Isothermal Amplification Method Research Articles

Traditional to technological advancements in Ganoderma detection methods in oil palm.

Ganoderma sp., the fungal agent causing basal stem rot (BSR), poses a severe threat to global oil palm production. Alarming increases in BSR occurrences within oil palm growing zones are attributed to varying effectiveness in its current management strategies. Asymptomatic progression of the disease and the continuous monoculture of oil palm pose challenges for prompt and effective management. Therefore, the development of precise, early, and timely detection techniques is crucial for successful BSR management. Conventional methods such as visual assessments, culture-based assays, and biochemical and physiological approaches prove time-consuming and lack specificity. Serological-based diagnostic methods, unsuitable for fungal diagnostics due to low sensitivity, assay affinity, cross-contamination which further underscores the need for improved techniques. Molecular PCR-based assays, utilizing universal, genus-specific, and species-specific primers, along with functional primers, can overcome the limitations of conventional and serological methods in fungal diagnostics. Recent advancements, including real-time PCR, biosensors, and isothermal amplification methods, facilitate accurate, specific, and sensitive Ganoderma detection. Comparative whole genomic analysis enables high-resolution discrimination of Ganoderma at the strain level. Additionally, omics tools such as transcriptomics, proteomics, and metabolomics can identify potential biomarkers for early detection of Ganoderma infection. Innovative on-field diagnostic techniques, including remote methods like volatile organic compounds profiling, tomography, hyperspectral and multispectral imaging, terrestrial laser scanning, and Red-Green-Blue cameras, contribute to a comprehensive diagnostic approach. Ultimately, the development of point-of-care, early, and cost-effective diagnostic techniques accessible to farmers is vital for the timely management of BSR in oil palm plantations.

Molecular detection of toxoplasmosis in wild rats using loop-mediated isothermal amplification assay.

Toxoplasmosis is caused by the parasite Toxoplasma gondii. Cats are the only known hosts that excrete resistant oocysts. Wild rats serve as crucial reservoirs and intermediate hosts for T. gondii's survival and dissemination. Consuming soil and water containing oocysts can lead to illness. This study aimed to estimate the prevalence of toxoplasmosis in wild rats through molecular detection as an indicator of environmental contamination in Surabaya. One hundred rats were collected from the three areas (housing, dense settlements, and traditional markets) and distributed into the five zones: West, East, Central, North, and South of Surabaya. Brain tissue samples were extracted using a Geneaid™ (New Taipei City, Taiwan) DNA isolation kit and analyzed through the loop-mediated isothermal amplification (LAMP) method. The study analyzed brain tissue from 100 wild rats, consisting of 77 Rattus tanezumi and 33 Rattus norvegicus, displaying 30% LAMP positivity. The study revealed that 30% (30/100) of wild rats tested were infected with T. gondii. The molecular prevalence rate in male rats was 32.35% (22/68), compared to females with 25% (8/32). 41.9% of the housing population, 33.3% of traditional markets, and 22.6% of dense settlements had the highest molecular prevalence. The high positive molecular rate at the trapping site can be attributed to cats and dense populations. Thirty percentage wild rats were tested positive for toxoplasmosis in Surabaya, East Java, Indonesia using LAMP method. Implementing strict control and monitoring is crucial in preventing the transmission of diseases from wild rats to humans. It is necessary to carry out further research related to genetic analysis of T. gondii to determine the type of T. gondii that infects animals and humans in Surabaya through bioassay and molecular test.

Direct detection of Aeromonas hydrophila from water and tissue samples by loop-mediated isothermal amplification (LAMP) method

Direct detection of Aeromonas hydrophila from water and tissue samples by loop-mediated isothermal amplification (LAMP) method

Development and Comparison of Visual LAMP and LAMP-TaqMan Assays for Colletotrichum siamense.

Strawberry anthracnose caused by Colletotrichum spp. has resulted in significant losses in strawberry production worldwide. Strawberry anthracnose occurs mainly at the seedling and early planting stages, and Colletotrichum siamense is the main pathogen in North China, where mycelia, anamorphic nuclei, and conidia produced in the soil are the main sources of infection. The detection of pathogens in soil is crucial for predicting the prevalence of anthracnose. In this study, a visualized loop-mediated isothermal amplification (LAMP) assay and a loop-mediated isothermal amplification method combined with a TaqMan probe (LAMP-TaqMan) assay were developed for the β-tubulin sequence of C. siamense. Both methods can detect Colletotrichum siamense genomic DNA at very low concentrations (104 copies/g) in soil, while both the visualized LAMP and LAMP-TaqMan assays exhibited a detection limit of 50 copies/μL, surpassing the sensitivity of conventional PCR and qPCR techniques, and both methods showed high specificity for C. siamense. The two methods were compared: LAMP-TaqMan exhibited enhanced specificity due to the incorporation of fluorescent molecular beacons, while visualized LAMP solely necessitated uncomplicated incubation at a constant temperature, with the results determined by the color change; therefore, the requirements for the instrument are relatively straightforward and user-friendly. In conclusion, both assays will help monitor populations of C. siamense in China and control strawberry anthracnose in the field.

The Establishment of the Multi-Visual Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Vibrio harveyi, Vibrio parahaemolyticus, and Singapore grouper iridovirus

Groupers are valuable economic fish in the southern sea area of China, but the threat of disease is becoming more and more serious. Vibrio harveyi, V. parahaemolyticus, and Singapore grouper iridovirus (SGIV) are three important pathogens that cause disease in groupers, and infection with either a single one or a mix of these pathogens poses a serious threat to the healthy development of grouper culture. To enhance the rapid diagnosis and screening in the early stages, it is necessary to develop rapid detection methods of these pathogens. To simultaneously and rapidly detect the three pathogens, in this study, we utilized the TolC of V. harveyi, DNAJ of V. parahaemolyticus, and RAD2 of SGIV as the target genes and established a triple visual loop-mediated isothermal amplification (LAMP) method. This LAMP method showed a detection time as fast as 30 min and a high sensitivity of 100 fg/μL. Moreover, this method exhibited strong specificity and no cross-reaction with seven types of Vibrio and Staphylococcus aureus, as well as five common viruses in aquatic animals. Then, the LAMP products were enzymically cut, and three characteristic strips were used to identify the pathogen species. The results of the clinical trials demonstrated that the method could accurately and specifically detect V. harveyi, V. parahaemolyticus, and SGIV in grouper tissues. In summary, this study successfully established a triple visual LAMP rapid detection method for V. harveyi, V. parahaemolyticus, and SGIV. The method offers several advantages including simple equipment, easy operation, rapid reaction, high specificity, high sensitivity, and visual results. It is suitable for the early and rapid diagnosis of groupers infected with V. harveyi, V. parahaemolyticus, and SGIV, thereby providing useful technical support for further application in the large-scale disease surveillance of aquaculture animals.

Two-Stage Mixed-Dye-Based Isothermal Amplification with Ribonuclease-Cleavable Enhanced Probes for Dual-Visualization Detection of SARS-CoV-2 Variants of Interest.

Rapid and visual detection of SARS-CoV-2 variants is vital for timely assessment of variant transmission in resource-limited settings. Here, a closed-tube, two-stage, mixed-dye-based isothermal amplification method with ribonuclease-cleavable enhanced probes (REP), termed REP-TMAP, for dual-visualization detection of SARS-CoV-2 variants including JN.1, BA.2, BA.4/5, and Delta is introduced. The first stage of REP-TMAP is reverse transcription recombinase polymerase amplification and the second stage is dual-visualization detection synergistically mediated by the REP and the mixed dyes of cresol red and hydroxy naphthol blue. In REP-TMAP reaction, the color change under ambient light indicates SARS-CoV-2 infection, while the fluorescence change under blue light excitation specifies variant type. On detecting transcribed RNA of SARS-CoV-2 spike gene, this assay is rapid (within 40min), highly sensitive (10-200 copies per reaction), and highly specific (identification of single-base mutations). Furthermore, the assay has been clinically validated to accurately detect JN.1, BA.2, and BA.4/5 variants from 102 human oropharyngeal swabs. The proposed assay therefore holds great potentials to provide a rapid, dual-visualization, sensitive, specific, point-of-care detection of SARS-CoV-2 variants and beyond.

Authentication of the Apis cerana honey with the proofreading enzyme-mediated probe cleavage combined with ladder-shape melting temperature isothermal amplification (Proofman-LMTIA) method

Honey adulteration has been on the rise with the increasing demand for high-quality honey, and the authentication of A. cerana honey is a major problem that the current bee industry needs to solve. The aim of this research was to establish the proofreading enzyme-mediated probe cleavage combined with ladder-shape melting temperature isothermal amplification (Proofman-LMTIA) method for the authentication of Apis cerana honey. The LMTIA primers were designed to target the specific 18S rRNA sequence of A. cerana, and the conditions for Proofman-LMTIA amplification were optimized. The specificity and sensitivity of the method were determined and compared with that of the droplet digital polymerase chain reaction (PCR). The optimal temperature of the established Proofman-LMTIA method was 62 °C. The method can specially to detect the A. cerana gene without cross-reactivity with the genomes from other animal and plant species, and the amplification reaction can be finished within 20 min with the sensitivity of 10 pg A. cerana genomic DNA, which was more sensitive than the droplet digital PCR method. The obtained results demonstrate that the established Proofman-LMTIA procedure was specific, sensitive, and rapid, and can be used for authentication of A. cerana honey.

Development of a Simple Method to Detect the Carbapenemase-Producing Genes blaNDM, blaOXA-48-like, blaIMP, blaKPC, and blaVIM Using a LAMP Method with Lateral Flow DNA Chromatography.

Infections by carbapenemase-producing Enterobacterales constitute a global public health threat. The rapid and efficient diagnosis of Enterobacterales infection is critical for prompt treatment and infection control, especially in hospital settings. We developed a novel loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography to identify five major groups of carbapenemase-producing genes (blaNDM, blaOXA-48-like, blaIMP, blaKPC, and blaVIM). This method uses DNA-DNA hybridization-based detection in which LAMP products can be easily visualized as colored lines. No specific technical expertise, expensive equipment, or special facilities are required for this method, allowing its broad application. Here, 73 bacteria collections including strains with carbapenemase-producing genes were tested. Compared to sequencing results, LAMP DNA chromatography for five carbapenemase-producing genes had a sensitivity and specificity of 100% and >97%, respectively. This newly developed method can be a valuable rapid diagnostic test to guide appropriate treatments and infection control measures, especially in resource-limited settings.

Development of a multiplex Loop-Mediated Isothermal Amplification (LAMP) for the diagnosis of bacterial periprosthetic joint infection.

Periprosthetic joint infection (PJI) is one of the most serious and debilitating complications that can occur after total joint arthroplasty. Therefore, early diagnosis and appropriate treatment are important for a good prognosis. Recently, molecular diagnostic methods have been widely used to detect the causative microorganisms of PJI sensitively and rapidly. The Multiplex Loop-Mediated Isothermal Amplification (LAMP) method eliminates the complex temperature cycling and delays caused by temperature transitions seen in polymerase chain reaction (PCR) methods, making it faster and easier to perform compared to PCR-based assays. Therefore, this study developed a multiplex LAMP assay for diagnosing bacterial PJI using LAMP technology and evaluated its analytical and clinical performance. We developed a multiplex LAMP assay for the detection of five bacteria: Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Pseudomonas aeruginosa, and Escherichia coli, frequently observed to be the causative agents of PJI. The method of analytical sensitivity and cross-reactivity were determined by spiking standard strains into the joint synovial fluid. The analytical sensitivity of the multiplex LAMP assay was compared with that of a quantitative real-time PCR (qPCR) assay. Clinical performance was evaluated using 20 joint synovial fluid samples collected from patients suspected of having bacterial PJI. The analytical sensitivity of the gram-positive bacterial multiplex LAMP assay and qPCR were 105/104 CFU/mL, 103/103 CFU/mL, and 105/104 CFU/mL against S. agalactiae, S. epidermidis, and S. aureus, respectively. For P. aeruginosa and E. coli, the analytical sensitivity of the multiplex LAMP and qPCR assays were 105/104 and 106/104 CFU/mL, respectively. The multiplex LAMP assay detects target bacteria without cross-reacting with other bacteria, and exhibited 100% sensitivity and specificity in clinical performance evaluation. This multiplex LAMP assay can rapidly detect five high-prevalence bacterial species causing bacterial PJI, with excellent sensitivity and specificity, in less than 1 h, and it may be useful for the early diagnosis of PJI.

Development of a multiplex one-tube loop-mediated isothermal amplification method for rapidly simultaneous detection of the mecA and nuc genes in methicillin-resistant Staphylococcus aureus

This study developed a multiplex one-tube loop-mediated isothermal amplification method using fluorescent dye-labeled DNA probes (mfLAMP) for the rapid simultaneous detection of the nuc (S. aureus-specific marker) and mecA genes (PBP2a-specific marker) in methicillin-resistant Staphylococcus aureus (MRSA). In the mfLAMP method, a graphene oxide (GO) was used as an effective adsorbent to eliminate free fluorescent dye-labeled DNA probes (mecAf-probe: Cy5-ssDNA-Biotin and nucf-probe: FAM-ssDNA-Biotin) present in negative results. The samples were then measured using a developed program that enable simultaneous detection of fluorescein (FAM) and Cyanine5 (Cy5). Green fluorescence represents the detection of the nuc gene, red fluorescence indicates the detection of the mecA gene, and yellow fluorescence signifies the detection of MRSA. The mfLAMP was specific to MRSA and could detect 10°CFU/mL of MRSA in standard buffer and artificially inoculated irrigation water. The recovery rate from contaminated irrigation water ranged from 100.41 to 109.85 %. This study is the first to report the mfLAMP using GO, which could be point-of-care testing for the rapid and simultaneous detection of MRSA's nuc and mecA genes. The results demonstrate that the mfLAMP will be a novel tool for simultaneously detecting more than two target genes or bacteria within 1 h without an enrichment step.

Identification of Salmonella Enterica Serovar Typhi DNA by loop-mediated isothermal amplification with fluorescent detection

Introduction. Real-time PCR may be used along with loop isothermal amplification method (LAMP) allowing to conduct the study in 30–40 minutes to diagnose typhoid fever. Several LAMP assay variations for detecting Salmonella enterica serovar Typhi have been described. The studies report that relevant primers were tested on strains specific for Malaysia and China. We attempted to evaluate the LAMP primers described above for identifying S. Typhi strains specific for the Russian Federation and compare their sensitivity and specificity with each other. Materials and methods. A comparative in silico analysis of target sequences was carried out both in the open NCBI database and among the genetic sequences of collection strains at the St. Petersburg Pasteur Institute. Several sets of primers for LAMP amplification of various S. Typhi genome regions were tested. The tested primers amplify the following fragments: SalTyp1 — region of the STY1607 gene, SalTyp2 — region of the STY2879 gene, SalTyp3 — region of the STBHUCCB_38510 gene of the PapD chaperone. Results. In silico analysis of LAMP primers showed that only the SalTyp 3 set has strict specificity for Salmonella enterica serovar Typhi. For the SalTyp 1 and SalTyp 2 an opportunity of false-positive reactions with some E. coli strains was shown. A LAMP method for DNA fluorescent detection of the typhoid fever causative agent was chosen. Assay has been based on a marker gene termed STBHUCCB_38510 and amplification with six specific primers. The detection limit was 20 copies/reaction in reference plasmids and the reaction time lasted for 35 minutes. The specificity of the method was tested on DNA specimens of 20 S. Typhi isolates and 90 strains of other heterologous bacteria from 24 different species. No false positive or false negative results were identified. Conclusion. The developed method can be used in clinical practice for laboratory confirmation of typhoid fever diagnosis as a part of epidemiological monitoring of environmental objects as well as food products.

Isolation of Vibrio cholerae and Vibrio vulnificus from Estuarine Waters, and Genotyping of V. vulnificus Isolates Using Loop-Mediated Isothermal Amplification.

Bacteria in the genus Vibrio are ubiquitous in estuarine and coastal waters. Some species (including Vibrio cholerae and Vibrio vulnificus) are known human pathogens causing ailments like cholera, diarrhea, or septicemia. Notably, V. vulnificus can also cause a severe systemic infection (known as vibriosis) in eels raised in aquaculture facilities. Water samples were periodically collected from the estuary of the Asahi River, located in the southern part of Okayama City, Japan. These samples were directly plated onto CHROMagar Vibrio plates, and colonies displaying turquoise-blue coloration were selected. Thereafter, polymerase chain reaction was used to identify V. cholerae and V. vulnificus. A total of 30 V. cholerae strains and 194 V. vulnificus strains were isolated during the warm season when the water temperature (WT) was higher than 20 °C. Concurrently, an increase in coliforms was observed during this period. Notably, V. vulnificus has two genotypes, designated as genotype 1 and genotype 2. Genotype 1 is pathogenic to humans, while genotype 2 is pathogenic to both humans and eels. The loop-mediated isothermal amplification method was developed to rapidly determine genotypes at a low cost. Of the 194 strains isolated, 80 (41.2%) were identified as genotype 1 strains. Among the 41 strains isolated when the WTs were higher than 28 °C, 25 strains (61.0%) belonged to genotype 1. In contrast, of the 32 strains isolated when the WTs were lower than 24 °C, 27 strains (84.4%) belonged to genotype 2. These results suggest that the distribution of the two genotypes was influenced by WT.

Molecular and epidemiological characterization of Staphylococcus aureus causing bovine mastitis in China

BackgroundStaphylococcus aureus is one of the most prevalent pathogens in bovine mastitis, which leads to substantial financial losses for the dairy industry. ResultsIn this study, S. aureus (n = 72) was isolated from 18 dairy farms in 15 provinces across China in 2021. The identification of these isolates at the species level was achieved by employing 16S rRNA sequencing. An isothermal amplification method for auxiliary detection of S. aureus was established, which can be employed not only for laboratory detection but also for point-of-care testing (POCT). Molecular characteristics of S. aureus mastitis in Chinese dairy cows were determined through MLST and spa typing. Finally, methicillin-resistant Staphylococcus aureus (MRSA) and MRSA resistance genes were detected using MIC and PCR amplification techniques. 72 isolates were identified as 12 sequence types (STs) and 7 clonal complexes (CC). ST1/CC1 was the dominant prevalent accounting for 33.3 % of the total, and exhibiting a wide distribution range. In terms of spa types, t114 was the dominant type, accounting for 31.9 % of the total, followed by t529 as the second major type. Four S. aureus strains were classified as MRSA according to their levels of oxacillin resistance (MIC ≥4 μg/mL). Among these four MRSA strains, one of them was found to be mecA positive. However, the presence of drug-resistance genes mecA and mecC was not detected in the remaining three MRSA strains, indicating the possible existence of new resistance genes. ConclusionsOur study investigated the prevalence of S. aureus mastitis in dairy cows in China, while also examined the molecular characteristics and MRSA strains. This information will help with the clinical monitoring, prevention, and control of S. aureus mastitis in dairy cattle.

DEVELOPMENT AND VALIDATION OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) FOR DETECTION OF LEPTOSPIR GENETIC MATERIAL

Primer systems were designed and tested and the method of loop-mediated isothermal amplification was validated for rapid diagnosis of leptospirosis. In general, five sets of primers were calculated and analyzed in situ and in vitro, which were ordered for synthesis and chosen for the future as the most promising. Indicators of analytical sensitivity and detection limits of the method were established, the specificity of the method in its tests with homologous and close to taxonomic characteristics heterologous samples of genetic material was proved and completed reproducibility of the method was established under the conditions of its staging on various laboratory equipment and with reagents from different manufacturers. The developed technique of loop-mediated amplification for the diagnosis of leptospirosis was tested on field samples and its diagnostic effectiveness was established.

Validation and optimization of the loop-mediated isothermal amplification (LAMP) technique for rapid detection of wheat stripe mosaic virus, a wheat-infecting pathogen

BackgroundWheat stripe mosaic virus (WhSMV) is a significant wheat pathogen that causes substantial yield losses in Brazil and other countries. Although several detection methods are available, reliable and efficient tools for on-site WhSMV detection are currently lacking. In this study, a Loop-Mediated Isothermal Amplification (LAMP) method was developed for rapid and reliable field detection of WhSMV. We designed WhSMV-specific primers for the LAMP assay and optimized reaction conditions for increased sensitivity and specificity using infected plant samples. ResultsWe have developed a diagnostic method utilizing the Loop-Mediated Isothermal Amplification (LAMP) technique capable of rapidly and reliably detecting WhSMV. The LAMP assay has been optimized to enhance sensitivity, specificity, and cost-effectiveness. ConclusionThe LAMP assay described here represents a valuable tool for early WhSMV detection, serving to mitigate the adverse economic and social impacts of this viral pathogen. By enabling swift and accurate identification, this assay can significantly improve the sustainability of cereal production systems, safeguarding crop yields against the detrimental effects of WhSMV.

Evaluation of the SARS-CoV-2 RNA detection reagent LAMPdirect Genelyzer KIT using nasopharyngeal swab and saliva samples

The LAMPdirect Genelyzer KIT allows for the detection of SARS-CoV-2 RNA in saliva samples with a loop-mediated isothermal amplification (LAMP) method and generates results within 20 min. It has been approved by the Pharmaceuticals and Medical Devices Agency in Japan. In this study, the performance of the LAMPdirect Genelyzer KIT was compared with that of the RT-qPCR reference method using 50 nasopharyngeal swabs and 100 saliva samples. In addition, we evaluated the applicability of an alternative reverse transcriptase and the effects of an inactivation buffer. The total agreement rates were 80.0 % and 82.0 % for nasopharyngeal and saliva samples, respectively. When considering samples at the detection limit (50 copies/reaction) that increases the chance of transmission between humans, the total agreement rates were 100% and 94.1% for nasopharyngeal and saliva samples, respectively. The LAMP method is simple, fast, and inexpensive, making it useful for small medical institutions or rural areas.

Large-volume RT-LAMP enables extraction-free amplification of HIV RNA from fingerstick plasma

BackgroundPoint-of-care (POC) nucleic acid amplification tests (NAAT) can significantly expand testing coverage, which is critical for infectious disease diagnostics and monitoring. The development of various isothermal amplification techniques greatly simplifies NAATs, but the cumbersome nucleic acid extraction step remains a bottleneck for the POC. Alternatively, extraction-free amplification, where crude samples are directly added into the assay, substantially simplifies the workflow. However, sample dilution is often needed in extraction-free amplification to reduce assay inhibition from sample matrices. Since NAATs are typically run at small volumes around 20 μL, the input sample quantity is therefore limited, resulting in an inevitable sensitivity loss. ResultsHere we explore the potential to perform isothermal amplification in larger reaction volumes to accommodate larger sample quantities, thereby improving sensitivity in extraction-free amplification. We demonstrated the approach by developing large-volume reverse transcription loop-mediated isothermal amplification (RT-LAMP) for HIV RNA detection from fingerstick plasma. We found that LAMP at reaction volumes up to 1 mL maintained the same performance. We then identified plasma dilution conditions needed to maintain the limit of detection in RT-LAMP. Subsequently, using inactivated HIV virus, we showed the successful detection of 24 HIV RNA copies in a 500 μL RT-LAMP reaction in the presence of 20 μL plasma (fingerstick volumes), translating to a viral load of 1200 copies per mL. To reduce the increased reagent cost with expanded reaction volumes, we further identified lower-cost reagents with maintained assay performance. Moreover, we showed that large-volume LAMP, compared to 20 μL reactions, could tolerate higher concentrations of various inhibitors in the sample, such as albumin and GuSCN. Significance and noveltyNAATs are conventionally conducted at small reaction volumes. Here we demonstrated that LAMP can be run at large reaction volumes (over 100 μL) with maintained assay performance, allowing sample inhibition to be mitigated while accommodating larger sample quantities. The same strategy of expanding reaction volumes could be applied to other isothermal amplification methods and various POC applications, to streamline test workflows and/or improve assay sensitivity.

Simple, sensitive, and visual detection of 12 respiratory pathogens with one-pot-RPA-CRISPR/Cas12a assay.

Respiratory infections pose a serious threat to global public health, underscoring the urgent need for rapid, accurate, and large-scale diagnostic tools. In recent years, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, combined with isothermal amplification methods, has seen widespread application in nucleic acid testing (NAT). However, achieving a single-tube reaction system containing all necessary components is challenging due to the competitive effects between recombinase polymerase amplification (RPA) and CRISPR/Cas reagents. Furthermore, to enable precision medicine, distinguishing between bacterial and viral infections is essential. Here, we have developed a novel NAT method, termed one-pot-RPA-CRISPR/Cas12a, which combines RPA with CRISPR molecular diagnostic technology, enabling simultaneous detection of 12 common respiratory pathogens, including sixbacteria and sixviruses. RPA and CRISPR/Cas12a reactions are separated by paraffin, providing an independent platform for RPA reactions to generate sufficient target products before being mixed with the CRISPR/Cas12a system. Results can be visually observed under LED blue light. The sensitivity of the one-pot-RPA-CRISPR/Cas12a method is 2.5 × 100copies/μL plasmids, with no cross-reaction with other bacteria or viruses. Additionally, the clinical utility was evaluated by testing clinical isolates of bacteria and virus throat swab samples, demonstrating favorable performance. Thus, our one-pot-RPA-CRISPR/Cas12a method shows immense potential for accurate and large-scale detection of 12 common respiratory pathogens in point-of-care testing.

Establishment of a real‐time fluorescence and visual colorimetric detection method for Staphylococcus aureus based on loop‐mediated isothermal amplification

AbstractA loop‐mediated isothermal amplification (LAMP) method combined with calcein was established for the detection of Staphylococcus aureus. LAMP primers were designed targeting the ASL18_04625 species‐specific gene screened through bioinformatics and PCR analysis. The target gene and detection method demonstrated 100% specificity for S. aureus and no cross‐reactions with other pathogens. For LAMP reactions, the results could be directly observed with the naked eye or evaluated by S‐shaped amplification curves of fluorescence signals. The assay's detection limit was 3.395 × 10−4 ng/μL genomic DNA or 3.21 CFU/mL pure bacterial culture. Furthermore, the target bacteria of 8.5 × 105–8.5 × 100 CFU/mL could be accurately detected in spiked milk samples, and the overall detection process could be completed within 40 min. This method improved the accuracy and convenience for S. aureus detection and provied a dependable tool for the rapid screening of S. aureus.

An Argonaute-mediated bio-barcode bioassay for one-tube and on-site detection of Staphylococcus aureus

Isothermal amplification method has enabled major advancement toward rapid and deployable detection for nucleic acid. While exciting, there are still a series of deficiencies facing their practical implementation, such as carryover cross-contamination, which are major bottlenecks that must be addressed. In addition, there is a lack of high-throughput detection for foodborne pathogens in foods. Here, we reported an Argonaute-mediated bio-barcode bioassay for one-step cleavage, on-site and high-throughput detection of S. aureus (Staphylococcus aureus) through introducing Tag sequence and recombinase aided amplification (RAA). Furthermore, we designed a single vessel via tube-in-tube manner for one-tube contamination-free and portable detection without opening the lid. With this strategy, the same detection limit as fluorescence detection could be obtained, 1 CFU/mL with high specificity for S. aureus. We further evaluated the performance of bio-barcode biosensing strategy using S. aureus-contaminated clinical and food samples. Argonaute-mediated bio-barcode bioassay had the advantages of modular, universal plug-and-play and high-throughput scanning. This work not only satisfied the requirement for convenient detection but also fulfilled the need for portable on-site nucleic acid detection with portability, adaptability, transferability and practicality.