Search Results (185)

Background/Objectives: Norovirus is a major cause of acute gastroenteritis worldwide. Considering its highly infectious and transmissible nature, rapid and accurate diagnostic tools are of utmost importance for the effective control of outbreaks in the context of point-of-care testing (POCT). In this study, we developed a genogroup-specific multiplex reverse transcriptase loop-mediated isothermal amplification assay to detect the human norovirus genogroups I and II (GI and GII, respectively). Methods: For the comprehensive detection of clinically relevant genotypes, two sets of primers were incorporated into the assays targeting the RdRp-VP1 junction: one against GI.1 and GI.3, and the other for GII.2 and GII.4. Following optimization of the reaction variables, we standardized the reaction conditions at 65 °C with 6 mM MgSO₄, 1.4 mM dNTPs, 7.5 U WarmStart RTx Reverse Transcriptase, and Bst DNA polymerase at 8 U and 10 U for GI and GII, respectively. Amplification was monitored in real-time using a thermocycler platform to ensure precise quantification and detection. Finally, the assay was evaluated through portable isothermal detection device to test its feasibility in on-site settings. Results: Both assays detected the template down to 10²–10³ copies per reaction and showed high target selectivity, yielding no non-specific amplification across 39 enteric pathogens. These assays enabled prompt detection of GI within 10–12 min and of GII within 12–17 min after the reaction was initiated. Onsite validation reveals all template detection below 15 min, demonstrating its potential feasibility in point-of-care applications. Including the sample preparation time, test results were obtained in less than 1 h. Conclusions: This method is a rapid, reliable, and scalable solution for detecting human norovirus in POCT settings for both clinical diagnosis and public health surveillance. Full article

Hendra virus (HeV) and Nipah virus (NiV) are two highly pathogenic RNA viruses with zoonotic potential, which can cause severe diseases with high mortality rates (50–100%) in humans and animals. Given this context, these viruses are classified as Biosafety Level 4 (BSL-4) pathogens, thus limiting research studies. Despite the high case fatalities, there are currently no human vaccines available for either virus, owing in part to the limitations in research and hesitancy in funding. In the absence of widespread vaccination, diagnostic tests are crucial for the rapid identification of cases and disease surveillance. This review synthesizes current knowledge on the epidemiology, transmission dynamics, and pathogenesis of NiV and HeV to contextualize a detailed assessment of the available diagnostic tools. We examined molecular and serological assays, including RT-PCR, ELISA, and LAMP, highlighting sample sources, detection windows, and performance. Diagnostic considerations across human and animal hosts are discussed, with emphasis on outbreak applicability and field-readiness, given the need for diagnostic assays that are suitable for use in low-income areas. Further development of diagnostic assays, including isothermal amplification tests and other next-generation approaches, is recommended to fill the gap in rapid, point-of-care diagnostics. Full article

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by the SFTS virus (SFTSV). A rapid and cost-effective point-of-care testing detection system is important for the early diagnosis of SFTS. Herein, we developed a ready-to-use dried reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the direct detection of SFTSV in clinical samples. The assay enables simple, RNA-extraction-free detection using heat-treated serum or plasma, followed by a 30 min incubation at 65 °C. The results are visually interpreted through the color emitted, which can be observed under LED light. The established assay demonstrated detection sensitivity for SFTSV at 10⁴ copies/µL and was effective in identifying infections in cats. Despite being less sensitive than real-time RT-PCR, this dried RT-LAMP method offers a rapid, cost-effective alternative suitable for point-of-care use, particularly in remote or resource-limited settings. The simplified workflow and visual readout make it a practical tool for the early detection and daily surveillance of SFTSV in animals. Full article

Accurate, rapid and inexpensive diagnosis of poultry respiratory pathogens remains a challenge, especially in many developing countries. Meanwhile, poultry respiratory pathogens are a major threat to poultry production worldwide, accounting for billions of dollars in economic loss to the sector. Early and accurate diagnosis of these diseases is critical for economic poultry production. Molecular diagnostic methods, including PCR-based techniques, have been developed and used to fill this gap, but unfortunately, these techniques require skilled technicians, relatively costly equipment and reagents and can only be performed in a laboratory setting. This warrants the development of other diagnostic tools, which can be used in the field even by unskilled personnel. In this review, we discussed the genesis, challenges, advances and prospects of loop-mediated isothermal amplification (LAMP) for the detection of poultry respiratory pathogens at the flock side, especially in resource-constrained countries. We highlighted the application of LAMP in routine poultry disease surveillance and early outbreak detection, underscoring its value as a transformative diagnostic tool in poultry production. The development and use of a point-of-care (POC) LAMP assay that can be used to screen for these poultry respiratory pathogens simultaneously enhance disease surveillance and diagnosis. Full article

The wide host range, potential lethality, and zoonotic potential of SARS-CoV-2 infection in animals highlights the need for additional surveillance strategies. We validated a commercial, pH-based, colorimetric RT-LAMP assay for the detection of SARS-CoV-2 RNA in animal feces. The comparator assay was rRT-PCR. The limit of detection of the RT-LAMP assay was 72 genome copies per reaction. RT-LAMP was highly specific for SARS-CoV-2 and did not detect other human or animal coronaviruses. RT-LAMP was robust, with valid results generated for incubation lengths of 30 to 45 min, incubation temperatures of 60 to 70 °C, and reaction volumes of 10 to 25 µL. The diagnostic sensitivity was 100% for clinical fecal samples with high viral loads (Ct ≤ 25), 97.4% for samples with moderate to high viral loads (Ct ≤ 33), and 62% overall (Ct ≤ 40). The diagnostic specificity was 97.9%. Blinded method testing organized by an independent laboratory confirmed the satisfactory reproducibility of the assay. To our knowledge, this study represents the first validation of RT-LAMP for SARS-CoV-2 detection in animals. RT-LAMP testing could detect SARS-CoV-2 infection more rapidly and at the point of care in animals with moderate to high viral loads, allowing for earlier implementation of control measures. Full article

The increasing prevalence of antimicrobial-resistant (AMR) bacteria in humans, animals, and the environment underscores the necessity for a rapid, sensitive, and specific method to identify resistance genes. Objectives: This study aims to develop a reliable detection tool for identifying the tetracycline-resistant gene tet(M) in Enterococcus species using a real-time loop-mediated isothermal amplification (RT-LAMP) assay. Real-time visualization through a turbidimeter enabled precise estimation of time-to-positivity for gene detection. Methodology: Six primers were designed using PrimerExplorer v.5, and the assay was optimized across different temperatures and incubation times. Validation was conducted by testing 52 tet(M)-positive clinical enterococci isolates and spiking urine samples from a healthy volunteer and a cow with tet(M)-positive Enterococcus species. Results: The tet(M) gene was detected as early as 33 min, with optimal amplification occurring within 60 min at 60 °C. The assay demonstrated 100% specificity with the established primers. The sigmoidal graphs were corroborated with visual confirmation methods, including a green color change (visible to the naked eye), green fluorescence (under UV light), and a 200 bp PCR product observed via agarose gel electrophoresis. Notably, the tet(M) RT-LAMP assay exhibited a detection limit of 0.001 pg/μL, significantly surpassing conventional PCR, which had a detection limit of 0.1 pg/μL. Conclusions: This rapid, cost-effective, highly sensitive, and specific tet(M) RT-LAMP assay holds significant promise as a surveillance tool for antimicrobial resistance monitoring within a One Health framework, particularly in low-resource countries. Full article

Avian influenza A viruses (AIV) significantly impact both animal and human health. Reliable diagnostics are crucial for controlling AIV, including the highly pathogenic strains like H5Nx. In this study, we developed and validated the on-site Alveo Sense Poultry Avian Influenza Tests to rapidly detect the AIV M-gene and subtypes H5, H7, and H9 in unprocessed samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) and impedance-based measurements. The Alveo Sense tests, using single-use microfluidic cartridges, deliver results within 45 min. Each cartridge includes assays for the AIV M gene and specific H5 and H7 or H9 subtypes, with internal process controls. The laboratory validation involved specificity, limit of detection (LoD), diagnostic sensitivity, reproducibility, and robustness tests using various AIV strains, other avian pathogens, and field samples. The assays showed 100% specificity for AIV subtypes without cross-reactivity with non-AIV pathogens. The LoD95 for H5, H7, and H9 ranged between RT-PCR Ct values of 29–33 in both cloacal and oropharyngeal samples and were able to detect avian influenza virus in both spiked samples and field samples. Reproducibility and repeatability studies showed perfect agreement across operators and laboratories and remained stable and accurate under different pre-analytical conditions. The Alveo Sense tests offer rapid, accurate, and reliable on-site diagnostics for AIV subtypes H5, H7, and H9 on samples from fresh dead and sick birds, valuable for early flock-level detection and outbreak control. Further field validation will improve the understanding of their diagnostic performance across various avian species. Full article

Background/Objective. Toxin-producing strains of Clostridioides difficile (C. diff) are the most commonly identified cause of healthcare-associated infection in the elderly. Risk factors include advanced age, hospitalization, prior or concomitant systemic antibacterial therapy, chemotherapy, and gastrointestinal surgery. Patients with unspecified and new-onset diarrhea with ≥3 unformed stools in 24 h are the target population for C. diff infection (CDI) testing. To present data on the risks of laboratory misdiagnosis in managing CDI. Materials. In two general hospitals, we examined 116 clinical stool specimens from hospitalized patients with acute diarrhea suspected of nosocomial or antibiotic-associated diarrhea (AAD) due to C. diff. Enzyme immunoassay (EIA) tests for the detection of C. diff toxins A (cdtA) and B (cdtB) in stool, automated CLIA assay for the detection of C. diff GDH antigen and qualitative determination of cdtA and B in human feces and anaerobic stool culture were applied for CDI laboratory diagnosis. MALDI-TOF (Bruker) was used to identify the presumptive anaerobic bacterial colonies. The following methods were used as confirmatory diagnostics: the LAMP method for the detection of Salmonella spp. and simultaneous detection of C. jejuni and C. coli, an E. coli Typing RT-PCR detection kit (ETEC, EHEC, STEC, EPEC, and EIEC), API 20E and aerobic stool culture methods. Results. A total of 40 toxigenic strains of C. diff were isolated from all 116 tested diarrheal stool samples, of which 38/40 produced toxin B and 2/40 strains were positive for both cdtA and cdtB. Of the stool samples positive for cdtA (6/50) and/or cdtB (44/50) by EIA, 33 were negative for C. diff culture but positive for the following diarrheal agents: Salmonella enterica subsp. arizonae (1/33, LAMP, culture, API 20E); C. jejuni (2/33, LAMP, culture, MALDI TOF); ETEC O142 (1/33), STEC O145 and O138 (2/33, E. coli RT-PCR detection kit, culture); C. perfringens (2/33, anaerobic culture, MALDI TOF); hypermycotic enterotoxigenic K. pneumonia (2/33) and enterotoxigenic P. mirabilis (2/33, culture; PCR encoding LT-toxin). Two of the sixty-six cdtB-positive samples (2/66) showed a similar misdiagnosis when analyzed using the CLIA method. However, the PCR analysis showed that they were cdtB-negative. In contrast, the LAMP method identified a positive result for C. jejuni in one sample, and another was STEC positive (stx1+/stx2+) by RT-PCR. We found an additional discrepancy in the CDI test results: EPEC O86 (RT-PCR eae+) was isolated from a fecal sample positive for GHA enzyme (CLIA) and negative for cdtA and cdtB (CLIA and PCR). However, the culture of C. diff was negative. These findings support the hypothesis that certain human bacterial pathogens that produce enterotoxins other than C. diff, as well as intestinal commensal microorganisms, including Klebsiella sp. and Proteus sp., contribute to false-positive EIA card tests for C. diff toxins A and B, which are the most widely used laboratory tests for CDI. Conclusions. CDI presents a significant challenge to clinical practice in terms of laboratory diagnostic management. It is recommended that toxin-only EIA tests should not be used as the sole diagnostic tool for CDI but should be limited to detecting toxins A and B. Accurate diagnosis of CDI requires a combination of laboratory diagnostic methods on which proper infection management depends. Full article

PEDV poses a significant threat to the global swine industry, necessitating rapid and accurate diagnostic methods for effective disease management. In this study, we developed a foldable, easy-to-use paper-based microfluidic analytical device (μPAD) for on-site detection of PEDV. The device seamlessly integrates paper-based nucleic acid enrichment, LAMP reaction, and visual lateral flow detection into a single platform. Key parameters, including nucleic acid extraction protocols, chromatographic channel configurations, colorimetric indicators, and reaction temperature and duration, were systematically optimized. The resulting LAMP-μPAD assay detects PEDV within 30 min at 60 °C, achieving a limit of detection of 4.82 × 10² copies/μL with no cross-reactivity against other viruses. When evaluated against RT-PCR using clinical specimens, the assay demonstrated a specificity of 100%, a sensitivity of 95.3%, and an overall concordance of 98.5%. This paper-based sensor offers a promising alternative for the rapid, on-site detection of PEDV and other highly transmissible pathogens. Full article

Porcine epidemic diarrhea virus (PEDV), a primary pathogen causing diarrhea in pigs, particularly in piglets, has a mortality rate of up to 100%. Field PEDV strains circulating in pig production can be phylogenetically divided into two genotypes, GI and GII. Differential diagnosis of clinical strains with different genotypes is helpful for understanding disease epidemiology, vaccine selection, and prevention and control measures. The loop-mediated isothermal amplification method (LAMP), a novel nucleic acid amplification technique, has been utilized to detect a variety of pathogens in practice. In this study, we developed a distinguished RT-LAMP method to identify genotypes GI and GII strains of PEDV. Two pairs of primers, PEDV-LM and PEDV-LS, were designed based on the membrane and spike genes of PEDV, respectively. PEDV-LM primers exhibited specificity for all PEDV strains, while PEDV-LS primers specifically targeted the PEDV genotype GI. The diagnostic sensitivity of both primers was 1 × 10² copies/reaction, which is 100 times more sensitive than RT-PCR. The RT-LAMP reaction process was completed at 65 °C for 40 min just in a water bath or metal bath. A cross-reactivity assay confirmed that this method is specific for PEDV GI and GII, with no cross-amplification observed with other swine-origin viruses such as PDCoV, PoRV, PRV, and PRRSV. Therefore, this refined LAMP technique offers a rapid, sensitive, and reliable method with which to detect and differentiate between PEDV GI and GII, making it a superior tool for the large-scale clinical surveillance of PEDV infections. Full article

In previous studies, we isolated a series of novel gentisides with nerve growth factor (NGF)-mimic activities from Gentiana rigescens Franch and conducted continuous structure–activity relationship (SAR) studies. Recently, a lead compound named TBG096 was discovered with significant NGF-mimic activity, low toxicity, and ability to pass through the blood–brain barrier (BBB). At the cell level, TBG096 exerts NGF-mimic activity by regulation of heat-shock cognate protein 70 (Hsc70) and downstream proteins. Subsequently, high-fat diet (HFD)-induced Alzheimer disease (AD) mouse models were used to evaluate the anti-AD efficacy of the compound. TBG096 significantly improved the memory dysfunction of AD mice at doses of 0.1, 5, and 20 mg/kg, respectively. In order to elucidate the mechanism of action of the compound against AD, the RNA-sequence analysis of transcriptomics, quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence staining, and Western blot analysis were performed using animal samples. TBG096 significantly increased the expression of the Wnt gene family (Wnt10b, Wnt5a, and Wnt1) and the number of mature neurons and newborn neurons in the hippocampus and cerebral cortex of AD mice, respectively. At the same time, it reduced the activity of microglia, astrocyte cells, and expression of inducible nitric oxide synthase (INOS) in the brain. Moreover, this compound significantly increased phosphorylated-adenosine 5′-monophosphate-activated protein kinase (AMPK), Hsc70, and lysosomal-associated membrane protein 2a (LAMP2A) and decreased the expression of hexokinase 2 (HK2), pyruvate kinase M2 (PKM2), amyloid precursor protein (APP), microtubule-associated protein tau (Tau), phosphoryl-Tau, and β-amyloid (Aβ) at the protein level. These results suggest that TBG096 produced the NGF-mimic activity and the anti-AD effect via promoting neurogenesis and modification of the Hsc70/HK2/PKM2/LAMP2A signaling pathway, proposing a potential novel approach to counteracting cognitive decline by developing small molecules that promote neurogenesis and the Hsc70 signaling pathway. Full article

Feline infectious peritonitis (FIP), a devastating disease with near-complete mortality, is caused by the feline coronavirus (FCoV) and affects domestic cats worldwide. Herein, we report the development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay incorporating xylenol orange (XO) as a visual indicator for FCoV detection. The assay employed six oligonucleotide primers targeting regions of the nucleocapsid (N) gene. Under optimized conditions (65 °C, 60 min), amplification products were detected through pH-dependent colour changes in the XO dye. The RT-LAMP-XO assay exhibited high specificity for FCoV, with no cross-reactivity against other common feline viral pathogens. While the detection limit (1.7 × 10¹ copies/µL) was an order of magnitude higher than that of qPCR, the method offered advantages in simplicity and speed compared to existing diagnostic approaches. Although less sensitive than qPCR, the RT-LAMP-XO assay may serve as a rapid screening tool when used in combination with additional primer sets. These findings demonstrate the potential utility of XO-based RT-LAMP as a simple, visual detection method for FCoV infection. Full article

(1) Background: There are many cases of human disease caused by the hepatitis A virus contamination of aquatic products, so the development of the rapid detection of hepatitis A virus in aquatic products is crucial. (2) Methods: In this study, we developed three visual loop-mediated isothermal amplification methods for the rapid and intuitive detection of hepatitis A virus in aquatic products. New specific LAMP primers were designed for the HAV-specific VP1 protein shell. (1) HNB dye was added to the LAMP reaction system. After the reaction, the color of the reaction mixture changed from violet to sky blue, showing a positive result. (2) Cresol red dye was added to the LAMP reaction system, and a positive result was indicated by orange, while a negative result was indicated by purple. (3) By labeling FIP with biotin and LF with 6-FAM, the amplified product simultaneously contained biotin and 6-FAM, which bound to the anti-biotin antibody on the gold nanoparticles on the lateral flow dipstick (LFD). Subsequently, biotin was further combined with the anti-fam antibody on the T-line of the test strip to form a positive test result. (3) Results: The three visual LAMP methods were highly specific for HAV. The sensitivity of the visual assay was 2.59 × 10⁰ copies/μL. The positive detection ratio for 155 bivalve shellfish samples was 8.39%, which was the same as that for RT-qPCR. The three visual LAMP methods established in our work have better sensitivity than the international gold standard, and their operation is simple and requires less time. (4) Conclusions: The results can be obtained by eye color comparison and lateral flow dipsticks. Without the use of large-scale instrumentation, the sensitivity is the same as that of RT-qPCR. The test strips are lightweight, small in size, and easy to carry; they are suitable for emergency detection, on-site monitoring, field sampling, or remote farms and other non-laboratory environments for rapid identification. Full article

Background: Dengue virus (DENV) infection is a significant public health concern in several tropical and subtropical regions, where early and rapid detection is crucial for effective patient management and controlling the spread of the disease. Particularly in resource-limited, rural healthcare settings where dengue is endemic, there exists a need for diagnostic methods that are both easy to perform and highly sensitive. Objective: This study focuses on the development and validation of a single-tube reverse transcription loop-mediated isothermal amplification termed TURN-RT-loop-mediated isothermal amplification (LAMP) for the detection of DENV. Methodology: The TURN-RT-LAMP assay designed in this study combines two sets of primers targeting the 5′- and 3′-UTR of DENV, with the aim to increase the sensitivity of detection. Results: Clinical validation of the TURN-RT-LAMP assay using samples collected from febrile individuals with a serological or antigenic diagnosis revealed a sensitivity of >96%. The performance of this assay was statistically compared with that of the standard diagnostic method, quantitative reverse transcription-polymerase chain reaction. Conclusions: The results support the potential of RT-LAMP as a rapid, sensitive, and specific tool for the diagnosis and surveillance of dengue, particularly suitable for field use in low-resource settings. Full article

Resource-limited settings and supply chain difficulties faced throughout the COVID-19 pandemic prompted the development of rapid and alternative methods of detecting SARS-CoV-2. These methods include reverse-transcription loop-mediated isothermal amplification (RT-LAMP), reverse-transcription recombinase polymerase amplification (RT-RPA), and CRISPR-Cas12a fluorescence detection. We describe RT-LAMP, RT-RPA, and CRISPR-Cas12a assays for the detection of the N and E-gene amplicons of SARS-CoV-2 and the optimization of various assay components, including incubation temperatures, Cas12a enzymes, reporter molecules, and the use of a lyophilized RT-LAMP master mix. We also describe the testing of a one-tube RT-LAMP-CRISPR-Cas12a assay. The one-tube assay showed promise in reducing hands-on time and improving time-to-result. We found no improvements in assay sensitivity with RT-RPA, but did achieve detection at a lower copy number with the lyophilized RT-LAMP master mix compared to liquid reagent (50 vs. 100 copies at 20 min). When used to detect the presence of SARS-CoV-2 RNA in clinical saliva samples from 75 infected patients, the discriminatory ability of the optimized RT-LAMP-CRISPR Cas12a assay was found to be comparable with RT-qPCR, with a minor reduction in sensitivity. Full article