Direct or DNA Extraction-Free Amplification and Quantification of Foodborne Pathogens.

The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The examples provided include the detection of Escherichia coli in milk samples and Salmonella in pork meat samples. This protocol includes relevant reagents and methods including obtaining target sequences, assay design, sample processing, and amplification. These methods, though used for specific example matrices, could be applied to many other foodborne pathogens and sample types.

Development of a visual detection method of porcine deltacoronavirus using loop-mediated isothermal amplification.

The emergence of porcine deltacoronavirus (PDCoV) presents a significant threat to both human and animal health due to its ability to cause highly contagious enteric diseases. This underscores the crucial need for timely and accurate diagnosis to facilitate effective epidemiological investigation and clinical management. This research aimed to establish a visual detection method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for PDCoV testing. In this study, six pairs of primers were designed according to the conserved sequences of PDCoV ORF1a/b genes. The primer sets and parameters that affect LAMP reaction were optimized. The visual RT-LAMP method was developed by incorporating methyl red into the optimized reaction system, it exclusively detected PDCoV without cross-reactivity with other viruses and the detection limits for PDCoV could reach 10 copies/µL. In comparison with RT-PCR for testing 132 clinical samples, the relative specificity and sensitivity of the visual RT-LAMP were found to be 99.2 and 100%, respectively, with a concordance rate of 99.2% and a kappa value of 0.959, indicating that the visual RT-LAMP is a reliable method for the application of PDCoV detection in clinical samples.

A novel platform for mutation detection in colorectal cancer using a PNA-LNA molecular switch.

Detection of KRAS mutation in colorectal cancer (CRC) is important in the prediction of response to target therapy. The study aims to develop a novel mutation detection platform called the "PNA-LNA molecular switch" for the detection of KRAS mutation in CRC. We employed the enhanced binding specificity of peptide nucleic acid (PNA) and locked nucleic acid (LNA) in conjunction with a loop-mediated isothermal amplification (LAMP) approach to identify the mutation status of KRAS oncogene codon 12 (c.35G>T/G12V and c.35G>A/G12D) using synthetic oligonucleotides and colon cancer cell lines (Caco-2 and SW480). This method specifically blocked the amplification of the wild-type sequences while substantially amplifying the mutated ones, which was visualized by both colorimetric and fluorescence assays. We then checked the mutation profile of KRAS codon 12 in the DNA derived from tumor tissue samples (number of samples, n = 30) and circulating tumor cells (n = 24) from CRC patients. Finally, we validated the results by comparing them with the data obtained from DNA sequencing of colon tumors (n = 21) of the same CRC patients. This method showed excellent sensitivity (1 DNA copy/µl), reproducibility [relative standard deviation (%RSD) < 5%, for n = 3], and linear dynamic range (1 ag/µl-10 pg/µl, R2 = 0.94). This platform is significantly faster, relatively cheaper, has superior sensitivity and specificity, and does not require any high-end equipment. To conclude, this method has the potential to be translated into clinical settings for the detection of mutations in diverse diseases and conditions.

Advancements in rapid diagnostics and genotyping of Piscirickettsia salmonis using Loop-mediated Isothermal Amplification.

Introduction: Piscirickettsia salmonis, the causative agent of Piscirickettsiosis, poses a significant threat to the Chilean aquaculture industry, resulting in substantial economic losses annually. The pathogen, first identified as specie in 1992, this pathogen was divided into two genogroups: LF-89 and EM-90, associated with different phenotypic mortality and pathogenicity. Traditional genotyping methods, such as multiplex PCR, are effective but limited by their cost, equipment requirements, and the need for specialized expertise. Methods: This study validates Loop-mediated Isothermal Amplification (LAMP) as a rapid and specific alternative for diagnosing P. salmonis infections. We developed the first qPCR and LAMP assay targeting the species-conserved tonB receptor gene (tonB-r, WP_016210144.1) for the specific species-level identification of P. salmonis. Additionally, we designed two genotyping LAMP assays to differentiate between the LF-89 and EM-90 genogroups, utilizing the unique coding sequences Nitronate monooxygenase (WP_144420689.1) for LF-89 and Acid phosphatase (WP_016210154.1) for EM-90. Results: The LAMP assays demonstrated sensitivity and specificity comparable to real-time PCR, with additional benefits including rapid results, lower costs, and simplified operation, making them particularly suitable for field use. Specificity was confirmed by testing against other salmonid pathogens, such as Renibacterium salmoninarum, Vibrio ordalii, Flavobacterium psychrophilum, Tenacibaculum maritimum, and Aeromonas salmonicida, with no cross-reactivity observed. Discussion: The visual detection method and precise differentiation between genogroups underscore LAMP's potential as a robust diagnostic tool for aquaculture. This advancement in the specie detection (qPCR and LAMP) and genotyping of P. salmonis represents a significant step forward in disease management within the aquaculture industry. The implementation of LAMP promises enhanced disease surveillance, early detection, and improved management strategies, ultimately benefiting the salmonid aquaculture sector.

Optimization of loop-mediated isothermal amplification assay for sunflower mildew disease detection.

Loop-Mediated Isothermal Amplification (LAMP) represents a valuable technique for DNA/RNA detection, known for its exceptional sensitivity, specificity, speed, accuracy, and affordability. This study focused on optimizing a LAMP-based method to detect early signs of Plasmopara halstedii, the casual pathogen of sunflower downy mildew, a severe threat to sunflower crops. Specifically, a set of six LAMP primers (two outer, two inner, and two loop) were designed from P. halstedii genomic DNA, targeting the ribosomal Large Subunit (LSU). These primers were verified by in silico analysis and experimental validation using both target and non-target species' DNAs. Optimizations encompassing reaction conditions (temperature, time) and component concentrations (magnesium, Bst DNA polymerase, primers, and dNTP) were determined. Validation of these optimizations was performed by agarose gel electrophoresis. Furthermore, various colorimetric chemicals (Neutral Red, Hydroxynaphthol Blue, SYBR Safe, Thiazole Green) were evaluated to facilitate method analysis, and the real-time analysis has been optimized, presenting multiple approaches for detecting sunflower downy mildew using the LAMP technique. The analytical sensitivity of the method was confirmed by detecting P. halstedii DNA concentrations as low as 0.5 pg/µl. This pioneering study, establishing P. halstedii detection through the LAMP method, stands as unique in its field. The precision, robustness, and practicality of the LAMP protocol make it an ideal choice for studies focusing on sunflower mildew, emphasizing its recommended use due to its operational ease and reliability.

Hybrid paper/PDMS microfluidic device integrated with RNA extraction and recombinase polymerase amplification for detection of norovirus in foods.

Human norovirus (HuNoV) is recognized as the leading causative agent of foodborne outbreaks of epidemic gastroenteritis. Consequently, there is a high demand for developing point-of-care testing for HuNoV. We developed an origami microfluidic device that facilitates rapid detection of murine norovirus 1 (MNV-1), a surrogate for HuNoV, encompassing the entire process from sample preparation to result visualization. This process includes RNA absorption via a paper strip, RNA amplification using recombinase polymerase amplification (RPA), and a lateral flow assay for signal readout. The on-chip detection of MNV-1 was completed within 35 min, demonstrating 100% specificity to MNV-1 in our settings. The detection limit of this microfluidic device for MNV-1 was 200 PFU/mL, comparable to the in-tube RPA reaction. It also successfully detected MNV-1 in lettuce and raspberries at concentrations of 170 PFU/g and 230 PFU/g, respectively, without requiring extra concentration steps. This device demonstrates high compatibility with isothermal nucleic acid amplification and holds significant potential for detecting foodborne viruses in agri-food products in remote and resource-limited settings. IMPORTANCE: HuNoV belongs to the family of Caliciviridae and is a leading cause of acute gastroenteritis that can be transmitted through contaminated foods. HuNoV causes around one out of five cases of acute gastroenteritis that lead to diarrhea and vomiting, placing a substantial burden on the healthcare system worldwide. HuNoV outbreaks can occur when food is contaminated at the source (e.g., wild mussels exposed to polluted water), on farms (e.g., during crop cultivation, harvesting, or livestock handling), during packaging, or at catered events. The research outcomes of this study expand the approaches of HuNoV testing, adding value to the framework for routine testing of food products. This microfluidic device can facilitate the monitoring of HuNoV outbreaks, reduce the economic loss of the agri-food industry, and enhance food safety.

Real-Time Visualization of HIV-1 RNA Detection Using Loop-Mediated Isothermal Amplification-Enabled Particle Diffusometry.

Isothermal nucleic acid amplification tests, NAATs, such as reverse transcription-loop-mediated isothermal amplification (RT-LAMP), offer promising capabilities to perform real-time semiquantitative detection of viral pathogens. These tests provide rapid results, utilize simple instrumentation for single-temperature reactions, support efficient user workflows, and are suitable for field use. Herein, we present a novel and robust method for real-time monitoring of HIV-1 RNA RT-LAMP utilizing a novel implementation of particle diffusometry (PD), a diffusivity quantification technique using fluorescent particles, to quantify viral concentration in nuclease-free water. We monitor changes in particle diffusion dynamics of 400 nm fluorescently labeled particles throughout the RT-LAMP of HIV-1 RNA in nuclease-free water, enabling measurement within 20 min and detection of concentrations as low as 25 virus particles per µL. Moreover, in a single-blind study, we demonstrate semiquantitative detection by accurately determining the initial concentration of an unknown HIV-1 RNA within a 10% absolute error margin. These results highlight the potential of real-time PD readout for quantifying HIV-1 RNA via RT-LAMP, offering promise for viral load monitoring of HIV and other chronic infections.

Advances in Malaria Diagnostic Methods in Resource-Limited Settings: A Systematic Review.

Malaria continues to pose a health challenge globally, and its elimination has remained a major topic of public health discussions. A key factor in eliminating malaria is the early and accurate detection of the parasite, especially in asymptomatic individuals, and so the importance of enhanced diagnostic methods cannot be overemphasized. This paper reviewed the advances in malaria diagnostic tools and detection methods over recent years. The use of these advanced diagnostics in lower and lower-middle-income countries as compared to advanced economies has been highlighted. Scientific databases such as Google Scholar, PUBMED, and Multidisciplinary Digital Publishing Institute (MDPI), among others, were reviewed. The findings suggest important advancements in malaria detection, ranging from the use of rapid diagnostic tests (RDTs) and molecular-based technologies to advanced non-invasive detection methods and computerized technologies. Molecular tests, RDTs, and computerized tests were also seen to be in use in resource-limited settings. In all, only twenty-one out of a total of eighty (26%) low and lower-middle-income countries showed evidence of the use of modern malaria diagnostic methods. It is imperative for governments and other agencies to direct efforts toward malaria research to upscale progress towards malaria elimination globally, especially in endemic regions, which usually happen to be resource-limited regions.

Monitoring Escherichia coli in Water through Real-Time Loop-Mediated Isothermal Amplification on Biochips.

Access to clean water is fundamental to public health and safety, serving as the cornerstone of well-being in communities. Despite the significant investments of millions of dollars in water testing and treatment processes, the United States continues to grapple with over 7 million waterborne-related cases annually. This persistent challenge underscores the pressing need for the development of a new, efficient, rapid, low-cost, and reliable method for ensuring water quality. The urgency of this endeavor cannot be overstated, as it holds the potential to safeguard countless lives and mitigate the pervasive risks associated with contaminated water sources. In this study, we introduce a biochip LAMP assay tailored for water source monitoring. Our method swiftly detects even extremely low concentrations of Escherichia coli (E. coli) in water, and 10 copies/µL of E. coli aqueous solution could yield positive results within 15 min on a PC-MEDA biochip. This innovation marks a significant departure from the current reliance on lab-dependent methods, which typically necessitate several days for bacterial culture and colony counting. Our multifunctional biochip system not only enables the real-time LAMP testing of crude E. coli samples but also holds promise for future modifications to facilitate on-site usage, thereby revolutionizing water quality assessment and ensuring rapid responses to potential contamination events.

Enzyme- and label-free cascade isothermal amplification aptasensor for the ultrasensitive detection of ochratoxin A.

BACKGROUND: Ultrasensitive detection is crucial for the early warning and intervention of risk factors, ultimately benefiting the environment and human health. Low levels of ochratoxin A (OTA) present a hidden yet significant threat, and rapid detection via high-performing biosensors is therefore essential. RESULTS: A cascade isothermal amplification aptasensor (CIA-aptasensor) was designed for OTA detection. On the surface of a magnetic bead probe, the OTA level was converted into positively correlated trigger cDNA through its competitive binding with OTA-Apt. The released trigger cDNA activated catalytic hairpin assembly followed by coupling with a hybridization chain reaction to achieve CIA. After adding graphene oxide and SYBR Green I, the background interference was eliminated to specifically obtain OTA-related fluorescence. The ultrasensitive limit of detection was 0.22 pg mL-1, an improvement of 1368-fold over conventional enzyme-linked aptamer sorbent assay by the same OTA-Apt, demonstrating satisfactory reliability and practicability. Thus, the CIA-aptasensor provides an enzyme- and label-free simplified homogeneous system with minimal background interference using isothermal conditions. SIGNIFICANCE: This study provides a polymerase chain reaction-like approach for enhancing the sensitivity and performance of a biosensor, which could be extended for the application of CIA and label-free signaling strategy to other risk factors.

Rapid detection of bat coronaviruses from fecal samples using loop-mediated isothermal amplification assay in the field.

The global impact of the COVID-19 pandemic has emphasized the critical need for effective viral diagnostics. Although polymerase chain reaction (PCR) is a well-established nucleotide amplification technique, its limitations, such as the need for expensive equipment and skilled technicians, have led to the exploration of alternative methods, including loop-mediated isothermal amplification (LAMP). Bats, as a crucial natural reservoir of coronaviruses (CoVs), particularly Scotophilus bat coronavirus 512 (Scotophilus bat-CoV 512) prevalent among Taiwan's bat population, are the focus of this study. We aimed to detect Scotophilus bat-CoV 512 from bats in field conditions using loop-mediated isothermal amplification (LAMP) assay for on-site detection. Therefore, our study delves into the specificity of the LAMP reaction, emphasizing the careful design of primers to prevent false positive results. A cross reactivity and primer specificity test involving seven different microorganisms, including closely related bat CoVs and two bacterial species typically found in feces, revealed that the LAMP assay uniquely detected Scotophilus bat-CoV 512. The developed colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was optimized for the primers targeting nucleocapsid (N) gene, and the sensitivity test revealed a detection limit of 2.4 × 103 copies/µL. Our findings indicate the potential of the RT-LAMP assay for on-site detection in the field and subsequent laboratory analysis for comprehensive sampling and further research on bat CoV isolation. The surveillance and monitoring of bat CoVs contribute substantially to mitigating human threats, particularly concerning the emergence of new pandemic variants.

Multiplex Loop-Mediated Isothermal Amplification Coupled Lateral Flow Assay for Point-of-Care Detection of Syphilis.

Syphilis is a re-emerging sexually transmitted disease caused by the pathogenic spirochete T. pallidum. Every year more than 5 million cases are reported globally. The current diagnostic methods are primarily based on serological assays, which are less sensitive at an early stage of infection. To improve the disease diagnosis, there is a need to develop a rapid, simple, sensitive, and cost-effective point-of-care application, which plays an effective role in the detection of syphilis infection. In this study, we developed a multiplex loop-mediated isothermal amplification coupled lateral flow assay (multiplex LAMP-LFA) for the detection of syphilis. Two different genes, the target amplicon (polA) and the internal control amplicon (human RNase P) were amplified using multiplex LAMP assay. The amplified products were detected using LFA strips coated with Anti-FITC and Anti-DIG antibodies within 5 minutes of flowthrough. Multiplex LAMP LFA detection limit was found to be 3.8 × 103 copies/mL with high specificity. The developed strip was tested with 130 clinically suspected cases and 50 healthy individuals. With the clinical samples, the method shows a sensitivity of 93.84% and a specificity of 100%. The Multiplex LAMP LFA has the potential to overcome the limitations of both Non Treponemal tests and Treponemal tests which are prone to prozone effects and expensive reagents respectively. The proposed method holds promise for sensitive, rapid, and visual detection of T. pallidum, thereby offering a facile and affordable alternative to existing diagnostic methods. This approach is poised to advance the development of point-of-care diagnostics, addressing a critical need in public healthcare, particularly in resource-limited settings. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01308-4.

Toward establishing a rapid constant temperature detection method for canine parvovirus based on endonuclease activities.

Canine parvovirus (CPV) can cause high morbidity and mortality rates in puppies, posing a significant threat to both pet dogs and the breeding industry. Rapid, accurate, and convenient detection methods are important for the early intervention and treatment of canine parvovirus. In this study, we propose a visual CPV detection system called nucleic acid mismatch enzyme digestion (NMED). This system combines loop-mediated isothermal amplification (LAMP), endonuclease for gene mismatch detection, and colloidal gold lateral chromatography. We demonstrated that NMED can induce the binding of the amplicon from the sample to the specific labeling probe, which in turn triggers digestion by the endonuclease. The sensitivity and visual visibility of LAMP were increased by combining endonuclease and colloidal gold lateral chromatography assisted by a simple temperature-controlled device. The sensitivity of the NMED assay was 1 copy/µL, which was consistent with quantitative PCR (qPCR). The method was validated with 20 clinical samples that potentially had CPV infection; 15 positive samples and 5 negative samples were evaluated; and the detection accuracy was consistent with that of qPCR. As a rapid, accurate, and convenient molecular diagnostic method, NMED has great potential for application in the field of pathogenic microorganism detection. IMPORTANCE: The NMED method has been established in the laboratory and used for CPV detection. The method has several advantages, including simple sampling, high sensitivity, intuitive results, and no requirement for expensive equipment. The establishment of this method has commercial potential and offers a novel approach and concept for the future development of clinical detection of pathogenic microorganisms.

Precise pathogen quantification by CRISPR-Cas: a sweet but tough nut to crack.

Pathogen detection is increasingly applied in medical diagnosis, food processing and safety, and environmental monitoring. Rapid, sensitive, and accurate pathogen quantification is the most critical prerequisite for assessing protocols and preventing risks. Among various methods evolved, those based on clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) have been developed as important pathogen detection strategies due to their distinct advantages of rapid target recognition, programmability, ultra-specificity, and potential for scalability of point-of-care testing (POCT). However, arguments and concerns on the quantitative capability of CRISPR-based strategies are ongoing. Herein, we systematically overview CRISPR-based pathogen quantification strategies according to the principles, properties, and application scenarios. Notably, we review future challenges and perspectives to address the of precise pathogen quantification by CRISPR-Cas. We hope the insights presented in this review will benefit development of CRISPR-based pathogen detection methods.

Assessment of LAMPAuris for Rapid Detection of Candida auris in Clinical Specimens.

Candida auris is a pathogenic yeast frequently exhibiting multidrug resistance and thus warrants special attention. The prompt detection and proper identification of this organism are needed to prevent its spread in healthcare facilities. The authors of this paper had previously developed LAMPAuris, a loop-mediated isothermal amplification assay, for the specific detection of C. auris. LAMPAuris is evaluated in this report for its ability to identify C. auris from five clades and to detect it from clinical specimens. A total of 103 skin swab samples were tested in comparison with a culture-based method and C. auris-specific SYBR green qPCR. The results show that the LAMPAuris assay had specificities ranging from 97 to 100% and sensitivities ranging from 66 to 86%. The lower sensitivity could be attributed to DNA degradation caused by the prolonged storage of the samples. In conclusion, LAMPAuris proved to be a rapid and reliable method for identifying C. auris and for detecting it in clinical specimens. Fresh specimens should ensure better yield and higher sensitivities.

Rapid real-time quantitative colorimetric LAMP methodology for field detection of Verticillium dahliae in crude olive-plant samples.

BACKGROUND: Verticilium dahliae is the most important wilt pathogen of olive trees with a broad host range causing devastating diseases currently without any effective chemical control. Traditional detection methodologies are based on symptoms-observation or lab-detection using time consuming culturing or molecular techniques. Therefore, there is an increasing need for portable tools that can detect rapidly V. dahliae in the field. RESULTS: In this work, we report the development of a novel method for the rapid, reliable and on-site detection of V. dahliae using a newly designed isothermal LAMP assay and crude extracts of olive wood. For the detection of the fungus, LAMP primers were designed targeting the internal transcribed spacer (ITS) region of the rRNA gene. The above assay was combined with a purpose-built prototype portable device which allowed real time quantitative colorimetric detection of V. dahliae in 35 min. The limit of detection of our assay was found to be 0.8 fg/µl reaction and the specificity 100% as indicated by zero cross-reactivity to common pathogens found in olive trees. Moreover, detection of V. dahliae in purified DNA gave a sensitivity of 100% (Ct < 30) and 80% (Ct > 30) while the detection of the fungus in unpurified crude wood extracts showed a sensitivity of 80% when multisampling was implemented. The superiority of the LAMP methodology regarding robustness and sensitivity was demonstrated when only LAMP was able to detect V. dahliae in crude samples from naturally infected trees with very low infection levels, while nested PCR and SYBR qPCR failed to detect the pathogen in an unpurified form. CONCLUSIONS: This study describes the development of a new real time LAMP assay, targeting the ITS region of the rRNA gene of V. dahliae in olive trees combined with a 3D-printed portable device for field testing using a tablet. The assay is characterized by high sensitivity and specificity as well as ability to operate using directly crude samples such as woody tissue or petioles. The reported methodology is setting the basis for the development of an on-site detection methodology for V. dahliae in olive trees, but also for other plant pathogens.

Protein-Induced DNA Dumbbell Amplification (PINDA) and its applications to food hazards detection.

Quantification of trace amounts of proteins is technically challenging because proteins cannot be directly amplified like nucleic acids. To improve the analytical sensitivity and to complement conventional protein analysis methods, we developed a highly sensitive and homogeneous detection strategy called Protein-Induced DNA Dumbbell Amplification (PINDA). PINDA combines protein recognition with exponential nucleic acid amplification by using protein binding probes made of DNA strands conjugated to protein affinity ligands. When a pair of probes bind to the same target protein, complementary nucleic acid sequences that are conjugated to each probe are brought into close proximity. The increased local concentration of the probes results in the formation of a stable dumbbell structure of the nucleic acids. The DNA dumbbell is readily amplifiable exponentially using techniques such as loop-mediated isothermal amplification. The PINDA assay eliminates the need for washing or separation steps, and is suitable for on-site applications. Detection of the model protein, thrombin, has a linear range of 10 fM-100 pM and detection limit of 10 fM. The PINDA technique is successfully applied to the analysis of dairy samples for the detection of ß-lactoglobulin, a common food allergen, and Salmonella enteritidis, a foodborne pathogenic bacterium. The PINDA assay can be easily modified to detect other targets by changing the affinity ligands used to bind to the specific targets.

Development and evaluation of a rapid visual loop-mediated isothermal amplification assay for the tcdA gene in Clostridioides difficile detection.

Background: The tcdA gene codes for an important toxin produced by Clostridioides difficile (C. difficile), but there is currently no simple and cost-effective method of detecting it. This article establishes and validates a rapid and visual loop-mediated isothermal amplification (LAMP) assay for the detection of the tcdA gene. Methods: Three sets of primers were designed and optimized to amplify the tcdA gene in C. difficile using a LAMP assay. To evaluate the specificity of the LAMP assay, C. difficile VPI10463 was used as a positive control, while 26 pathogenic bacterial strains lacking the tcdA gene and distilled water were utilized as negative controls. For sensitivity analysis, the LAMP assay was compared to PCR using ten-fold serial dilutions of DNA from C. difficile VPI10463, ranging from 207 ng/µl to 0.000207 pg/µl. The tcdA gene of C.difficile was detected in 164 stool specimens using both LAMP and polymerase chain reaction (PCR). Positive and negative results were distinguished using real-time monitoring of turbidity and chromogenic reaction. Results: At a temperature of 66 °C, the target DNA was successfully amplified with a set of primers designated, and visualized within 60 min. Under the same conditions, the target DNA was not amplified with the tcdA12 primers for 26 pathogenic bacterial strains that do not carry the tcdA gene. The detection limit of LAMP was 20.700 pg/µl, which was 10 times more sensitive than that of conventional PCR. The detection rate of tcdA in 164 stool specimens using the LAMP method was 17% (28/164), significantly higher than the 10% (16/164) detection rate of the PCR method (X2 = 47, p < 0.01). Conclusion: LAMP method is an effective technique for the rapid and visual detection of the tcdA gene of C. difficile, and shows potential advantages over PCR in terms of speed, simplicity, and sensitivity. The tcdA-LAMP assay is particularly suitable for medical diagnostic environments with limited resources and is a promising diagnostic strategy for the screening and detection of C. difficile infection in populations at high risk.

ERA-CRISPR/Cas12a system: a rapid, highly sensitive and specific assay for Mycobacterium tuberculosis.

Introduction: Mycobacterium tuberculosis, the causative agent of human tuberculosis, poses a significant threat to global public health and imposes a considerable burden on the economy. However, existing laboratory diagnostic methods for M. tuberculosis are time-consuming and have limited sensitivity levels. Methods: The CRISPR/Cas system, commonly known as the "gene scissors", demonstrates remarkable specificity and efficient signal amplification capabilities. Enzymatic recombinase amplification (ERA) was utilized to rapidly amplify trace DNA fragments at a consistent temperature without relying on thermal cyclers. By integrating of CRISPR/Cas12a with ERA, we successfully developed an ERA-CRISPR/Cas12a detection system that enables rapid identification of M. tuberculosis. Results: The sensitivity of the ERA-CRISPR/Cas12a fluorescence and lateral flow systems was 9 copies/µL and 90 copies/µL, respectively. Simultaneously, the detection system exhibited no cross-reactivity with various of respiratory pathogens and non-tuberculosis mycobacteria, demonstrating a specificity of 100%. The positive concordance rate between the ERA-CRISPR/Cas12a fluorescence system and commercial qPCR was 100% in 60 clinical samples. Meanwhile, the lateral flow system showed a positive concordance rate of 93.8% when compared to commercial qPCR. Both methods demonstrated a negative concordance rate of 100%, and the test results can be obtained in 50 min at the earliest. Discussion: The ERA-CRISPR/Cas12a system offers a rapid, sensitive, and specific method that presents a novel approach to laboratory diagnosis of M. tuberculosis.

Near zero background noise photoelectrochemical sensor based on sensitized signal probe CdS QDs-Dox intercalating double-stranded DNA for PEDV detection.

The highly sensitive detection method for porcine epidemic diarrhea virus (PEDV) is crucial for promptly identify infected pigs and effectively control the spread of the virus. In this study, the sensitization enhancement of organic photoactive material was combined with near zero background noise strategy for PEDV sensitive detection. A novel sensitized signal probe CdS quantum dots-doxycycline complex (CdS QDs-Dox) was prepared serving as a photoelectrochemical (PEC) probe embedded in dsDNA. Subsequently, a thiol-modified upstream inner primer (SH-FIP) was immobilized on the surface of electrode modified with gold nanoparticles (Au NPs) via Au-S bonding, enabling the loop-mediated isothermal amplification (LAMP) of PEDV on the electrode surface. The PEC probe (CdS QDs-Dox) embedded in the amplified dsDNA groove showed an increasing photocurrent signal with the rise of PEDV concentration, establishing a near-zero background LAMP-PEC sensing platform for PEDV detection. Under optimized conditions, the photocurrent intensity of this platform exhibited a good linear relationship with PEDV concentrations ranging from 0.0005 pg/µL to 10 pg/µL, achieving a detection limit as low as 0.17 fg/µL. This platform demonstrates outstanding specificity and sensitivity, thereby enabling precise quantitative detection of diverse pathogens.