Immunoglobulin A coating identifies colitogenic bacteria in inflammatory bowel disease.

Specific members of the intestinal microbiota dramatically affect inflammatory bowel disease (IBD) in mice. In humans, however, identifying bacteria that preferentially affect disease susceptibility and severity remains a major challenge. Here, we used flow-cytometry-based bacterial cell sorting and 16S sequencing to characterize taxa-specific coating of the intestinal microbiota with immunoglobulin A (IgA-SEQ) and show that high IgA coating uniquely identifies colitogenic intestinal bacteria in a mouse model of microbiota-driven colitis. We then used IgA-SEQ and extensive anaerobic culturing of fecal bacteria from IBD patients to create personalized disease-associated gut microbiota culture collections with predefined levels of IgA coating. Using these collections, we found that intestinal bacteria selected on the basis of high coating with IgA conferred dramatic susceptibility to colitis in germ-free mice. Thus, our studies suggest that IgA coating identifies inflammatory commensals that preferentially drive intestinal disease. Targeted elimination of such bacteria may reduce, reverse, or even prevent disease development.

The source of the Black Death in fourteenth-century central Eurasia.

The origin of the medieval Black Death pandemic (AD 1346-1353) has been a topic of continuous investigation because of the pandemic's extensive demographic impact and long-lasting consequences1,2. Until now, the most debated archaeological evidence potentially associated with the pandemic's initiation derives from cemeteries located near Lake Issyk-Kul of modern-day Kyrgyzstan1,3-9. These sites are thought to have housed victims of a fourteenth-century epidemic as tombstone inscriptions directly dated to 1338-1339 state 'pestilence' as the cause of death for the buried individuals9. Here we report ancient DNA data from seven individuals exhumed from two of these cemeteries, Kara-Djigach and Burana. Our synthesis of archaeological, historical and ancient genomic data shows a clear involvement of the plague bacterium Yersinia pestis in this epidemic event. Two reconstructed ancient Y. pestis genomes represent a single strain and are identified as the most recent common ancestor of a major diversification commonly associated with the pandemic's emergence, here dated to the first half of the fourteenth century. Comparisons with present-day diversity from Y. pestis reservoirs in the extended Tian Shan region support a local emergence of the recovered ancient strain. Through multiple lines of evidence, our data support an early fourteenth-century source of the second plague pandemic in central Eurasia.

Viscoelastic control of spatiotemporal order in bacterial active matter.

Active matter consists of units that generate mechanical work by consuming energy1. Examples include living systems (such as assemblies of bacteria2-5 and biological tissues6,7), biopolymers driven by molecular motors8-11 and suspensions of synthetic self-propelled particles12-14. A central goal is to understand and control the self-organization of active assemblies in space and time. Most active systems exhibit either spatial order mediated by interactions that coordinate the spatial structure and the motion of active agents12,14,15 or the temporal synchronization of individual oscillatory dynamics2. The simultaneous control of spatial and temporal organization is more challenging and generally requires complex interactions, such as reaction-diffusion hierarchies16 or genetically engineered cellular circuits2. Here we report a simple technique to simultaneously control the spatial and temporal self-organization of bacterial active matter. We confine dense active suspensions of Escherichia coli cells and manipulate a single macroscopic parameter-namely, the viscoelasticity of the suspending fluid- through the addition of purified genomic DNA. This reveals self-driven spatial and temporal organization in the form of a millimetre-scale rotating vortex with periodically oscillating global chirality of tunable frequency, reminiscent of a torsional pendulum. By combining experiments with an active-matter model, we explain this behaviour in terms of the interplay between active forcing and viscoelastic stress relaxation. Our findings provide insight into the influence of bacterial motile behaviour in complex fluids, which may be of interest in health- and ecology-related research, and demonstrate experimentally that rheological properties can be harnessed to control active-matter flows17,18. We envisage that our millimetre-scale, tunable, self-oscillating bacterial vortex may be coupled to actuation systems to act a 'clock generator' capable of providing timing signals for rhythmic locomotion of soft robots and for programmed microfluidic pumping19, for example, by triggering the action of a shift register in soft-robotic logic devices20.

Thymic development of gut-microbiota-specific T cells.

Humans and their microbiota have coevolved a mutually beneficial relationship in which the human host provides a hospitable environment for the microorganisms and the microbiota provides many advantages for the host, including nutritional benefits and protection from pathogen infection1. Maintaining this relationship requires a careful immune balance to contain commensal microorganisms within the lumen while limiting inflammatory anti-commensal responses1,2. Antigen-specific recognition of intestinal microorganisms by T cells has previously been described3,4. Although the local environment shapes the differentiation of effector cells3-5 it is unclear how microbiota-specific T cells are educated in the thymus. Here we show that intestinal colonization in early life leads to the trafficking of microbial antigens from the intestine to the thymus by intestinal dendritic cells, which then induce the expansion of microbiota-specific T cells. Once in the periphery, microbiota-specific T cells have pathogenic potential or can protect against related pathogens. In this way, the developing microbiota shapes and expands the thymic and peripheral T cell repertoire, allowing for enhanced recognition of intestinal microorganisms and pathogens.

Human placenta has no microbiome but can contain potential pathogens.

We sought to determine whether pre-eclampsia, spontaneous preterm birth or the delivery of infants who are small for gestational age were associated with the presence of bacterial DNA in the human placenta. Here we show that there was no evidence for the presence of bacteria in the large majority of placental samples, from both complicated and uncomplicated pregnancies. Almost all signals were related either to the acquisition of bacteria during labour and delivery, or to contamination of laboratory reagents with bacterial DNA. The exception was Streptococcus agalactiae (group B Streptococcus), for which non-contaminant signals were detected in approximately 5% of samples collected before the onset of labour. We conclude that bacterial infection of the placenta is not a common cause of adverse pregnancy outcome and that the human placenta does not have a microbiome, but it does represent a potential site of perinatal acquisition of S. agalactiae, a major cause of neonatal sepsis.

Serum Cell-Free DNA-based Detection of Mycobacterium avium Complex Infection.

Rationale: Mycobacterium avium complex (MAC) is the most common cause of nontuberculous mycobacterial (NTM) pulmonary disease (PD), which exhibits increasing global incidence. Current microbiologic methods routinely used in clinical practice lack sensitivity and have long latencies, leading to delays in diagnosis and treatment initiation and evaluation. A clustered regularly interspaced short palindromic repeats (CRISPR)-based assay that measures MAC cell-free DNA (cfDNA) concentrations in serum could provide a rapid means to detect MAC infection and monitor response to antimicrobial treatment. Objectives: To develop and optimize a CRISPR MAC assay for MAC infection detection and to evaluate its diagnostic and prognostic performance in two MAC disease cohorts. Methods: MAC cfDNA serum concentrations were measured in individuals with diagnoses of MAC disease or who had bronchiectasis or chronic obstructive pulmonary disease diagnoses without histories of NTM PD or NTM-positive sputum cultures. Diagnostic performance was analyzed using pretreatment serum from two cohorts. Serum MAC cfDNA changes during MAC PD treatment were evaluated in a subset of patients with MAC PD who received macrolide-based multidrug regimens. Measurements and Main Results: The CRISPR MAC assay detected MAC cfDNA in MAC PD with 97.6% (91.6-99.7%) sensitivity and 97.6% (91.5-99.7%) specificity overall. Serum MAC cfDNA concentrations markedly decreased after MAC-directed treatment initiation in patients with MAC PD who demonstrated MAC culture conversion. Conclusions: This study provides preliminary evidence for the utility of a serum-based CRISPR MAC assay to rapidly detect MAC infection and monitor the response to treatment.

Field-Deployable Colorimetric Array for On-Site Diagnosis of Urinary Tract Infection and Identification of Causative Pathogens.

Urinary tract infection (UTI) is a common and prevalent disease caused by a spectrum of pathogens. Lack of access to rapid, portable, and high-quality diagnostics in resource-limited settings aggravates the improper treatment of UTIs, which is also a major driver of antibiotic misuse worldwide. Here, we describe a custom-made portable colorimetric array (PoCA) for reading out polymerase chain reaction (PCR) amplicons, the rationale of which is to transfer the previously developed dsDNA-based photosensitization colorimetric assay (solution) onto paper discs for detection. By integrating mini-LED irradiation and paper discs, the PoCA can read out 96 PCR tests in one pot, thus allowing diagnosis and identification of 12 prevailing UTI pathogens in less than 2 h, coupled with a portable thermal cycler for PCR. After analyzing 200 clinical urine samples, the pathogen profiling accuracy of the PoCA was demonstrated to be higher than the standard urine culture (confirmed with metagenomic next-generation sequencing). The PoCA platform could be used in primary care for rapid UTI diagnosis and pathogen identification.

A New SPQC Biosensor for the Detection of a New Target of Escherichia/Shigella Genera Based on a Novel Method of Synthesizing Long-Range DNA.

The rapid and sensitive detection of Escherichia/Shigella genera is crucial for human disease and health. This study introduces a novel series of piezoelectric quartz crystal (SPQC) sensors for detecting Escherichia/Shigella genera. In this innovative biosensor, we propose a new target and novel method for synthesizing long-range DNA. The method relies on the amplification of two DNA probes, referred to as H and P amplification (HPA), resulting in the products of long-range DNA named Sn. The new target was screened from the 16S rRNA gene and utilized as a biomarker. The SPQC sensor operates as follows: the Capture probe is modified on the electrodes. In the presence of a Displace probe and target, the Capture can form a complex with the Displace probe. The resulting complex hybridizes with Sn, bridging the gap between the electrodes. Finally, silver wires are deposited between the electrodes using Sn as a template. This process results in a sensitive response from the SPQC. The detection limit of the SPQC sensor is 1 CFU/mL, and the detection time is within 2 h. This sensor would be of great benefit for food safety monitoring and clinical diagnosis.

Ultrasensitive Dual ELONA/SERS-RPA Multiplex Diagnosis of Antimicrobial Resistance.

Antimicrobial resistance (AMR) is a significant global health threat concern, necessitating healthcare practitioners to accurately prescribe the most effective antimicrobial agents with correct doses to combat resistant infections. This is necessary to improve the therapeutic outcomes for patients and prevent further increase in AMR. Consequently, there is an urgent need to implement rapid and sensitive clinical diagnostic methods to identify resistant pathogenic strains and monitor the efficacy of antimicrobials. In this study, we report a novel proof-of-concept magnetic scaffold-recombinase polymerase amplification (RPA) technique, coupled with an enzyme-linked oligonucleotide assay (ELONA) and surface-enhanced Raman scattering (SERS) detection, aimed at selectively amplifying and detecting the DNA signature of three resistant carbapenemase genes, VIM, KPC, and IMP. To achieve this, streptavidin-coated magnetic beads were functionalized with biotin-modified forward primers. RPA was conducted on the surface of the beads, resulting in an immobilized duplex amplicon featuring a single overhang tail specific to each gene. These tails were subsequently hybridized with recognition HRP probes conjugated to a complementary single-stranded oligonucleotide and detected colorimetrically. Additionally, they underwent hybridization with similar selective SERS probes and were measured using a handheld Raman spectrometer. The resulting quantification limits were at subpicomolar level for both assays, allowing the potential for early diagnosis. Moreover, we demonstrated the platform capability to conduct a multiplex RPA-SERS detection of the three genes in a single tube. Compared to similar approaches like PCR, RPA offers advantages of speed, affordability, and isothermal operation at 37 °C, eliminating the need for a thermal cycler. The whole assay was completed within <2 h. Therefore, this novel magnetic scaffold ELONA/SERS-RPA platform, for DNA detection, demonstrated excellent capability for the rapid monitoring of AMR in point-of-care applications, in terms of sensitivity, portability, and speed of analysis.

Pontiella agarivorans sp. nov., a novel marine anaerobic bacterium capable of degrading macroalgal polysaccharides and fixing nitrogen.

Marine macroalgae produce abundant and diverse polysaccharides, which contribute substantially to the organic matter exported to the deep ocean. Microbial degradation of these polysaccharides plays an important role in the turnover of macroalgal biomass. Various members of the Planctomycetes-Verrucomicrobia-Chlamydia (PVC) superphylum are degraders of polysaccharides in widespread anoxic environments. In this study, we isolated a novel anaerobic bacterial strain NLcol2T from microbial mats on the surface of marine sediments offshore Santa Barbara, CA, USA. Based on 16S ribosomal RNA (rRNA) gene and phylogenomic analyses, strain NLcol2T represents a novel species within the Pontiella genus in the Kiritimatiellota phylum (within the PVC superphylum). Strain NLcol2T is able to utilize various monosaccharides, disaccharides, and macroalgal polysaccharides such as agar and É©-carrageenan. A near-complete genome also revealed an extensive metabolic capacity for anaerobic degradation of sulfated polysaccharides, as evidenced by 202 carbohydrate-active enzymes (CAZymes) and 165 sulfatases. Additionally, its ability of nitrogen fixation was confirmed by nitrogenase activity detected during growth on nitrogen-free medium, and the presence of nitrogenases (nifDKH) encoded in the genome. Based on the physiological and genomic analyses, this strain represents a new species of bacteria that may play an important role in the degradation of macroalgal polysaccharides and with relevance to the biogeochemical cycling of carbon, sulfur, and nitrogen in marine environments. Strain NLcol2T (= DSM 113125T = MCCC 1K08672T) is proposed to be the type strain of a novel species in the Pontiella genus, and the name Pontiella agarivorans sp. nov. is proposed.IMPORTANCEGrowth and intentional burial of marine macroalgae is being considered as a carbon dioxide reduction strategy but elicits concerns as to the fate and impacts of this macroalgal carbon in the ocean. Diverse heterotrophic microbial communities in the ocean specialize in these complex polymers such as carrageenan and fucoidan, for example, members of the Kiritimatiellota phylum. However, only four type strains within the phylum have been cultivated and characterized to date, and there is limited knowledge about the metabolic capabilities and functional roles of related organisms in the environment. The new isolate strain NLcol2T expands the known substrate range of this phylum and further reveals the ability to fix nitrogen during anaerobic growth on macroalgal polysaccharides, thereby informing the issue of macroalgal carbon disposal.

Simple tape-stripping method for highly reliable and quantitative analysis of skin microbiome.

The composition of human skin microbiome profoundly impacts host skin health and disease. However, the relationship between skin homeostasis or the development of skin diseases and daily changes in skin microbial composition is poorly understood. Longitudinal samplings at more frequent intervals would address this issue, while conventional sampling methods have technical difficulties, leading to limitations in sampling opportunities. Here, we developed a simple and stable tape-stripping method regardless of the operator's skill. Our method enables skin microbial sampling within 30 seconds and taking multiple skin microbial samples from the same body site. The amount of microbial DNA among multiple sampling sites could be measured within 13.5%. The sequencing results of multiple sampling showed high consistency, Pearson's correlation coefficient between multiple samples of 0.98. Furthermore, these results were comparable to those collected by the conventional swabbing method. These results demonstrate that our tape-stripping method enables simple microbiome collection and highly reliable quantitative skin microbiome analysis. These features of our method would lead to a further understanding of skin disease development or diagnosis of skin conditions in clinical research by increasing the opportunities for microbial sampling.

Portable loop-mediated isothermal amplification device with spectrometric detection for rapid pathogen identification.

With the rise in extreme weather due to global warming, coupled with globalization facilitating the spread of infectious diseases, there's a pressing need for portable testing platforms offering simplicity, low cost, and remote transmission, particularly beneficial in resource-limited and non-urban areas. We have developed a portable device using loop-mediated isothermal amplification (LAMP) with spectrometric detection to identify Salmonella Typhimurium DNA. The device utilizes the LinkIt 7697 microcontroller and a microspectrometer to capture and transmit spectral signals in real-time, allowing for improved monitoring and analysis of the reaction progress. We built a hand-held box containing a microspectrometer, thermoelectric cooler, ultraviolet LED, disposable reaction tube, and homemade thermal module, all powered by rechargeable batteries. Additionally, we conducted thorough experiments to ensure temperature accuracy within 1 °C under thermal control, developed a heating module with a LinkIt 7697 IoT development board to heat the DNA mixture to the reaction temperature within 3 min, and integrated foam insulation and a 3D-printed frame to enhance the device's thermal stability. We successfully demonstrated the amplification of Salmonella Typhimurium DNA with an impressive sensitivity of 2.83 × 10-4 ng/µL. A remote webpage interface allows for monitoring the temperature and fluorescence during the LAMP process, improving usability. This portable LAMP device with real-time detection offers a cost-effective solution for detecting Salmonella Typhimurium in food products. Its unique design and capabilities make it a promising tool for ensuring food safety.

Standard addition method for rapid, cultivation-independent quantification of Legionella pneumophila cells by qPCR in biotrickling filters.

Cultivation-independent molecular biological methods are essential to rapidly quantify pathogens like Legionella pneumophila (L. pneumophila) which is important to control aerosol-generating engineered water systems. A standard addition method was established to quantify L. pneumophila in the very complex matrix of process water and air of exhaust air purification systems in animal husbandry. Therefore, cryopreserved standards of viable L. pneumophila were spiked in air and water samples to calibrate the total bioanalytical process which includes cell lysis, DNA extraction, and qPCR. A standard addition algorithm was employed for qPCR to determine the initial concentration of L. pneumophila. In mineral water, the recovery rate of this approach (73%-134% within the concentration range of 100-5000 Legionella per mL) was in good agreement with numbers obtained from conventional genomic unit (GU) calibration with DNA standards. In air samples of biotrickling filters, in contrast, the conventional DNA standard approach resulted in a significant overestimation of up to 729%, whereas our standard addition gave a more realistic recovery of 131%. With this proof-of-principle study, we were able to show that the molecular biology-based standard addition approach is a suitable method to determine realistic concentrations of L. pneumophila in air and process water samples of biotrickling filter systems. Moreover, this quantification strategy is generally a promising method to quantify pathogens in challenging samples containing a complex microbiota and the classical GU approach used for qPCR leads to unreliable results.

Multiplex detection of bacterial pathogens by PCR/SERS assay.

Bacterial infections are a leading cause of death globally. The detection of DNA sequences correlated to the causative pathogen has become a vital tool in medical diagnostics. In practice, PCR-based assays for the simultaneous detection of multiple pathogens currently rely on probe-based quantitative strategies that require expensive equipment but have limited sensitivity or multiplexing capabilities. Hence, novel approaches to address the limitations of the current gold standard methods are still in high demand. In this study, we propose a simple multiplex PCR/SERS assay for the simultaneous detection of four bacterial pathogens, namely P. aeruginosa, S. aureus, S. epidermidis, and M. smegmatis. Wherein, specific primers for amplifying each target gDNA were applied, followed by applying SERS nanotags functionalized with complementary DNA probes and Raman reporters for specific identification of the target bacterial pathogens. The PCR/SERS assay showed high specificity and sensitivity for genotyping bacterial pathogen gDNA, whereby as few as 100 copies of the target gDNA could be detected. With high sensitivity and the convenience of standard PCR amplification, the proposed assay shows great potential for the sensitive detection of multiple pathogen infections to aid clinical decision-making.

A multi-channel electrochemical biosensor based on polyadenine tetrahedra for the detection of multiple drug resistance genes.

Antimicrobial resistance poses a serious threat to human health due to the high morbidity and mortality caused by drug-resistant microbial infections. Therefore, the development of rapid, sensitive and selective identification methods is key to improving the survival rate of patients. In this paper, a sandwich-type electrochemical DNA biosensor based on a polyadenine-DNA tetrahedron probe was constructed. The key experimental conditions were optimized, including the length of polyadenine, the concentration of the polyadenine DNA tetrahedron, the concentration of the signal probe and the hybridization time. At the same time, poly-avidin-HRP80 was used to enhance the electrochemical detection signal. Finally, excellent biosensor performance was achieved, and the detection limit for the synthetic DNA target was as low as 1 fM. In addition, we verified the practicability of the system by analyzing E. coli with the MCR-1 plasmid and realized multi-channel detection of the drug resistance genes MCR-1, blaNDM, blaKPC and blaOXA. With the ideal electrochemical interface, the polyA-based biosensor exhibits excellent stability, which provides powerful technical support for the rapid detection of antibiotic-resistant strains in the field.

FARPA-based tube array coupled with quick DNA extraction enables ultra-fast bedside detection of antibiotic-resistant pathogens.

Rapid and accurate detection of pathogens and antimicrobial-resistant (AMR) genes of the pathogens are crucial for the clinical diagnosis and effective treatment of infectious diseases. However, the time-consuming steps of conventional culture-based methods inhibit the precise and early application of anti-infection therapy. For the prompt treatment of pathogen-infected patients, we have proposed a novel tube array strategy based on our previously reported FARPA (FEN1-aided recombinase polymerase amplification) principle for the ultra-fast detection of antibiotic-resistant pathogens on site. The entire process from "sample to result" can be completed in 25 min by combining quick DNA extraction from a urine sample with FARPA to avoid the usually complicated DNA extraction step. Furthermore, a 36-tube array made from commercial 384-well titre plates was efficiently introduced to perform FARPA in a portable analyser, achieving an increase in the loading sample throughput (from several to several tens), which is quite suitable for the point-of-care testing (POCT) of multiple pathogens and multiple samples. Finally, we tested 92 urine samples to verify the performance of our proposed method. The sensitivities for the detection of E. coli, K. pneumoniae, E. faecium, and E. faecalis were 92.7%, 93.8%, 100% and 88.9%, respectively. The specificities for the detection of the four pathogens were 100%. Consequently, our rapid, low-cost and user-friendly POCT method holds great potential for guiding the rational use of antibiotics and reducing bacterial resistance.

A Dual-mode platform for the rapid detection of Escherichia coli O157:H7 based on CRISPR/Cas12a and RPA.

Escherichia coli O157:H7 (E. coli O157:H7) is a foodborne pathogenic microorganism that is commonly found in the environment and poses a significant threat to human health, public safety, and economic stability worldwide. Thus, early detection is essential for E. coli O157:H7 control. In recent years, a series of E. coli O157:H7 detection methods have been developed, but the sensitivity and portability of the methods still need improvement. Therefore, in this study, a rapid and efficient testing platform based on the CRISPR/Cas12a cleavage reaction was constructed. Through the integration of recombinant polymerase amplification and lateral flow chromatography, we established a dual-interpretation-mode detection platform based on CRISPR/Cas12a-derived fluorescence and lateral flow chromatography for the detection of E. coli O157:H7. For the fluorescence detection method, the limits of detection (LODs) of genomic DNA and E. coli O157:H7 were 1.8 fg/µL and 2.4 CFU/mL, respectively, within 40 min. Conversely, for the lateral flow detection method, LODs of 1.8 fg/µL and 2.4 × 102 CFU/mL were achieved for genomic DNA and E. coli O157:H7, respectively, within 45 min. This detection strategy offered higher sensitivity and lower equipment requirements than industry standards. In conclusion, the established platform showed excellent specificity and strong universality. Modifying the target gene and its primers can broaden the platform's applicability to detect various other foodborne pathogens.

Raman-enhanced sensor based on CRISPR-SERS technology for the rapid and hypersensitive detection of Mycobacterium tuberculosis.

Tuberculosis is a highly infectious disease caused by the bacterium Mycobacterium tuberculosis, and the spread of this agent has caused serious health problems worldwide. The rapid and accurate detection of M. tuberculosis is essential for controlling the spread of infection and for preventing the emergence of multidrug-resistant strains. In this study, the powerful trans-cleavage ability of CRISPR-Cas12a for ssDNA was combined with a surface-enhanced Raman spectroscopy (SERS)-based strategy to establish a CRISPR-SERS sensor for the hypersensitive detection of M. tuberculosis DNA. We observed a linear relationship between the concentration of M. tuberculosis DNA and the output signal over the range of 5 to 100 pM. The equation describing the standard curve was y = 24.10x + 1594, with R2 = 0.9914. The limit of detection was as low as 4.42 pM for genomic DNA, and a plasmid containing an M. tuberculosis-specific sequence was detected at 5 copy/µL. A detection accuracy of 100% was achieved in the analysis of DNA isolated from the sputum of hospitalized patients with tuberculosis. The entire detection process is simple to deploy and only takes 50 min and results in the sensitive and specific detection of M. tuberculosis DNA. This study provides a new method for the detection of tuberculosis. The tool is stable and can be utilized on-site, and it thus broadens the diagnostic application of CRISPR-Cas12a-based sensor technology.

A molecular beacon design for a colorimetric loop-mediated isothermal amplification assay.

In this study, a molecular beacon (MB) was designed for colorimetric loop-mediated isothermal amplification (cLAMP). The length of complementary bases on the MB, guanine and cytosine content (GC content), and hybridization sites of complementary bases were investigated as key factors affecting the design of the MB. We designed MBs consisting of 10, 15, and 20 complementary bases located at both ends of the HRPzyme. In the case of the long dumbbell DNA structure amplified from the hlyA gene of Listeria monocytogenes, possessing a flat region (F1c-B1) of 61 base pairs (bp), an MB was designed to intercalate into the flat region between the F1c and B1 regions of the LAMP amplicons. In the case of the short dumbbell DNA structure amplified from the bcfD gene of Salmonella species possessing a flat region (F1c-B1) length of 6 bp, another MB was designed to intercalate into the LoopF or LoopB regions of the LAMP amplicons. The results revealed that the hybridization site of the MB on the LAMP amplicons was not crucial in designing the MB, but the GC content was an important factor. The highest hybridization efficiencies for LAMP amplicons were obtained from hlyA gene-specific and bcfD gene-specific MBs containing 20- and 15-base complementary sequences, respectively, which exhibited the highest GC content. Therefore, designing MBs with a high GC content is an effective solution to overcome the low hybridization efficiency of cLAMP assays. The results obtained can be used as primary data for designing MBs to improve cLAMP accessibility.

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 detection method, 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 the 5' end of 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 10 min faster than several other probe-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.