Accuracy of cell-free Mycobacterium tuberculosis DNA testing in pleural effusion for diagnosing tuberculous pleurisy: a multicenter cross-sectional study.

BACKGROUND: The diagnosis of tuberculous pleurisy (TP) presents a significant challenge due to the low bacterial load in pleural effusion (PE) samples. Cell-free Mycobacterium tuberculosis DNA (cf-TB) in PE samples is considered an optimal biomarker for diagnosing TP. This study aimed to evaluate the applicability of cf-TB testing across diverse research sites with a relatively large sample size. METHODS: Patients suspected of TP and presenting with clinical symptoms and radiological evidence of PE were consecutively enrolled by treating physicians from 11 research sites across 6 provinces in China between April 2020 and August 2022. Following centrifugation, sediments obtained from PE were used for Xpert MTB/RIF (Xpert) and mycobacterial culture, while the supernatants were subjected to cf-TB testing. This study employed a composite reference standard to definite TP, which was characterized by any positive result for Mycobacterium tuberculosis (MTB) through either PE culture, PE Xpert, or pleural biopsy. RESULTS: A total of 1412 participants underwent screening, and 1344 (95.2%) were subsequently enrolled in this study. Data from 1241 (92.3%) participants were included, comprising 284 with definite TP, 677 with clinically diagnosed TP, and 280 without TP. The sensitivity of cf-TB testing in definite TP was 73.6% (95% CI 68.2-78.4), significantly higher than both Xpert (40.8%, 95% CI 35.3-46.7, P < 0.001) and mycobacterial culture (54.2%, 95% CI 48.4-59.9, P < 0.001). When clinically diagnosed TP was incorporated into the composite reference standard for sensitivity analysis, cf-TB testing showed a sensitivity of 46.8% (450/961, 95% CI 43.7-50.0), significantly higher than both Xpert (116/961, 12.1%, 95% CI 10.2-14.3, P < 0.001) and mycobacterial culture (154/961, 16.0%, 95% CI 13.8-18.5, P < 0.001). The specificities of cf-TB testing, Xpert, and mycobacterial culture were all 100.0%. CONCLUSIONS: The performance of cf-TB testing is significantly superior to that of Xpert and mycobacterial culture methods, indicating that it can be considered as the primary diagnostic approach for improving TP detection. Trial registration The trial was registered on Chictr.org.cn (ChiCTR2000031680, https://www.chictr.org.cn/showproj.html?proj=49316 ).

Analysis of the microbial community diversity in various regions of the healthy oral cavity.

BACKGROUND: Microbiomics offers new methods for conducting epidemiological surveys of oral microbiota in large populations. Compared to curette sampling, swab sampling is more convenient and less technically sensitive, making it more suitable for such surveys. To verify the feasibility of using swabs for buccal mucosa sampling in large-scale studies, we collected samples from the buccal mucosa and tooth surfaces of healthy individuals using both swabs and curettes. Microbiomics was employed to analyze and compare microbial abundance and diversity between these two methods. METHODS: Four sites were assessed: the buccal mucosa on both sides and the buccal surfaces of the left and right mandibular first molars. Two sampling methods, swab and curette, were used to collect bacterial communities from healthy individuals. Specifically, buccal mucosa samples (n = 10) and tooth surface samples (n = 20) were analyzed using 16 S rDNA gene sequencing. Bacterial signals were detected through fluorescence in situ hybridization (FISH), targeting the bacterial 16 S rDNA gene. Metastats analysis and Wilcoxon test were used. RESULTS: A total of 383 OTUs were detected in the 30 samples, which belonged to 1 kingdom (bacteria), 11 phyla, 23 classes, 40 orders, 75 families, 143 genus, and 312 species. Among them, 223 OTUs were found on both the buccal mucosa and tooth surfaces. The statistics suggest that although there were no significant differences in colony composition, there were differences in the abundance and distribution of colonies on the dental and buccal mucosal surfaces. When detecting oral disease-causing pathogens such as Enterococcus faecalis and Porphyromonas gingivalis, the efficiency of detection is higher when using curette sampling. Compared to right tooth sampling with a curette, the swab sampling group had higher levels of Firmicutes, while Fusobacteria and Bacteroidetes were more prevalent in the curette tissues. CONCLUSIONS: In oral health individuals, there is no difference in the bacterial composition of the oral buccal mucosa and the dental surface, differing only in abundance. Thus, the buccal mucosa can act as a substitute for the teeth in epidemiological investigations exploring the bacterial composition of the oral cavity.

Detection and quantification of Brucella abortus DNA in water buffaloes (bubalus bubalis) using droplet digital polymerase chain reaction.

Brucellosis represents a major public health concern worldwide. Human transmission is mainly due to the consumption of unpasteurized milk and dairy products of infected animals. The gold standard for the diagnosis of Brucella spp in ruminants is the bacterial isolation, but it is time-consuming. Polymerase Chain Reaction (PCR) is a quicker and more sensitive technique than bacterial culture. Droplet digital PCR (ddPCR) is a novel molecular assay showing high sensitivity in samples with low amount of DNA and lower susceptibility to amplification inhibitors. Present study aimed to develop a ddPCR protocol for the detection of Brucella abortus in buffalo tissue samples. The protocol was validated using proficiency test samples for Brucella spp by real time qPCR. Furthermore, 599 tissue samples were examined. Among reference materials, qPCR and ddPCR demonstrated same performance and were able to detect up to 225 CFU/mL. Among field samples, ddPCR showed higher sensitivity (100%), specificity and accuracy of 93.4% and 94.15%, respectively. ddPCR could be considered a promising technique to detect B. abortus in veterinary specimens, frequently characterized by low amount of bacteria, high diversity in matrices and species and poor storage conditions.

Detection of Mycobacterium tuberculosis from tongue swabs using sonication and sequence-specific hybridization capture.

Tongue swabs hold promise as a non-invasive sample for diagnosing tuberculosis (TB). However, their utility as replacements for sputum has been limited by their varied diagnostic performance in PCR assays compared to sputum. The use of silica-based DNA extraction methods may limit sensitivity due to incomplete lysis of Mycobacterium tuberculosis (MTB) cells and co-extraction of non-target nucleic acid, which may inhibit PCR. Specificity may also be compromised because these methods are labor-intensive and prone to cross-contamination. To address these limitations, we developed a sample preparation method that combines sonication for MTB lysis and a sequence-specific MTB DNA capture method using hybridization probes immobilized on magnetic beads. In spiked tongue swabs, our hybridization capture method demonstrated a 100-fold increase in MTB DNA yield over silica-based Qiagen DNA extraction and ethanol precipitation. In a study conducted on clinical samples from South Africa, our protocol had 74% (70/94) sensitivity and 98% (41/42) specificity for detecting active pulmonary TB with sputum Xpert MTB/RIF Ultra as the reference standard. While hybridization capture did not show improved sensitivity over Qiagen DNA extraction and ethanol precipitation, it demonstrated better specificity than previously reported methods and was easier to perform. With integration into point-of-care platforms, these strategies have the potential to help enable rapid non-sputum-based TB diagnosis across key underserved patient populations.

Using Next-Generation Sequencing to Understand Infection Prevention in Surgical Treatment of Upper Extremity Fractures-A Prospective Cohort Study.

INTRODUCTION: Postoperative fracture site infection can lead to notable patient morbidity, increase cost of care, and further contribute to healthcare disparities globally. Dogma suggests surgical blades as a vehicle for introducing bacteria into the surgical site; however, there is a paucity of literature to support this claim. This study uses advanced DNA sequencing to detect bacterial DNA on surgical blades used in upper extremity fracture surgeries. METHODS: This was a prospective study, conducted at a high-volume level 1 trauma center. All acute, closed upper extremity fractures requiring surgical stabilization were consecutively enrolled in a prospective fashion. The primary end point was the presence of bacterial DNA on the surgical blade using next-generation sequencing (NGS). At the time of surgery, two blades were sterilely opened. One blade served as the control while the other was used for the initial skin incision. Two negative control blades were opened directly into a sterile container. Two positive control blades were used for skin incision through known infections. All samples were sent for NGS analysis. RESULTS: Forty patients were enrolled in this study. The median age was 33.5 years, and 30% were female; the median body mass index was 26.52. Humerus fractures were the most common injury (N = 17, 42.5%), followed by clavicle fractures (13, 32.5%) and radius/ulna fractures (10, 25.0%). NGS analysis revealed no contamination of test blades used for skin incision. Three control blades tested positive for bacterial DNA. Negative control blades tested negative for bacterial DNA (0/2); the positive control blades resulted positive for bacterial DNA contamination (2/2). CONCLUSION: Surgical blades used for skin incision in the upper extremity are not contaminated with bacterial DNA as analyzed by NGS. This finding challenges previous surgical dogma regarding surgical blade contamination and supports that the same surgical blade can safely be used for deeper dissection. LEVEL OF EVIDENCE: Level II study: IRB approval-IRB#848938.

Investigation of potentially pathogenic Vibrionaceae in Saint-Louis city, Senegal.

Introduction: as cholera, due to toxigenic bacteria Vibrio cholera (serogroups O1 and O139), is a major public health threat in Africa, the aim of this work was to investigate potentially pathogenic Vibrionaceae bacteria firstly from human stool samples, and secondly from various environmental water points of Saint-Louis city in Senegal. Methods: a hospital-based study was conducted between 2013 and 2015. Stool samples were taken and cultured from daily incoming patients or hospitalized for acute diarrhea at Saint-Louis´ regional hospital. For environment, a monthly longitudinal sampling from January to October 2016 was carried out at 10 sites in the city. We used total DNA extracted from APW (alkaline peptone water) broth solutions and on suspect bacterial colonies to run PCR Multiplex targeting specific DNA fragments to detect Vibrio genus and specific species. In case of positivity, a simplex PCR was performed to test for cholera toxins Ctx, and V. parahaemolyticus TRH and TDH. Results: for 43 patients screened, bacterial culture was positive in 6% of cases but no strain of V. cholerae or other Vibrio sp. was isolated. PCR on 90 APW solutions were positive for Vibrio sp.(n = 43), V. cholera(n = 27), V. mimicus(n = 16), V. parahaemolyticus(8), V. alginolyticus(n = 4), and V. vulnificus(n = 2). Unlike for those on suspected colonies which were positive for a majority of V. parahaemolyticus (n = 40) and V. cholerae non-O1 / O139 (n = 35). Six strains of V. parahaemolyticus carried TRH gene, 3 of which expressed simultaneously virulence TRH and TDH genes. For physicochemical parameters, all temperatures varied similarly according to a unimodal seasonality, as well as salinity. Conclusion: despite the presence of natural populations of Vibrionaceae, even toxigenic ones, was noted in water environment, along with favorable habitat conditions that could play a role in transmission of Vibriosis in the Saint Louis population, we did not isolate any of them from patients screened at the hospital.

Polymerase Chain Reaction and Dot-Blot Hybridization for Leptospira Detection in Water Samples.

The dot-blot is a simple, fast, sensitive, and versatile technique that enables the identification of minimal quantities of DNA specifically targeted by probe hybridization in the presence of carrier DNA. It is based on the transfer of a known amount of DNA onto an inert solid support, such as a nylon membrane, utilizing the dot-blot apparatus and without electrophoretic separation. Nylon membranes have the advantage of high nucleic acid binding capacity (400 µg/cm2), high strength, and are positively or neutrally charged. The probe used is a highly specific ssDNA fragment of 18 to 20 bases long labeled with digoxigenin (DIG). The probe will conjugate with the Leptospira DNA. Once the probe has hybridized with the target DNA, it is detected by an anti-digoxigenin antibody, allowing its easy detection through its emissions revealed in an X-ray film. The dots with an emission will correspond to the DNA fragments of interest. This method employs the non-isotopic labeling of the probe, which may have a very long half-life. The drawback of this standard immuno-label is a lower sensitivity than isotopic probes. Nevertheless, it is mitigated by coupling polymerase chain reaction (PCR) and dot-blot assays. This approach enables the enrichment of the target sequence and its detection. Additionally, it may be used as a quantitative application when compared against a serial dilution of a well-known standard. A dot-blot application to detect Leptospira from the three main clades in water samples is presented here. This methodology can be applied to large amounts of water once they have been concentrated by centrifugation to provide evidence of the presence of Leptospiral DNA. This is a valuable and satisfactory tool for general screening purposes, and may be used for other non-culturable bacteria that may be present in water, enhancing the comprehension of the ecosystem.

Droplet digital molecular beacon-LAMP assay via pico-injection for ultrasensitive detection of pathogens.

A pico-injection-aided digital droplet detection platform is presented that integrates loop-mediated isothermal amplification (LAMP) with molecular beacons (MBs) for the ultrasensitive and quantitative identification of pathogens, leveraging the sequence-specific detection capabilities of MBs. The microfluidic device contained three distinct functional units including droplet generation, pico-injection, and droplet counting. Utilizing a pico-injector, MBs are introduced into each droplet to specifically identify LAMP amplification products, thereby overcoming issues related to temperature incompatibility. Our methodology has been validated through the quantitative detection of Escherichia coli, achieving a detection limit as low as 9 copies/µL in a model plasmid containing the malB gene and 3 CFU/µL in a spiked milk sample. The total analysis time was less than 1.5 h. The sensitivity and robustness of this platform further demonstrated the potential for rapid pathogen detection and diagnosis, particularly when integrated with cutting-edge microfluidic technologies.

One-pot electrochemical detection of foodborne pathogen based on in situ nucleic acid amplification and wash-free assay.

A signal amplification electrochemical biosensor chip was developed to integrate loop-mediated isothermal amplification (LAMP) based on in situ nucleic acid amplification and methyl blue (MB) serving as the hybridization redox indicator for sensitive and selective foodborne pathogen detection without a washing step. The electrochemical biosensor chip was designed by a screen-printed carbon electrode modified with gold nanoparticles (Au NPs) and covered with polydimethylsiloxane membrane to form a microcell. The primers of the target were immobilized on the Au NPs by covalent attachment for in situ amplification. The electroactive MB was used as the electrochemical signal reporter and embedded into the double-stranded DNA (dsDNA) amplicons generated by LAMP. Differential pulse voltammetry was introduced to survey the dsDNA hybridization with MB, which differentiates the specifically electrode-unbound and -bound labels without a washing step. Pyrene as the back-filling agent can further improve response signaling by reducing non-specific adsorption. This method is operationally simple, specific, and effective. The biosensor showed a detection linear range of 102-107 CFU mL-1 with the limit of detection of 17.7 CFU mL-1 within 40 min. This method showed promise for on-site testing of foodborne pathogens and could be integrated into an all-in-one device.

Development of a multiplex PCR assay and quantification of microbial markers by ddPCR for identification of saliva and vaginal fluid.

The identification of biological fluids at crime scenes contributes to crime scene reconstruction and provides investigative leads. Traditional methods for body fluid identification are limited in terms of sensitivity and are mostly presumptive. Emerging methods based on mRNA and DNA methylation require high quality template source. An exploitable characteristic of body fluids is their distinct microbial profiles allowing for the discrimination of body fluids based on microbiome content. Microbial DNA is highly abundant within the body, robust and stable and can persist in the environment long after human DNA has degraded. 16S rRNA sequencing is the gold standard for microbial analysis; however, NGS is costly, and requires intricate workflows and interpretation. Also, species level resolution is not always achievable. Based on the current challenges, the first objective of this study was to develop a multiplex conventional PCR assay to identify vaginal fluid and saliva by targeting species-specific 16S rRNA microbial markers. The second objective was to employ droplet digital PCR (ddPCR) as a novel approach to quantify bacterial species alone and in a mixture of body fluids. Lactobacillus crispatus and Streptococcus salivarius were selected because of high abundance within vaginal fluid and saliva respectively. While Fusobacterium nucleatum and Gardnerella vaginalis, though present in healthy humans, are also frequently found in oral and vaginal infections, respectively. The multiplex PCR assay detected L. crispatus and G. vaginalis in vaginal fluid while F. nucleatum and S. salivarius was detected in saliva. Multiplex PCR detected F. nucleatum, S. salivarius and L. crispatus in mixed body fluid samples while, G. vaginalis was undetected in mixtures containing vaginal fluid. For samples exposed at room temperature for 65 days, L. crispatus and G. vaginalis were detected in vaginal swabs while only S. salivarius was detected in saliva swabs. The limit of detection was 0.06 copies/µl for F. nucleatum (2.5 ×10-9 ng/µl) and S. salivarius (2.5 ×10-6 ng/µl). L. crispatus and G. vaginalis had detection limits of 0.16 copies/µl (2.5 ×10-4 ng/µl) and 0.48 copies/µl (2.5 ×10-7 ng/µl). All 4 bacterial species were detected in mixtures and aged samples by ddPCR. No significant differences were observed in quantity of bacterial markers in saliva and vaginal fluid. The present research reports for the first time the combination of the above four bacterial markers for the detection of saliva and vaginal fluid and highlights the sensitivity of ddPCR for bacterial quantification in pure and mixed body fluids.

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.

Specific detection of gut pathogens for one-pot chip based on RPA-CRISPR/Cas12a.

BACKGROUND: There are billions of bacteria in the intestine, most of which are harmless and play important roles in humans. Although only a very small number of bacteria can cause diseases, once the pathogenic bacteria are ingested into the body and multiply in large quantities, it can lead to inflammatory diseases in the intestines and even other organs. Although polymerase chain reaction can specifically detect bacterial nucleic acid. However, the demand for temperature cycling limits its portability. Therefore, it is hoped to establish a high-throughput, highly specific and portable detection platform for directly detecting nucleic acid of intestinal pathogens. RESULTS: Herein, a one-pot chip based on RPA-CRCISPR/Cas12a platform was developed. The chip is the same size as a glass slide and allows detection at the same temperature. Multiple samples could be detected simultaneously on the one chip, achieved high-throughput detection and improved the integration of detection. The specific recognition of CRISPR/Cas12a avoided the influence of non-specific amplification of RPA and enhanced the specificity of the analysis. At the same time, the one-pot chip avoided secondary contamination when the lid was opened during the analysis process. And the bacterial concentration showed good linearity at 102-108 cfu mL-1. The limit of detection could be as low as 0.43 cfu mL-1. This method has been successfully used to detect pollution samples. It can provide a reliable platform for early screening of gastrointestinal and other inflammatory diseases. SIGNIFICANCE: The one-pot chip based on the RPA-CRISPR/Cas12a platform established can directly detect the nucleic acid of intestinal pathogens, with portability and specificity. It is worth noting that the platform has good programmability, can be used for other target detection by changing crRNA and RPA primers, it can achieve multi sample detection on the one chip.

Efficacy of TrueNat cartridge extracted DNA for detecting drug resistant tuberculosis by line probe assay.

BACKGROUND: Currently for diagnosing Mycobacterium tuberculosis and its drug resistance, two sputum samples are required. One of them is subjected to TrueNat™ and if positive the other sample is subjected to line probe assay (LPA). This study was done to evaluate whether TrueNat extracted DNA can be directly used for performing LPA in a diagnostic laboratory setting to decrease patient turn-around time. METHODS: Total 45 smear positive sputum samples were subjected to TrueNat™ MTB detection and first and second line (FL and SL) LPA testing in parallel. DNA extracted by Trueprep® Cartridge was also tested by LPA and results were compared. Further, TrueNat extracted DNA from 20 samples was divided into 6 aliquots each, two of which were stored at 4 °C, 37 °C and 55 °C (under humidification) each. One aliquot from stored DNA at each temperature was used for FL & SL LPA on day three and the other on day eight. The blots thus obtained were compared with those of conventional LPA at day 1. RESULTS: For FL-LPA, TrueNat extracted DNA gave valid results for all 45 (100%) samples but conventionally extracted DNA could give results for 44 (97.8%) samples. Likewise, for SL-LPA, valid results were obtained for 40 (88.9%) and 35 (77.8%) samples respectively using TrueNat extracted DNA and conventionally extracted DNA respectively. All samples with invalid LPA results had Ct values ≥ 28 by TrueNat PCR. LPA results were obtained for all the 20 samples using stored DNA at all temperatures and duration. CONCLUSIONS: TrueNat extracted DNA can be used for performing LPA under field conditions for selected samples.

Poly(3,4-dihydroxyphenylalanine)-modified cellulose paper for the extraction of deoxyribonucleic acid by a laboratory-built automated extraction device.

The success of polymerase chain reaction (PCR) depends on the quality of deoxyribonucleic acid (DNA) templates. This study developed a cost-effective and eco-friendly DNA extraction system utilizing poly(3,4-dihydroxyphenylalanine)-modified cellulose paper (polyDOPA@paper). PolyDOPA@paper was prepared by oxidatively self-polymerizing DOPA under weak alkaline conditions and utilizing the adhesive property of polyDOPA on different materials. Compared to the uncoated cellulose paper, polyDOPA coating significantly enhances DNA adsorption owing to its abundant amino, carboxyl, and hydroxyl moieties. The DNA extraction mechanism using polyDOPA@paper was discussed. The maximum adsorption capacity of polyDOPA@paper for DNA was 20.7 µg cm-2. Moreover, an automated extraction system was designed and fabricated using 3D printing technology. The device simplifies the operation and ensures the reproducibility and consistency of the results. More importantly, it eliminates the need for specialized training of operators. The feasibility of the polyDOPA@paper-based automated extraction system was evaluated by quantitatively detecting Escherichia coli in spiked milk samples via a real-time PCR. The detection limit was 102 cfu mL-1. The results suggest that the system would have significant potential in detecting pathogens.

Comparative analysis of microbiome in coronal and root caries.

BACKGROUND: The global rise in the elderly population has increased the prevalence of root caries. Streptococcus mutans, Lactobacilli and Actinomyces are considered the primary pathogens of dental caries in culture-based studies. This study aimed to investigate bacterial profiles in coronal and root caries lesions and determine the association of specific bacterial genera at each site. METHODS: Dentine samples from carious lesions were collected from 22 extracted teeth using an excavator. Microbial DNA was extracted from the samples using a protocol developed for this study. 16S rRNA gene amplicon sequencing was employed for microbial analysis. PCR amplification targeted the V3-V4 region of the bacterial 16S rRNA, and the amplicon sequencing used an Illumina MiSeq system (2 × 300 bp paired-end reads). Statistical analysis was performed by the Phyloseq and DESeq2 packages in R. RESULTS: In coronal caries, Olsenella, Lactobacillus and Prevotella were the most prevalent genera, comprising approximately 70% of the microbiome community. In the root caries, however, although Olsenella, Prevotella and Lactobacillus remained the dominant genera, they accounted for only half of the microbiome community. This study identified significant differences in alpha diversity indices between the coronal and root caries. LEfSE analysis revealed several unique genera in each caries lesion. CONCLUSION: The microbiome of root caries lesions was richer and more complex than the coronal caries microbiota. The results suggest that lesion-related variations in the oral microflora may be detected in carious dentine.

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.

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.

A polymerase chain reaction experiment using Escherichia coli and Mars sand simulant for detection and analysis of extraterrestrial life.

In this study, we conducted polymerase chain reaction (PCR) experiments using Escherichia coli (E. coli) and a Mars sand simulant (Mars Global Simulant MGS-1, Exolith Lab) to detect and analyze potential extraterrestrial life. The targeted DNA sequence is common among the bacterial kingdom on Earth. PCR experiments conducted after alkaline heat extraction, wherein samples with varying amounts of Mars sand simulant were compared, revealed that the simulant interfered with DNA detection. We then conducted PCR experiments following treatment with a sand DNA extraction kit on samples with various E. coli densities. DNA bands for a minimum E. coli density of 900 cells/(g sand) were confirmed, while no DNA bands were visible in the 90 cells/(g sand) sample with and without the Mars sand simulant. The total DNA mass contained in 900 cells was calculated to be 15.3 pg (i.e., 1.53 pg in 0.1 g sand sample we evaluated). We tested and compared the influence of the eluate of Mars sand simulant and DNA adsorption onto Mars sand simulant based on optical absorbance measurements. Our findings suggest that the mechanism by which the Mars sand simulant prevents PCR is through the adsorption of DNA onto the Mars sand simulant.

Highly sensitive detection of Salmonella typhimurium via gold and magnetic nanoparticle-mediated sandwich hybridization coupled with ICP-MS.

Foodborne pathogens including Salmonella typhimurium (S. typhimurium) are responsible for over 600 million global incidences of illness annually, posing a significant threat to public health. Inductively coupled plasma mass spectrometry (ICP-MS), coupled with element labeling strategies, has emerged as a promising platform for multivariate and accurate pathogen detection. However, achieving high specificity and sensitivity remains a critical challenge. Herein, we synthesize clustered magnetic nanoparticles (MNPs) and popcorn-shaped gold nanoparticles (AuNPs) to conjugate capture and report DNA probes for S. typhimurium, respectively. These engineered nanoparticles facilitate the identification of S. typhimurium DNA through a sandwich hybridization technique. ICP-MS quantification of Au within the sandwich-structure complexes allows for precise S. typhimurium detection. The unique morphology of the AuNPs and MNPs increases the available sites for probe attachment, enhancing the efficiency of S. typhimurium DNA capture, broadening the detection range to 101-1010 copies mL-1, and achieving a low detection limit of 1 copy mL-1, and the overall assay time is 70 min. The high specificity of this method is verified by anti-interference assays against ten other pathogens. The recovery was 96.8-102.8% for detecting S. typhimurium DNA in biological samples. As these specially designed nanoparticles may facilitate the attachment of various proteins and nucleic acid probes, they may become an effective platform for detecting multiple pathogens.

A portable label-free electrochemical DNA biosensor for rapid detection of Salmonella Typhi.

A highly accurate, rapid, portable, and robust platform for detecting Salmonella enterica serovar Typhi (S. Typhi) is crucial for early-stage diagnosis of typhoid to avert and control the outbreaks of this pathogen, which threaten global public health. This study presents a proof-of-concept for our developed label-free electrochemical DNA biosensor system for S. Typhi detection, which employs a printed circuit board gold electrode (PCBGE), integrated with a portable potentiostat reader. Initially, the functionalized DNA biosensor and target detection were characterized using cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) methods using a benchtop potentiostat. Interestingly, the newly developed DNA biosensor can identify target single-stranded DNA concentrations ranging from 10 nM to 20 µM, achieving a detection limit of 7.6 nM within a brief 5 minute timeframe. Under optimal detection conditions, the DNA biosensor exhibits remarkable selectivity, capable of distinguishing a single mismatch base pair from the target single-stranded DNA sequence. We then evaluated the feasibility of the developed DNA biosensor system as a diagnostic tool by detecting S. Typhi in 50 clinical samples using a portable potentiostat reader based on the DPV technique. Remarkably, the developed biosensor can distinctly distinguish between positive and negative samples, indicating that the miniaturised DNA biosensor system is practical for detecting S. Typhi in real biological samples. The developed DNA biosensor device in this work proves to be a promising point-of-care (POC) device for Salmonella detection due to its swift detection time, uncomplicated design, and streamlined workflow detection system.