Isothermal Amplification Method Research Articles

Development and Validation of a Loop-Mediated Isothermal Amplification (LAMP) Method for the Rapid, Sensitive, and Specific Detection of Hazelnut as an Allergen in Food Matrix.

Analytical verification of hazelnut in food supports risk-based approaches for proper allergen labeling to prevent unwanted allergic reactions or to quality control diagnostic or therapeutic allergen preparations for allergic subjects. We present the development and validation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of hazelnut by targeting the internal transcribed spacer 2 gene. The qualitative method requires neither sophisticated analytical equipment nor antibodies, allowing an easy-to-use application with no ethical concern related to the use of animals. It demonstrated a limit of detection at or below 10 mg/kg hazelnut in various food matrices, making it also suitable for verifying hazelnut at levels of clinically relevant eliciting doses. Validation against proficiency test samples and testing of applicability with commercial food items confirmed its usefulness in processed foods. The simplicity of the method, including visual colorimetric detection, combined with high specificity and sensitivity, represents an advancement over existing qualitative methods.

CRISPR/Cas12a dual-mode biosensor for Staphylococcus aureus detection via enzyme-free isothermal amplification

Accurate and reliable detection of Staphylococcus aureus (S. aureus) is essential for preventing infections, particularly in healthcare and food safety contexts. This work presents a novel dual-mode biosensor that integrates the CRISPR/Cas12a system with an enzyme-free isothermal amplification method for detecting S. aureus. Hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) amplify the aptamer-triggered response, significantly enhancing sensitivity. CRISPR/Cas12a′s nuclease activity is utilized in two modes: cis cleavage generates a fluorescence signal, while trans cleavage produces an electrochemical signal, enabling dual-mode detection. The biosensor demonstrates outstanding performance, with a limit of detection (LOD) as low as 5.7 CFU mL−1 in electrochemical mode and 133.7 CFU mL−1 in fluorescence mode, showcasing excellent accuracy, stability, and sensitivity. It has been successfully applied to detecting actual samples, confirming its practical applicability. This innovative approach offers a powerful tool for the swift and precise identification of S. aureus and paves the way for developing next-generation dual-mode biosensors for various analytes. Future research will aim to simplify the detection process further, making it more accessible for use in resource-limited settings.

B-224 Rapid, Extraction-Free, and Portable Molecular Detection of HIV-1 and HCV Directly From Whole Blood

Abstract Background Early diagnosis of human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) is key to preventing viral transmission and improving linkage to treatment. Isothermal amplification methods, such as reverse transcription looped-mediated amplification (RT-LAMP), have enabled molecular diagnostics to expand beyond centralized laboratories. Despite the advances in molecular point-of-care (POC) testing, many of these tests are costly and still depend on a reliable power-source, specialized equipment, and cold-chain storage, making them impractical for resource-limited settings. To overcome these barriers, there is a need for innovative molecular diagnostics for blood-borne pathogens. Here, we propose a simple, extraction-free, portable workflow for detecting HIV-1 and HCV from whole blood within 40 minutes. Methods We developed singleplex RT-LAMP-based tests using modified published primer sets. Contrived whole blood samples containing HIV-1 or HCV virions were diluted in equal parts water and loaded directly into optimized RT-LAMP master mixes. To mitigate cold-chain storage dependence, RT-LAMP reactions were performed using a lyophilized master mix. The reactions were heated for 30 minutes using a hand-held, battery-powered heating device for simultaneous virion lysis and amplification. For simple visual detection, amplification was coupled with a lateral flow-based dipstick test. The analytical sensitivity of each detection test was evaluated using contrived whole blood samples ranging in HIV-1 and HCV 1a viral loads. Additionally, HCV detection was evaluated on genotypes 1b, 2b, and 3a. For result confirmation, a custom CRISPR-Cas12a-based assay was used to verify the presence or absence of pathogen-specific amplicons following amplification. Results The diagnostic workflow can be completed within 40 minutes, including less than 10 minutes of hands-on time. At optimal conditions, the HIV-1 and HCV 1a singleplex tests had lower limits of detections of 4.89 log10 cp/mL and 3.77 log10 IU/mL, respectively. Evaluation of the published literature showed that these viral loads are within the ranges observed during acute HIV-1 and HCV infection. HCV detection was also observed for genotypes 1b and 3a, albeit at a lower sensitivity than genotype 1a. No detection was observed for genotype 2b, presumably due to the lower viral loads of the samples. No cross-reactivity between the two tests was observed. Conclusions Our proposed workflow allows for rapid detection of blood-borne pathogens without the need for complex equipment or cold-chain storage, showing potential for application in resource-limited settings. Additional validation within a clinical setting using fingerstick blood remains to be tested.

Development of a rapid LFA test based on direct RT-LAMP for diagnosis of SARS-CoV-2

IntroductionIn response to the rapid spread of the SARS-CoV-2 virus, we developed a rapid molecular approach to diagnose COVID-19 without the need for RNA extraction. MethodsThe study utilized two molecular methods, RT-qPCR, and colorimetric RT-LAMP, to diagnose the RdRp and ORF8 genes, respectively, in oropharyngeal swabs. Due to the high sequence diversity of ORF8 in SARS-CoV and SARS-CoV-2, it has been identified as a suitable target for virus detection. The RT-LAMP method was also carried out directly on heat-treated swab samples. The strip tests were made using gold nanoparticles and combined with the RT-LAMP for further analysis. ResultsThe results showed that the isothermal amplification method had a sensitivity of 95% (95% C.I.: 86.08% to 98.96%) and a specificity of 75% (95% C.I.: 19.41% to 99.37%). The RT-LAMP-LFA method was able to distinguish positive and negative samples with 100% sensitivity (95% C.I.: 91.96 - 100) and 77.27% specificity (95% C.I.: 54.63 - 92.18). This method only required heating swab samples for 10 min at 65 ºC before the RT-LAMP reaction. ConclusionBy utilizing the RT-LAMP in combination with the LFA, it is possible to diagnose SARS-CoV-2 rapidly without the need for RNA extraction. The entire process from sample collection to test interpretation takes only 75-90 min, and the results can be interpreted by untrained individuals with the naked eye. By employing the ORF8 gene as a diagnostic target and eliminating the need for RNA extraction, the direct RT-LAMP-LFA method achieves a significant breakthrough that was not previously reported.

CRISPR/Cas13a-mediated triple signal amplification strategy for viral RNA detection

CRISPR/Cas13a system has been combined with different isothermal amplification strategies for RNA analysis. Nevertheless, expensive enzymes are usually indispensable in these isothermal amplification methods, which increases the detection cost. In this study, a CRISPR/Cas13a-mediated triple signal amplification strategy has been designed for selective, sensitive and inexpensive detection of viral RNA. SARS-CoV-2 S RNA activated CRISPR/Cas13a to cut hairpin DNA, triggering catalytic hairpin assembly (CHA) and subsequent hybridization chain reaction (HCR) to form a long-stranded Y-type DNA. G-quadruplexes were distributed on the branch of the long-stranded DNA and bound with N-methyl mesoporphyrin IX (NMM) to enhance the fluorescence signal. SARS-CoV-2 S RNA was specifically detected with a limit of detection (LOD) of 285 fM. Notably, no additional expensive enzymes were required in the entire detection process. The CRISPR/Cas13a-mediated triple signal amplification strategy holds great potential for specific and sensitive viral RNA detection with low cost.

A colorimetric reverse-transcription loop-mediated isothermal amplification method targeting the L452R mutation to detect the Delta variant of SARS-CoV-2

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered global difficulties for both individuals and economies, with new variants continuing to emerge. The Delta variant of SARS-CoV-2 remains most prevalent worldwide, and it affects the efficacy of coronavirus disease 2019 (COVID-19) vaccination. Expedited testing to detect the Delta variant of SARS-CoV-2 and monitor viral transmission is necessary. This study aimed to develop and evaluate a colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) technique targeting the L452R mutation in the S gene for the specific detection of the Delta variant. In the test, positivity was indicated as a color change from purple to yellow. The assay’s 95% limit of detection was 57 copies per reaction for the L452R (U1355G)-specific standard plasmid. Using 126 clinical samples, our assay displayed 100% specificity, 97.06% sensitivity, and 98.41% accuracy in identifying the Delta variant of SARS-CoV-2 compared to real-time RT-PCR. To our knowledge, this is the first colorimetric RT-LAMP assay that can differentiate the Delta variant from its generic SARS-CoV-2, enabling it as an approach for studying COVID-19 demography and facilitating proper effective control measure establishment to fight against the reemerging variants of SARS-CoV-2 in the future.

Development of machine learning algorithm for loop-mediated isothermal amplification including influence of temperature

To predict amplification in the Loop-Mediated Isothermal Amplification (LAMP) method, a binary model has been developed. This model assesses whether the combination of partial template binding facilitated by multiple primers aligns with a specific signature indicating amplification. For this model, 1512 settings were adjusted, encompassing variations in the extraction oligomer position from the primer, the oligomer length, the number of allowed mismatched bases, and the stringency of the homology position. The optimal set was determined based on the F1 value at a reaction temperature of 60°C, resulting in a value of 0.58. To implement machine learning by amalgamating multiple models, duplicate models were eliminated through cluster analysis. Machine learning using AutoML was performed on 108 distinct models. Consequently, the F1 values were 0.7119 at a reaction temperature of 60°C and 0.7210 at 68°C. The weak correlation between feature importance and the F1 value of the model alone suggests that machine learning utilizing this model incorporates numerous models to enhance prediction accuracy.

Development of a real-time loop-mediated isothermal amplification method with toothpick sampling for non-destructive detection of Ustilago esculenta in Zizania latifolia

Development of a real-time loop-mediated isothermal amplification method with toothpick sampling for non-destructive detection of Ustilago esculenta in Zizania latifolia

Development and evaluation of a loop-mediated isothermal amplification (LAMP) method for Candida glabrata detection.

Loop-mediated isothermal amplification (LAMP) is a simple and rapid nucleic acid method for DNA amplification at a constant temperature. The "gold standard" culture method for yeast detection, has low sensitivity with severe consequences, increasing morbidity and mortality rates. Here, we report the development of a LAMP method for the specific detection of C. glabrata. The specific LAMP primers for C. glabrata detection were designed and evaluated. The LAMP assay accurately detected C. glabrata with no cross-reactivity with other Candida species. The developed molecular method would be a promising tool in the management of invasive candidiasis.

A new exploration of the amplification mechanism of saltatory rolling circle amplification (SRCA) for food safety detection

A new property of Bst DNA polymerase was discovered by our team, which led to the establishment of a new isothermal amplification method for SRCA nucleic acid, which was applied to food safety detection, investigated the amplification mechanism, and the SRCA version 1.0 amplification mechanism was proposed. This study proposes a new SRCA amplification mechanism (version 2.0) by using the autosynthetic sequences CDG2040 and CDG2068, as well as the Listeria monocytogenes hlyA gene and Vibrio parahaemolyticus toxR gene as target sequences to further investigate the amplification mechanism. The initiation of the SRCA reaction depends primarily on the spatial structure of the non-closed circle formed by the discontinuous complementation of the bases on both sides of the target sequence. The DNA fragments that connect the target sequences in the SRCA reaction are the sum of the complementary sequences of adjacent partial sequences on both sides of the target sequence.

Development and application of a rapid visual detection technique for VanA gene in vancomycin-resistant Enterococcus faecium.

One of the key approaches to treating and controlling vancomycin-resistant Enterococcus faecium (VREFm) is an accurate and rapid diagnosis. To achieve this goal, a simple and rapid method must be constructed for immediate detection in the field. Multienzyme isothermal rapid amplification (MIRA) is an isothermal rapid amplification method that allows amplification reactions to be completed under room temperature conditions. When combined with lateral flow strips (LFSs), MIRA-LFS enables the rapid detection of pathogenic microorganisms. However, the MIRA method often produces false signals. These false signals are eliminated by using base mismatches introduced in primers and probes. The MIRA-LFS system was constructed with high specificity and sensitivity for the detection of VREfm, without the limitation of sophisticated instruments. This enables the prompt formulation of diagnostic and therapeutic decisions.

Development of a highly specific LAMP assay for detection of Sarcocystis tenella and Sarcocystis gigantea in sheep.

Sarcocystis infection in sheep has caused significant economic losses in the livestock industry, and the genetic similarity among Sarcocystis species highlights the need for precise diagnostic methods in sheep. This study developed a loop-mediated isothermal amplification (LAMP) method targeting COX-1 and 28S rRNA genes to detect Sarcocystis tenella and Sarcocystis gigantea, respectively. The LAMP method exhibited high specificity, selectively amplifying target DNA sequences without cross-reactivity with closely related protozoa, such as Toxoplasma gondii and Neospora caninum. Detection limits were determined as 3 × 105 copies/L for S. tenella and 6 × 104 copies/L for S. gigantea, enabling sensitive identification of low-level infections. Comparative analysis with conventional PCR on sheep cardiac tissues demonstrated a higher LAMP detection rate (80.0% vs 66.7%). In conclusion, the LAMP method offers superior sensitivity to conventional PCR, allows visual confirmation of results, and provides a rapid diagnostic tool for identifying S. tenella and S. gigantea infection in sheep. However, due to the limitation of sample availability, we were unable to assess all Sarcocystis species that use sheep as intermediate hosts, which warrants further research.

Advances in plant pathogen detection: integrating recombinase polymerase amplification with CRISPR/Cas systems.

Plant pathogens are causing substantial economic losses and thus became a significant threat to global agriculture. Effective and timely detection methods are prerequisite for combating the damages caused by the plant pathogens. In the realm of plant pathogen detection, the isothermal amplification techniques, e.g., recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), have emerged as a fast, precise, and most sensitive alternative to conventional PCR but they often comprise high rates of non-specific amplification and operational complexity. In recent advancements, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease Cas systems, particularly Cas12, have emerged as powerful tools for highly sensitive, specific, and rapid pathogen detection. Exploiting the collateral activities of Cas12, which selectively cleaves single-stranded DNA (ssDNA), novel detection platforms have been developed. The mechanism employs the formation of a triple complex molecule comprising guide RNA, Cas12 enzyme, and the substrate target nucleotide sequence. Upon recognition of the target, Cas12 indiscriminately cleaves the DNA strand, leading to the release of fluorescence from the cleaved ssDNA reporter. Integration of isothermal amplification methods with CRISPR/Cas12 enables one-step detection assays, facilitating rapid pathogen identification within 30min at a single temperature. This integrated RPA-CRISPR/Cas12a approach eliminates the need for RNA extraction and cDNA conversion, allowing direct use of crude plant sap as a template. With an affordable fluorescence visualization system, this portable method achieves 100-fold greater sensitivity than conventional techniques. This review summarizes recent advances in RPA-CRISPR/Cas12a for detecting plant pathogens, covering primer design, field-level portability, and enhanced sensitivity.

Rapid and simultaneous detection of common childhood diarrhea viruses by microfluidic-FEN1-assisted isothermal amplification with ultra-high specificity and sensitivity

Rapid and accurate diagnostic methods are crucial for managing viral gastroenteritis in children, a leading cause of global childhood morbidity and mortality. This study introduces a novel microfluidic-Flap endonuclease 1 (FEN1)-assisted isothermal amplification (MFIA) method for simultaneously detecting major viral pathogens associated with childhood diarrhea—rotavirus, norovirus, and adenovirus. Leveraging the specificity-enhancing properties of FEN1 with a universal dspacer-modified flap probe and the adaptability of microfluidic technology, MFIA demonstrated an exceptional detection limit (5 copies/μL) and specificity in the simultaneous detection of common diarrhea pathogens in clinical samples. Our approach addresses the limitations of current diagnostic techniques by offering a rapid (turn around time <1 h), cost-effective, easy design steps (universal flap design), and excellent detection performance method suitable for multiple applications. The validation of MFIA against the gold-standard PCR method using 150 actual clinical samples showed no statistical difference in the detection performance of the two methods, positioning it as a potential detection tool in pediatric diagnostic virology and public health surveillance. In conclusion, the MFIA method promises to transform pediatric infectious disease diagnostics and contribute significantly to global health efforts combating viral gastroenteritis.

Multiplex competitive annealing mediated isothermal amplification with high fidelity DNA polymerase (HiFi-CAMP)

Various isothermal amplification methods have been developed for point-of-care testing (POCT) of various infectious diseases. Here, we proposed a novel isothermal amplification method, named as 5′-half complementary primers mediated isothermal amplification (HCPA). Because of the similarity of our method to the previous method competitive annealing mediated isothermal amplification (CAMP) in primer design, we also use the name CAMP for our method. We demonstrated that CAMP is mediated by both a linear isothermal amplification pattern and a loop-mediated isothermal amplification pattern. To improve the specificity and enable multiplex detection, we further developed HiFi-CAMP method that uses a small amount of high-fidelity DNA polymerase to cut HFman probe to release fluorescent signal. The HiFi-CAMP method was demonstrated to have a good specificity and sensitivity, and fast amplification speed in detection of three human respiratory viruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus A (RSV-A) and influenza A viruses (IAV). When compared with gold standard RT-qPCR assays, the HiFi-CAMP assays showed sensitivities of 90.0 %, 71.4 % and 78.1 %, specificities of 100 %, 100 % and 95.5 %, and consistencies of 93.0 %, 93.3 % and 88.2 % for SARS-CoV-2, RSV-A and IAV, respectively. Furthermore, a duplex HiFi-CAMP assay was also developed to simultaneously detect RSV-A and SARS-CoV-2. The HiFi-CAMP will provide a promising candidate for POCT diagnosis in resource-limited settings.

Identification of Pathogen Causing Bulb Rot in Fritillaria taipaiensis P. Y. Li and Establishment of Detection Methods.

Fritillaria taipaiensis P. Y. Li (F. taipaiensis) is a traditional Chinese herbal medicine that has been used for over two millennia to treat cough and expectoration. However, the increasing cultivation of F. taipaiensis has led to the spread of bulb rot diseases. In this study, pathogens were isolated from rotten F. taipaiensis bulbs. Through molecular identification, pathogenicity testing, morphological assessment, and microscopy, Fusarium solani was identified as the pathogen causing bulb rot in F. taipaiensis. The colonization of F. solani in the bulbs was investigated through microscopic observation. The rapid and accurate detection of this pathogen will contribute to better disease monitoring and control. Loop-mediated isothermal amplification (LAMP) and qPCR methods were established to quickly and specifically identify this pathogen. These results provide valuable insights for further research on the prediction, rapid detection, and effective prevention and control of bulb rot in F. taipaiensis.

Rapid Detection of Staphylococcus Epidermidis Using Electric-Double-Layer (EDL) Field-Effect Transistor (FET) Biosensors with Loop-Mediated Isothermal Amplification

Staphylococcus epidermidis is a Gram-positive bacterium which commonly grows in human skin, nasal passages, respiratory tract, intestinal tract, air, and water. Although it is harmless to healthy individuals, immunocompromised patients are at risk of infection, which can lead to severe symptoms. With the increased reliance on antibiotics in hospitals, the antimicrobial resistance of Staphylococcus epidermidis has also risen, pointing out a challenge in clinical treatment.Therefore, the diagnosis of this bacterium has gained significant attention. The aim of this experiment is to detect bloodstream infections (BSIs) in the shortest possible time. The study is intended to be implemented in hospitals to provide a more convenient method for medical professionals to conduct screenings. The approach involves screening for the most common disease sequence, ssDNA, followed by sequential detection of corresponding cDNA, plasmids, and bacteria. To detect bacteria with the least amount of bacteerial load, a novel isothermal amplification method called Loop-mediated isothermal amplification (LAMP) is employed to increase the concentration of DNA of BSIs bacteria before measurement. The overall time required for this process doesn’t exceed one day, significantly increasing speed for the detection of blood-borne bacteria in hospitals. The core mechanism of this platform involves the use of electric-double-layer-gated field-effect-transistors (EDL-gated FETs) to amplify the gate voltage changes generated when the ssDNA binds with complementary DNA. These voltage changes are then converted into numerical values for analysis and interpretation.Currently, the research results show that this biomedical sensor can detect concentrations ranging from 1fM to 1pM. With the assistance of LAMP, it has successfully achieved a concentration of approximately 1yM (10-24 M) and detected with EDL-gated FETs. The experiment was also carried out step by step on simulated clinical specimens. The experiments have found successful serum nucleic acid amplification methods that provide excellent detection limits. Compared with other biomedical sensors in the past, it not only has lower detection limits but also brings features such as faster, more convenient, and more portable.Keywords: EDL, FET, Staphylococcus epidermidis, Biosensor, LAMP Figure 1

EasyNAT Malaria: a simple, rapid method to detect Plasmodium species using cross-priming amplification technology.

This study established and validated EasyNAT Malaria Assay as a promising molecular detection tool for malaria screening of high-risk populations in clinical practice. This novel isothermal amplification method may effectively facilitate the rapid diagnosis of malaria and prevent its transmission.

Rapid cyprinid herpesvirus 3 detection using loop-mediated isothermal amplification (LAMP) combined with gold nanoprobes and SYBR safe

As an etiological agent of a notifiable and emerging disease, Cyprinid herpesvirus 3 (CyHV-3 or koi herpesvirus, KHV) is a highly risky factor that affects koi and common carp yield through increasing the mortality rates. In the current study, a highly rapid and sensitive technique was established to detect CyHV-3 with the help of loop-mediated isothermal amplification (LAMP) method with dual visualizing indicators (SYBR safe and gold nanoparticle probes; AuNP-probes). Six specific primers were used to amplify the thymidine kinase (TK) of CyHV-3 using a LAMP reaction of 45 min at 65 °C along with detecting the products through visual inspection via hybridization at 65 °C for 20 min with a functionalized thiol-AuNP probe. A closed-tube LAMP-SYBR safe assay was also developed under the same condition without AuNP probe. The amplification of KHV-LAMP products were evaluated by the naked eye, through fluorescent emission of LAMP-SYBR safe dye-complex and colorimetric aggregate of LAMP product in the presence of AuNP probe. Furthermore, quantitative measurement was applied to the LAMP-AuNPs assay, using the spectral shift assay. The detection limit was 1.25 fentograms DNA which can compete the latest techniques available. The proposed technique yielded negative outcomes with DNA template from other viruses such as CyHV-1 and CEV. The LAMP-SYBR safe and LAMP-AuNPs assays introduced in this article were very sensitive, fast, and reliable diagnostic tools for the detection of CyHV-3.

SERS biosensors based on catalytic hairpin self-assembly and hybridization chain reaction cascade signal amplification strategies for ultrasensitive microRNA-21 detection.

A highly sensitive surface-enhanced Raman scattering (SERS) biosensor has been developedfor the detection of microRNA-21 (miR-21) using an isothermal enzyme-free cascade amplification method involving catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR). The CHA reaction is triggered by the target miR-21, which causes hairpin DNA (C1 and C2) to self-assemble into CHA products. After AgNPs@Capture captures the resulting CHA product, the HCR reaction is started, forming long-stranded DNA on the surface of AgNPs. A strong SERS signal is generated due to the presence of a large amount of the Raman reporter methylene blue (MB) in the vicinity of the SERS "hot spot" on the surface of AgNPs. The monitoring of the SERS signal changes of MB allows for the highly sensitive and specific detection of miR-21. In optimal conditions, the biosensor exhibits a satisfactory linear range and a low detection limit for miR-21 of 42.3 fM. Additionally, this SERS biosensor shows outstanding selectivity and reproducibility. The application of this methodology to clinical blood samples allows for the differentiation of cancer patients from healthy controls. As a result, the CHA-HCR amplification strategy used in this SERS biosensor could be a useful tool for miRNA detection and early cancer screening.