Isothermal Amplification Detection Research Articles

A new simplified sequence-dependent loop-mediated isothermal amplification (LAMP) detection method.

Fluorescence dye-based loop-mediated isothermal amplification (LAMP) is a sensitive nucleic acid detectionmethod, but is limited to single-plex detection and may yield non-specific signals. In this study, we propose a bifunctional probe-based real-time LAMP amplification method for single-plexed or multiplexed detection. The bifunctional probe is derived by modifying the5' endof the fluorophore and an internal quencher on one of the LAMP primers; therefore, it can simultaneously be involved in the LAMP process and signal amplification. The fluorescence intensity undergoes a cumulative exponential increase during the incorporation of the bifunctional probe into double-stranded DNA amplicons. The bifunctional probe-based LAMP method is simplified and cost-effective, as the primer design and experimental operations align entirely with the ordinary LAMP. Different from other current probe-based methods, this method does not require additional enzymes, sequences, or special probe structures. Also, it is 10min faster than several otherprobe-based LAMP methods. The bifunctional probe-based LAMP method allows the simultaneous detection of the target Vibrio parahaemolyticus DNA and the internal amplification control in a one-pot reaction, demonstrating its potential for multiplexed detection.

A Portable, Integrated, Sample-In Result-Out Nucleic Acid Diagnostic Device for Rapid and Sensitive Chikungunya Virus Detection.

Chikungunya virus, a mosquito-borne virus that causes epidemics, is often misdiagnosed due to symptom similarities with other arboviruses. Here, a portable and integrated nucleic acid-based diagnostic device, which combines reverse transcription-loop-mediated isothermal amplification and lateral-flow detection, was developed. The device is simple to use, precise, equipment-free, and highly sensitive, enabling rapid chikungunya virus identification. The result can be obtained by the naked eye within 40 min. The assay can effectively distinguish chikungunya virus from dengue virus, Japanese encephalitis virus, Zika virus, and yellow fever virus with high specificity and sensitivity as low as 598.46 copies mL-1. It has many benefits for the community screening and monitoring of chikungunya virus in resource-limited areas because of its effectiveness and simplicity. The platform has great potential for the rapid nucleic acid detection of other viruses.

Rapid and highly sensitive colorimetric LAMP assay and integrated device for visual detection of monkeypox virus

BackgroundThe monkeypox virus (MPXV) is a linear double-stranded DNA virus with a large genome that causes tens of thousands of infections and hundreds of deaths in at least 40 countries and regions worldwide. Therefore, timely and accurate diagnostic testing could be an important measure to prevent the ongoing spread of MPXV and widespread epidemics. ResultsHere, we designed multiple sets of primers for the target region of MPXV for loop-mediated isothermal amplification (LAMP) detection and identified the optimal primer set. Then, the specificity in fluorescent LAMP detection was verified using the plasmids containing the target gene, pseudovirus and other DNA/RNA viruses. We also evaluated the sensitivity of the colorimetric LAMP detection system using the plasmid and pseudovirus samples, respectively. Besides, we used monkeypox pseudovirus to simulate real samples for detection. Subsequent to the establishment and introduction of a magnetic beads (MBs)-based nucleic acid extraction technique, an integrated device was developed, characterized by rapidity, high sensitivity, and remarkable specificity. This portable system demonstrated a visual detection limit of 137 copies/mL, achieving sample-to-answer detection within 1 h. SignificanceThe device has the advantages of integration, simplicity, miniaturization, and visualization, which help promote the realization of accurate, rapid, portable, and low-cost testing. Meanwhile, this platform could facilitate efficient, cost-effective and easy-operable point-of-care testing (POCT) in diverse resource-limited settings in addition to the laboratory.

Sensitive and visual detection of SARS-CoV-2 using RPA-Cas12a one-step assay with ssDNA-modified crRNA

BackgroundCRISPR-Cas12a based one-step assays are widely used for nucleic acid detection, particularly for pathogen detection. However, the detection capability of the one-step assay is reduced because the Cas12a protein competes with the isothermal amplification enzymes for the target DNA and cleaves it. Therefore, the key to improving the sensitivity of the one-step assay is to address the imbalance between isothermal amplification and CRISPR detection. In previous study, we developed a Cas12a one-step assay using single-stranded DNA (ssDNA)-modified crRNA (mD-crRNA) and applied this method for the detection of pathogenic DNA. ResultsHere, we utilized mD-crRNA to establish a sensitive one-step assay that enables the visual detection of SARS-CoV-2 under ultraviolet light, achieving a detection limit of 5 aM without cross-reactivity. The sensitivity of mD-crRNA in the one-step assay was 100-fold higher than that of wild-type crRNA. Mechanistic studies revealed that the addition of ssDNA at the 3′ end of mD-crRNA attenuates the binding affinity between the Cas12a-mD-crRNA complex and the target DNA. Consequently, this reduction in binding affinity decreases the cis-cleavage activity of Cas12a, mitigating its cleavage of the target DNA in the one-step assay. As a result, there is an augmentation in the amplification and accumulation of target DNA, thereby enhancing detection sensitivity. In the clinical testing of 40 SARS-CoV-2 RNA samples, the concordance between the results of the one-step assay and known qPCR results was 97.5 %. SignificanceThe one-step assay using mD-crRNA proves to be highly sensitive and specificity and visually effective for the detection of SARS-CoV-2. Our study delves into the application of the mD-crRNA-mediated one-step assay in nucleic acid detection and its associated reaction mechanism. This holds great significance in addressing the inherent incompatibility issues between isothermal amplification and CRISPR detection.

Palm-Sized Lab-In-A-Magnetofluidic Tube Platform for Rapid and Sensitive Virus Detection.

Simple, sensitive, and accurate molecular diagnostics are critical for preventing rapid spread of infection and initiating early treatment of diseases. However, current molecular detection methods typically rely on extensive nucleic acid sample preparation and expensive instrumentation. Here, a simple, fully integrated, lab-in-a-magnetofluidic tube (LIAMT) platform is presented for "sample-to-result" molecular detection of virus. By leveraging magnetofluidic transport of micro/nano magnetic beads, the LIAMT device integrates viral lysis, nucleic acid extraction, isothermal amplification, and CRISPR detection within a single engineered microcentrifuge tube. To enable point-of-care molecular diagnostics, a palm-sized processor is developed for magnetofluidic separation, nucleic acid amplification, and visual fluorescence detection. The LIAMT platform is applied to detect SARS-CoV-2 and HIV viruses, achieving a detection sensitivity of 73.4 and 63.9 copiesµL-1, respectively. Its clinical utility is further demonstrated by detecting SARS-CoV-2 and HIV in clinical samples. This simple, affordable, and portable LIAMT platform holds promise for rapid and sensitive molecular diagnostics of infectious diseases at the point-of-care.

Iso‐E‐Codelock: A Rebuilding‐free Electrochemical Chip with a Customizable Decoding Probe for Real‐Time and Portable Pathogen Diagnostics

AbstractIn order to realize portable pathogen diagnostics with easier quantitation, digitization and integration, we develop a ready‐to‐use electrochemical sensing strategy (Iso‐E‐Codelock) for real‐time detection of isothermal nucleic acid amplification. Bridged by a branched DNA as codelock, the isothermal amplicon is transduced into increased current of an electrochemical probe, holding multiple advantages of high sensitivity, high selectivity, signal‐on response, “zero” background and one‐pot operation. Through a self‐designed portable instrument (BioAlex PHE−T), the detection can be implemented on a multichannel microchip and output real‐time amplification curves just like an expensive commercial PCR machine. The microchip is a rebuilding‐free and disposable component. The branch codelock probe can be customized for different targets and designs. Such high performance and flexibility have been demonstrated utilizing four virus (SARS‐CoV‐2, African swine fever, FluA and FluB) genes as targets, and two branch (3‐way and 4‐way) DNAs as codelock probes.

Iso-E-Codelock: A Rebuilding-free Electrochemical Chip with a Customizable Decoding Probe for Real-Time and Portable Pathogen Diagnostics.

In order to realize portable pathogen diagnostics with easier quantitation, digitization and integration, we develop a ready-to-use electrochemical sensing strategy (Iso-E-Codelock) for real-time detection of isothermal nucleic acid amplification. Bridged by a branched DNA as codelock, the isothermal amplicon is transduced into increased current of an electrochemical probe, holding multiple advantages of high sensitivity, high selectivity, signal-on response, "zero" background and one-pot operation. Through a self-designed portable instrument (BioAlex PHE-T), the detection can be implemented on a multichannel microchip and output real-time amplification curves just like an expensive commercial PCR machine. The microchip is a rebuilding-free and disposable component. The branch codelock probe can be customized for different targets and designs. Such high performance and flexibility have been demonstrated utilizing four virus (SARS-CoV-2, African swine fever, FluA and FluB) genes as targets, and two branch (3-way and 4-way) DNAs as codelock probes.

Comparative study on the target sequences of thaumatin-like protein (TLP) and internal transcribed spacer (ITS) in the detection of corn component in edible oil by ladder-shape melting temperature isothermal amplification (LMTIA)

Single copy thaumatin-like protein (TLP) genes and multiple copy internal transcribed spacer (ITS) genes from the nucleus of corn were selected as targets, designed and screened primers. The impact of target sequence selection on ladder-shape melting temperature isothermal amplification (LMTIA) detection of corn components in edible oil from aspects such as specificity, sensitivity, and stability were studied. The results showed that the LMTIA system targeting TLP has an optimal reaction temperature of 58 °C and can detect a minimum of 100 pg/μL of corn DNA, and the LMTIA system established with ITS as the target has higher sensitivity, with an optimal temperature of 59 °C, and can detect corn DNA as low as 1 pg/μL. Both systems have good specificity, stable reactions, and no false positive results were observed. Conclusion: ITS is more suitable as a target sequence for LMTIA detection of corn components in edible oil.

Prevalence of S. aureus and/or MRSA in hospitalized patients with diabetic foot and establishment of LAMP methods for rapid detection of the SCCmec gene

BackgroundPatients with diabetic feet are prone to be infected due to the impaired immune system. However, the prognostic outcome of different microbial infections remains controversial. Identification and rapid screening of the pathogenic microorganisms that pose the greatest threat to the prognosis of patients with diabetic foot infections (DFIs) is critical.MethodsClinical data were statistically analyzed, which were obtained from 522 patients with DFIs, including pathogenic bacterial culture results and treatment outcomes at the last return visit. In addition, a loop-mediated isothermal amplification (LAMP) detection method was developed to identify the prevalent subtype of methicillin-resistant Staphylococcus aureus (MRSA) in DFIs patients. This study was approved by the Ethics Committee of Nanfang Hospital (NFEC-202012-K6) and registered on ClinicalTrials.gov (NCT04916457) on June 1, 2021.ResultsWe found that the proportion of patients with infections of Staphylococcus aureus (S. aureus) and MRSA was 27.7% (145/522) and 33.7% (49/145), respectively. Additionally, the incidence of osteomyelitis was 46.9% (23/49) and amputation/disability was 40.8% (20/49) in patients with MRSA infection, which were significantly higher compared to patients with other types of bacterial infections such as methicillin-susceptible Staphylococcus aureus (MSSA). Notably, we demonstrated that the main prevalent subtype of MRSA in DFIs patients in our hospital was Staphylococcal chromosomal cassettes mec (SCCmec) type II. In addition, it only takes 1.5 h to complete the entire experimental procedure in this LAMP assay, providing high sensitivity (100%) and specificity (77.8%) in hospitalized patients with DFIs.ConclusionsWe demonstrated there is a very high rate of MRSA isolation in patients with DFIs and revealed that patients infected with MRSA are at a higher risk of developing osteomyelitis, and amputation or disability. Importantly, we have developed a method for quickly screening newly admitted patients for MRSA.

Rapid Visual Detection of Spiroplasma eriocheiris by Loop-Mediated Isothermal Amplification with Hydroxynaphthol Blue Dye.

In recent years, a new type of Spiroplasma has been found that can cause "tremor disease" of the Chinese mitten crab Eriocheir sinensis. The outbreak of epidemic tremor disease has caused a serious setback in the Chinese mitten crab farming industry, with an incidence rate of more than 30% and mortality rates of 80-100%. Therefore, finding a sensitive method to detect tremor disease in E. sinensis has become a current research focus. In this research, a loop-mediated isothermal amplification detection method coupled with hydroxynaphthol blue dye (LAMP-HNB) was developed and used to rapidly detect Spiroplasma eriocheiris. First, we designed and synthesized specific outer primers, inner primers and loop primers based on the 16S ribosomal RNA gene of S. eriocheiris. Second, the LAMP-HNB detection method for S. eriocheiris was successfully established by screening the primers, adjusting the temperature and time of the reaction, and optimizing the concentrations of Mg2+ and dNTPs. In the specific tests, only samples infected with S. eriocheiris showed positive results, and other infections caused by bacteria and parasites tested negative, proving that the test has high specificity. Moreover, the detection limit was 2.5 × 10-6ng/µL, indicating high sensitivity. This method for detecting S. eriocheiris provides rapid visual output based on LAMP-HNB detection and is a simple, fast, sensitive, and inexpensive method that can be applied to a wide range of field investigations.

Designing hybrid CRISPR-Cas12 and LAMP detection systems for treatment-resistant Plasmodium falciparum with in silico method.

Genes associated with drug resistance of first line drugs for Plasmodium falciparum have been identified and characterized of which three genes most commonly associated with drug resistance are P. falciparum chloroquine resistance transporter gene (PfCRT), P. falciparum multidrug drug resistance gene 1 (PfMDR1), and P. falciparum Kelch protein K13 gene (PfKelch13). Polymorphism in these genes could be used as molecular markers for identifying drug resistant strains. Nucleic acid amplification test (NAAT) along with DNA sequencing is a powerful diagnostic tool that could identify these polymorphisms. However, current NAAT and DNA sequencing technologies require specific instruments which might limit its application in rural areas. More recently, a combination of isothermal amplification and CRISPR detection system showed promising results in detecting mutations at a nucleic acid level. Moreover, the Loop-mediated isothermal amplification (LAMP)-CRISPR systems offer robust and straightforward detection, enabling it to be deployed in rural and remote areas. The aim of this study was to develop a novel diagnostic method, based on LAMP of targeted genes, that would enable the identification of drug-resistant P. falciparum strains. The methods were centered on sequence analysis of P. falciparum genome, LAMP primers design, and CRISPR target prediction. Our designed primers are satisfactory for identifying polymorphism associated with drug resistant in PfCRT, PfMDR1, and PfKelch13. Overall, the developed system is promising to be used as a detection method for P. falciparum treatment-resistant strains. However, optimization and further validation the developed CRISPR-LAMP assay are needed to ensure its accuracy, reliability, and feasibility.

A novel tailed primer nucleic acid test for detection of HPV 16, 18 and 45 DNA at the point of care

Cervical cancer is a leading cause of death for women in low-resource settings despite being preventable through human papillomavirus (HPV) vaccination, early detection, and treatment of precancerous lesions. The World Health Organization recommends high-risk HPV (hrHPV) as the preferred cervical cancer screening strategy, which is difficult to implement in low-resource settings due to high costs, reliance on centralized laboratory infrastructure, and long sample-to-answer times. To help meet the need for rapid, low-cost, and decentralized cervical cancer screening, we developed tailed primer isothermal amplification and lateral flow detection assays for HPV16, HPV18, and HPV45 DNA. We translated these assays into a self-contained cartridge to achieve multiplexed detection of three hrHPV genotypes in a disposable cartridge. The developed test achieves clinically relevant limits of detection of 50–500 copies per reaction with extracted genomic DNA from HPV-positive cells. Finally, we performed sample-to-answer testing with direct lysates of HPV-negative and HPV-positive cell lines and demonstrated consistent detection of HPV16, HPV18, and HPV45 with 5000–50,000 cells/mL in < 35 min. With additional optimization to improve cartridge reliability, incorporation of additional hrHPV types, and validation with clinical samples, the assay could serve as a point-of-care HPV DNA test that improves access to cervical cancer screening in low-resource settings.

Pipette-Free and Fully Integrated Paper Device Employing DNA Extraction, Isothermal Amplification, and Carmoisine-Based Colorimetric Detection for Determining Infectious Pathogens.

A pipette-free and fully integrated device that can be used to accurately recognize the presence of infectious pathogens is an important and useful tool in point-of-care testing, particularly when aiming to decrease the unpredictable threats posed by disease outbreak. In this study, a paper device is developed to integrate the three main processes required for detecting infectious pathogens, including DNA extraction, loop-mediated isothermal amplification (LAMP), and detection. All key reagents, including sodium dodecyl sulfate (SDS), NaOH, LAMP reagents, and carmoisine, are placed on the paper device. The paper device is operated simply via sliding and folding without using any bulky equipment, and the results can be directly observed by the naked eye. The optimized concentrations of sodium dodecyl sulfate (SDS), sodium hydroxide (NaOH), and carmoisine were found to be 0.1%, 0.1 M, and 0.5 mg/mL, respectively. The paper device was used to detect Enterococcus faecium at concentrations as low as 102 CFU/mL within 60 min. Also, E. faecium spiked in milk was successfully detected using the paper device, demonstrating the feasible application in real sample analysis.

Utility of a loop-mediated isothermal amplification detection kit to diagnose chronic pulmonary aspergillosis

BackgroundDiagnosing chronic pulmonary aspergillosis (CPA) is still challenging, and infection with Aspergillus spp. must be detected early. The aim of this study was to evaluate, for the first time, the usefulness of a new loop-mediated isothermal amplification (LAMP) test kit in diagnosing CPA. MethodsThis cross-sectional study was performed at the National Hospital Organization Kinki-Chuo Chest Medical Center. We prospectively enrolled patients with and without CPA. The primary outcomes of this study were the sensitivity and specificity of LAMP for the diagnosis of CPA. ResultsIn total, 34 patients with CPA and 21 without CPA (the control group) were enrolled between October 2019 and December 2021. Among the 34 patients with CPA, cultures were positive for CPA in 16 and negative for 18. The majority of fungi isolated in sputum were A. fumigatus in 15 specimens; Aspergillus flavus was isolated in 1) LAMP demonstrated 55.9% sensitivity (95% confidence interval [CI] 37.9–72.8) and 100.0% specificity (95% CI 77.2–100.0). ConclusionLAMP is an acceptable tool for diagnosing CPA, and its application in clinical practice necessitates more studies.

High specific microRNA detection based on G-quadruplex sensitive fluorescence under isothermal amplification

MicroRNAs (miRNAs) are crucial for various basic cellular processes and have been regarded as an important indicator for pathogenesis and development of cancer. While miRNAs are characterized by short sequence length, large single cell copy number variation, low abundance in vivo, and high homologous sequence similarity. The development of miRNA detection methods with high sensitivity, good selectivity and low cost is the key to study the function of miRNA. In this article, a method combining isothermal chain replacement amplification and G-quadruplex (G4) specific detection is developed for highly specific and sensitive detection of miRNA. By designing proper DNA sequences, a three-stranded DNA complex was formed and produced a lot of G4 structure sequences, which can be stained by specific fluorescence dye and quantified. In the experiment, the fluorescence dye shows extremely high specificity, which brings excellent signal discrimination, so that the target miRNAs can be accurately detected. Experiments under high background noise also verified this method is highly selective for target miRNAs. This method holds great imagination in expanding the detection methods to more platforms, such as nanopore sensors, because it alters the secondary structure of DNA sequences.

Research Progress of Nucleic Acid Detection Technology for Genetically Modified Maize.

Genetically modified (GM) maize is one of the earliest GM crops to have achieved large-scale commercial cultivation globally, and it is of great significance to excel in the development and implementation of safety policy regarding GM, and in its technical oversight. This article describes the general situation regarding genetically modified maize, including its varieties, applications, relevant laws and regulations, and so on. From a technical point of view, we summarize and critically analyze the existing methods for detecting nucleic acid levels in genetically modified maize. The nucleic acid extraction technology used for maize is explained, and the introduction of traditional detection techniques, which cover variable-temperature and isothermal amplification detection technology and gene chip technology, applications in maize are described. Moreover, new technologies are proposed, with special attention paid to nucleic acid detection methods using sensors. Finally, we review the current limitations and challenges of GM maize nucleic acid testing and share our vision for the future direction of this field.

Phenol red as electrochemical indicator for highly sensitive quantification of SARS-CoV-2 by loop-mediated isothermal amplification detection

The current COVID-19 pandemic has made patent the need for rapid and cost-effective diagnostic tests, crucial for future infectious outbreaks. Loop-mediated isothermal amplification (LAMP) is a promising and decentralized alternative to qPCR. In this work we have developed a sensitive, fast, and simple innovative methodology for quantification of SARS-CoV-2 RNA copies, combining reverse-transcription LAMP with electrochemical detection. This is based on the oxidation of phenol red (PR), a visual and electroactive LAMP indicator, which oxidation peak potential (Ep) changes with the progress of the LAMP reaction. Using that Ep shift as analytical signal, a calibration curve was obtained for fragment N1 copies of SARS-CoV2 (which provided better results than N or S fragments), with a potential shift of 16.2 mV per order of magnitude, and a practical limit of detection of 21 copies·μL−1. Moreover, the precision of Ep is excellent (RSD < 2%): 557 ± 5 mV for negative and 602 ± 7 mV for positive (2148 N fragment RNA copies·µL-1·-1) LAMP controls. This methodology has been applied to the analysis of nasopharyngeal swab samples, resulting in total concordance with clinical RT-qPCR results. Advances towards fully decentralization have been achieved by designing and fabricating a small portable heater for isothermal procedures, obtaining comparable results to those from a commercial benchtop thermal cycler.

Host Range and Loop-Mediated Isothermal Amplification Detection of Globisporangium sylvaticum from Guizhou, China.

Globisporangium, especially G. sylvaticum, causes devastating root rot, blight, and other diseases in various species of cash crops. To investigate the distribution and host range of G. sylvaticum in Guizhou, a suitable habitat for this pathogen, we collected 156 root-diseased samples, isolated the pathogens, and found that G. sylvaticum is widespread and has eleven host plants, including four novel hosts. Furthermore, to effectively identify G. sylvaticum, we developed a simple and dependable method based on loop-mediated isothermal amplification (LAMP), which used a primer set designed from the internal transcribed spacer sequences with high specificity and sensitivity of 1 pg/μL. Additionally, to perform field identification, we used the "Plant-LAMP" method with crude DNA extraction to detect the pathogen in 45 root samples from nine species of plants. Our results showed that this method could effectively detect G. sylvaticum in diseased roots. Therefore, our findings not only enrich existing research on the diversity of pathogenic Globisporangium in Guizhou but also present an efficient LAMP field detection method that could significantly contribute to plant disease management and prevention.

Visual isothermal amplification detection of ASFV based on trimeric G-quadruplex cis-cleavage activity of Cas-12a

African swine fever virus (ASFV) is a kind of DNA virus and can infect both domestic pigs and wild boars with fatality up to 100%. The contaminated meat products mainly led to the worldwide transmission of ASFV. The outbreak of ASF greatly affects the supply stability of meat products as well as the development of the global pig industry. In this study, a visual isothermal amplification detection assay for ASFV based on trimeric G-quadruplex cis-cleavage activity of Cas12a was developed. The introduction of Cas12a could discriminate the specific amplification from the non-specific amplification and improve the sensitivity. The detection limit was as low as 0.23 copies/μL. This assay had good potential in the detection of ASFV and would be helpful for the stability of meat production and supply.

Rapid Fluorescence-Free Detection of DNA Loop-Mediated Isothermal Amplification on Bare ITO Surface Under EG-FET Scheme

An extended gate field effect transistor (EG-FET)-based chip with a precision temperature control system is developed for real-time and fluorescence-free loop-mediated isothermal amplification (LAMP) detection. A label-free, non-optical instant method was used to detect the hydrogen ions released during nucleic acid amplification, rather than indirect measurements. The system comprises an EG-FET sensor, a temperature sensor, a heating block, and a signal processing system. Industrial grade bare indium tin oxide (ITO) is firstly demonstrated as the sensing material for nucleic acid amplification detection, such that stable and high hydrogen ion detection performance of -80.1±0.03 mV/pH with R <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> of 0.998 is achieved with the developed sensor. Moreover, a printed circuit board (PCB) with high thermal conductivity was used as the sensor base to provide a uniform temperature distribution during the reaction. The results show that the system maintains a stable LAMP assay temperature of 63°C with a variation of just 0.25°C. Moreover, in a pH-sensitive LAMP assay, the developed sensor achieves a limit-of-detection down to 10 genomic copies per reaction of lambda phage DNA within 17 minutes. The novel and performance iso-thermal EG-FET system is capable of real-time detecting the LAMP DNA amplification, and thus has significant potential for future biomedical applications.