trained personnel, and significant time. These resources are not available in POC settings, driving researchers to instead utilize isothermal amplification, conducted at a single temperature, as an alternative. Common isothermal amplification methods include loop-mediated isothermal amplification, recombinase polymerase amplification, rolling circle amplification, nucleic acid sequence-based amplification, and helicase-dependent amplification. There has been a growing interest in combining such amplification methods with POC detection methods to enable the development of diagnostic tests that are well suited for resource-limited settings as well as developed countries performing mass screenings. Exciting developments have been made in the integration of these two research areas due to the significant impact that such approaches can have on healthcare. This review will primarily focus on advances made by North American research groups between 2015 and June 2020, and will emphasize integrated approaches that reduce user steps, reliance on expensive equipment, and the system's time-to-result.

Exploring the Use of High-Resolution Melting Analysis and Helicase-Dependent Amplification for Discrimination of Bemisia tabaci (Hemiptera: Aleyrodidae) Cryptic Species and Trialeurodes vaporariorum.

The sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera; Aleyrodidae), and greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae), are highly problematic plant pests and virus vectors with worldwide distributions. Identification of whitefly species is typically accomplished by observation of distinct morphological characters; however, because of morphological inconsistency and indistinguishability, the discrimination of B. tabaci species variants is dependent on molecular techniques based on genetic differences. New assays were designed for the detection of B. tabaci A, B, and Q mitotype groups, and T. vaporariorum. Specific primer sets were designed for amplification of the mitochondrial cytochrome c oxidase I gene of the four targets to perform in end-point PCR, real-time PCR coupled to high-resolution melting analysis (HRM), and the isothermal helicase-dependent amplification (HDA). Primer specificities were validated using end-point PCR, then tested in HRM and HDA. Bemisia tabaci A, B, and Q mitotypes, and T. vaporariorum-targeted primer sets discriminately amplified specimens of different populations within their target whitefly group. These tests provide three novel discrimination assays for the high-consequence, exotic B. tabaci B and Q groups, along with the native B. tabaci A group and T. vaporariorum.