Sign in to use this feature.

Years

Between: -

Subjects

remove_circle_outline
remove_circle_outline
remove_circle_outline
remove_circle_outline
remove_circle_outline
remove_circle_outline

Journals

Article Types

Countries / Regions

Search Results (60)

Search Parameters:
Keywords = nucleic acid lateral flow assay

Order results
Result details
Results per page
Select all
Export citation of selected articles as:
21 pages, 2191 KiB  
Review
Heavy Metal Ion Detection Based on Lateral Flow Assay Technology: Principles and Applications
by Xiaobo Xie, Xinyue Hu, Xin Cao, Qianhui Zhou, Wei Yang, Ranran Yu, Shuaiqi Liu, Huili Hu, Ji Qi and Zhiyang Zhang
Biosensors 2025, 15(7), 438; https://doi.org/10.3390/bios15070438 - 7 Jul 2025
Viewed by 589
Abstract
Heavy metal ions pose a significant threat to the environment and human health due to their high toxicity and bioaccumulation. Traditional instrumentations, although sensitive, are often complex, costly, and unsuitable for on-site rapid detection of heavy metal ions. Lateral flow assay technology has [...] Read more.
Heavy metal ions pose a significant threat to the environment and human health due to their high toxicity and bioaccumulation. Traditional instrumentations, although sensitive, are often complex, costly, and unsuitable for on-site rapid detection of heavy metal ions. Lateral flow assay technology has emerged as a research hotspot due to its rapid, simple, and cost-effective advantages. This review summarizes the applications of lateral flow assay technology based on nucleic acid molecules and antigen–antibody interactions in heavy metal ion detection, focusing on recognition mechanisms such as DNA probes, nucleic acid enzymes, aptamers, and antigen–antibody binding, as well as signal amplification strategies on lateral flow testing strips. By incorporating these advanced technologies, the sensitivity and specificity of lateral flow assays have been significantly improved, enabling highly sensitive detection of various heavy metal ions, including Hg2+, Cd2+, Pb2+, and Cr3+. In the future, the development of lateral flow assay technology for detection of heavy metal ions will focus on multiplex detection, optimization of signal amplification strategies, integration with portable devices, and standardization and commercialization. With continuous technological advancements, lateral flow assay technology will play an increasingly important role in environmental monitoring, food safety, and public health. Full article
Show Figures

Figure 1

16 pages, 5722 KiB  
Article
Development of a Paper-Based Microfluidic Chip for Point-of-Care Detection of PEDV
by Renfeng Li, Xiangqin Tian, Wenyan Cao, Jiaxin Jiang, Jiakang Yuan, Linyue Li, Yonghe You, Yanlin Zhou, Ziliang Wang and Fangyu Wang
Vet. Sci. 2025, 12(5), 427; https://doi.org/10.3390/vetsci12050427 - 30 Apr 2025
Viewed by 624
Abstract
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 [...] Read more.
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
Show Figures

Graphical abstract

14 pages, 2999 KiB  
Article
Nucleic Acid Lateral Flow Assay Implemented with Isothermal Gene Amplification of SARS-CoV-2 RNA
by Kangwuk Kyung, Hyojin Lee, Soo-Kyung Kim and Dong-Eun Kim
Biosensors 2024, 14(12), 585; https://doi.org/10.3390/bios14120585 - 1 Dec 2024
Viewed by 2417
Abstract
We developed a rapid and sensitive diagnostic platform that integrates isothermal viral gene amplification with a nucleic acid lateral flow assay (NALFA) to detect SARS-CoV-2 RNA. Isothermal gene amplification was performed by combining reverse transcription of viral RNA with recombinase polymerase amplification (RPA). [...] Read more.
We developed a rapid and sensitive diagnostic platform that integrates isothermal viral gene amplification with a nucleic acid lateral flow assay (NALFA) to detect SARS-CoV-2 RNA. Isothermal gene amplification was performed by combining reverse transcription of viral RNA with recombinase polymerase amplification (RPA). In our diagnostic platform, DNA primers for the RPA reaction were modified by appending DNA tails, enabling the synthesis of tailed amplicon DNAs. These tailed amplicon DNAs were subsequently annealed to the complementary capture DNA probe affixed to the lateral flow strip during the NALFA of the reaction samples. The other side of each amplicon DNA tail was annealed to the reporter probe DNA conjugated with gold nanoparticles to visually detect the test line in the strip. This diagnostic platform reduces the time required to obtain readouts to within 1 h and can detect viral RNA concentrations as low as 3.1 cp/μL. Furthermore, when applied to nasopharyngeal clinical samples, our NALFA diagnostic platform yielded highly reliable molecular diagnostic readouts that were 100% consistent with the results of conventional RT-qPCR. Full article
Show Figures

Figure 1

14 pages, 958 KiB  
Review
Current Analytical Methods and Challenges for the Clinical Diagnosis of Invasive Pulmonary Aspergillosis Infection
by Madeline C. R. Schwarz, Alex E. Moskaluk, Joshua B. Daniels, Sue VandeWoude and Melissa M. Reynolds
J. Fungi 2024, 10(12), 829; https://doi.org/10.3390/jof10120829 - 28 Nov 2024
Viewed by 1850
Abstract
In the last decade, pulmonary fungal infections such as invasive pulmonary aspergillosis (IPA) have increased in incidence due to the increased number of immunocompromised individuals. This increase is especially problematic when considering mortality rates associated with IPA are upwards of 70%. This high [...] Read more.
In the last decade, pulmonary fungal infections such as invasive pulmonary aspergillosis (IPA) have increased in incidence due to the increased number of immunocompromised individuals. This increase is especially problematic when considering mortality rates associated with IPA are upwards of 70%. This high mortality rate is due to, in part, the length of time it takes to diagnose a patient with IPA. When diagnosed early, mortality rates of IPA decrease by as much as 30%. In this review, we discuss current technologies employed in both medical and research laboratories to diagnose IPA, including culture, imaging, polymerase chain reaction, peptide nucleic acid–fluorescence in situ hybridization, enzyme-linked immunosorbent assay, lateral flow assay, and liquid chromatography mass spectrometry. For each technique, we discuss both promising results and potential areas for improvement that would lead to decreased diagnosis time for patients suspected of contracting IPA. Further study into methods that offer increased speed and both analytical and clinical sensitivity to decrease diagnosis time for IPA is warranted. Full article
(This article belongs to the Special Issue Diagnosis of Human Pathogenic Fungi)
Show Figures

Figure 1

10 pages, 1706 KiB  
Article
MNAzyme-Assisted Nucleic Acid Lateral Flow Assay for Cost-Effective, On-Site Mercury Detection
by Seok Hyeon Kim, Yujun Kim, Seokjoon Kim, Eun Sung Lee, Byung Seok Cha and Ki Soo Park
Biosensors 2024, 14(10), 454; https://doi.org/10.3390/bios14100454 - 25 Sep 2024
Cited by 1 | Viewed by 1693
Abstract
Mercury ions (Hg2+) are toxic heavy metals present in the environment that pose significant health risks. An advanced detection system could allow for a prompt response and alleviate serious damage to humans. In this study, we developed a cost-effective, on-site detection [...] Read more.
Mercury ions (Hg2+) are toxic heavy metals present in the environment that pose significant health risks. An advanced detection system could allow for a prompt response and alleviate serious damage to humans. In this study, we developed a cost-effective, on-site detection method for Hg2+ using a multicomponent nucleic acid enzyme (MNAzyme)-assisted nucleic acid lateral flow assay (NALFA). The MNAzyme, which was engineered to contain thymine–thymine mismatches, is responsive only to the presence of Hg2+ and exerts efficient cleavage activity on substrates that can be captured by the NALFA strip, and thus the proposed system enables the visual detection of Hg2+ in the NALFA strip. Our assay demonstrated sufficient detection sensitivity and specificity to meet the WHO standards, offering a good practical alternative for rapid environmental and public health monitoring. Full article
(This article belongs to the Special Issue Probes for Biosensing and Bioimaging)
Show Figures

Figure 1

19 pages, 20339 KiB  
Article
Enhancing Colorimetric Detection of Nucleic Acids on Nitrocellulose Membranes: Cutting-Edge Applications in Diagnostics and Forensics
by Nidhi Subhashini, Yannick Kerler, Marcus M. Menger, Olga Böhm, Judith Witte, Christian Stadler and Alexander Griberman
Biosensors 2024, 14(9), 430; https://doi.org/10.3390/bios14090430 - 5 Sep 2024
Cited by 2 | Viewed by 2446
Abstract
This study re-introduces a protein-free rapid test method for nucleic acids on paper based lateral flow assays utilizing special multichannel nitrocellulose membranes and DNA-Gold conjugates, achieving significantly enhanced sensitivity, easier protocols, reduced time of detection, reduced costs of production and advanced multiplexing possibilities. [...] Read more.
This study re-introduces a protein-free rapid test method for nucleic acids on paper based lateral flow assays utilizing special multichannel nitrocellulose membranes and DNA-Gold conjugates, achieving significantly enhanced sensitivity, easier protocols, reduced time of detection, reduced costs of production and advanced multiplexing possibilities. A protein-free nucleic acid-based lateral flow assay (NALFA) with a limit of detection of 1 pmol of DNA is shown for the first time. The total production duration of such an assay was successfully reduced from the currently known several days to just a few hours. The simplification and acceleration of the protocol make the method more accessible and practical for various applications. The developed method supports multiplexing, enabling the simultaneous detection of up to six DNA targets. This multiplexing capability is a significant improvement over traditional line tests and offers more comprehensive diagnostic potential in a single assay. The approach significantly reduces the run time compared to traditional line tests, which enhances the efficiency of diagnostic procedures. The protein-free aspect of this assay minimizes the prevalent complications of cross-reactivity in immunoassays especially in cases of multiplexing. It is also demonstrated that the NALFA developed in this study is amplification-free and hence does not rely on specialized technicians, nor does it involve labour-intensive steps like DNA extraction and PCR processes. Overall, this study presents a robust, efficient, and highly sensitive platform for DNA or RNA detection, addressing several limitations of current methods documented in the literature. The advancements in sensitivity, cost reduction, production time, and multiplexing capabilities mark a substantial improvement, holding great potential for various applications in diagnostics, forensics, and molecular biology. Full article
(This article belongs to the Section Biosensors and Healthcare)
Show Figures

Figure 1

14 pages, 2789 KiB  
Article
Specific and Sensitive Visual Proviral DNA Detection of Major Pathogenic Avian Leukosis Virus Subgroups Using CRISPR-Associated Nuclease Cas13a
by Qingqing Xu, Yaoyao Zhang, Yashar Sadigh, Na Tang, Jiaqian Chai, Ziqiang Cheng, Yulong Gao, Aijian Qin, Zhiqiang Shen, Yongxiu Yao and Venugopal Nair
Viruses 2024, 16(7), 1168; https://doi.org/10.3390/v16071168 - 20 Jul 2024
Cited by 3 | Viewed by 1960
Abstract
Avian leukosis viruses (ALVs) include a group of avian retroviruses primarily associated with neoplastic diseases in poultry, commonly referred to as avian leukosis. Belonging to different subgroups based on their envelope properties, ALV subgroups A, B, and J (ALV-A, ALV-B, and ALV-J) are [...] Read more.
Avian leukosis viruses (ALVs) include a group of avian retroviruses primarily associated with neoplastic diseases in poultry, commonly referred to as avian leukosis. Belonging to different subgroups based on their envelope properties, ALV subgroups A, B, and J (ALV-A, ALV-B, and ALV-J) are the most widespread in poultry populations. Early identification and removal of virus-shedding birds from infected flocks are essential for the ALVs’ eradication. Therefore, the development of rapid, accurate, simple-to-use, and cost effective on-site diagnostic methods for the detection of ALV subgroups is very important. Cas13a, an RNA-guided RNA endonuclease that cleaves target single-stranded RNA, also exhibits non-specific endonuclease activity on any bystander RNA in close proximity. The distinct trans-cleavage activity of Cas13 has been exploited in the molecular diagnosis of multiple pathogens including several viruses. Here, we describe the development and application of a highly sensitive Cas13a-based molecular test for the specific detection of proviral DNA of ALV-A, B, and J subgroups. Prokaryotically expressed LwaCas13a, purified through ion exchange and size-exclusion chromatography, was combined with recombinase polymerase amplification (RPA) and T7 transcription to establish the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) molecular detection system for the detection of proviral DNA of ALV-A/B/J subgroups. This novel method that needs less sample input with a short turnaround time is based on isothermal detection at 37 °C with a color-based lateral flow readout. The detection limit of the assay for ALV-A/B/J subgroups was 50 copies with no cross reactivity with ALV-C/D/E subgroups and other avian oncogenic viruses such as reticuloendotheliosis virus (REV) and Marek’s disease virus (MDV). The development and evaluation of a highly sensitive and specific visual method of detection of ALV-A/B/J nucleic acids using CRISPR-Cas13a described here will help in ALV detection in eradication programs. Full article
(This article belongs to the Special Issue Recent Advances of Avian Viruses Research)
Show Figures

Figure 1

11 pages, 4450 KiB  
Article
A Portable, Integrated, Sample-In Result-Out Nucleic Acid Diagnostic Device for Rapid and Sensitive Chikungunya Virus Detection
by Changping Xu, Yalin Chen, Guiying Zhu, Huan Wu, Qi Jiang, Rui Zhang, Beibei Yu, Lei Fang and Zhiwei Wu
Micromachines 2024, 15(5), 663; https://doi.org/10.3390/mi15050663 - 19 May 2024
Viewed by 1999
Abstract
Chikungunya virus, a mosquito-borne virus that causes epidemics, is often misdiagnosed due to symptom similarities with other arboviruses. Here, a portable and integrated nucleic acid-based diagnostic device, which combines reverse transcription-loop-mediated isothermal amplification and lateral-flow detection, was developed. The device is simple to [...] Read more.
Chikungunya virus, a mosquito-borne virus that causes epidemics, is often misdiagnosed due to symptom similarities with other arboviruses. Here, a portable and integrated nucleic acid-based diagnostic device, which combines reverse transcription-loop-mediated isothermal amplification and lateral-flow detection, was developed. The device is simple to use, precise, equipment-free, and highly sensitive, enabling rapid chikungunya virus identification. The result can be obtained by the naked eye within 40 min. The assay can effectively distinguish chikungunya virus from dengue virus, Japanese encephalitis virus, Zika virus, and yellow fever virus with high specificity and sensitivity as low as 598.46 copies mL−1. It has many benefits for the community screening and monitoring of chikungunya virus in resource-limited areas because of its effectiveness and simplicity. The platform has great potential for the rapid nucleic acid detection of other viruses. Full article
(This article belongs to the Special Issue Micro/Nanofluidics Devices for Nucleic Acids and Cell Analysis)
Show Figures

Figure 1

11 pages, 2335 KiB  
Article
Introducing Triplex Forming Oligonucleotide into Loop-Mediated Isothermal Amplification for Developing a Lateral Flow Biosensor for Streptococci Detection
by Wei Chang, Po-Hao Chou, Cai-Tong Wu, Jheng-Da Song, Kun-Nan Tsai and Chiuan-Chian Chiou
Biosensors 2024, 14(5), 257; https://doi.org/10.3390/bios14050257 - 17 May 2024
Cited by 1 | Viewed by 2295
Abstract
Loop-mediated isothermal amplification (LAMP) technology is extensively utilized for the detection of infectious diseases owing to its rapid processing and high sensitivity. Nevertheless, conventional LAMP signaling methods frequently suffer from a lack of sequence specificity. This study integrates a triplex-forming oligonucleotide (TFO) probe [...] Read more.
Loop-mediated isothermal amplification (LAMP) technology is extensively utilized for the detection of infectious diseases owing to its rapid processing and high sensitivity. Nevertheless, conventional LAMP signaling methods frequently suffer from a lack of sequence specificity. This study integrates a triplex-forming oligonucleotide (TFO) probe into the LAMP process to enhance sequence specificity. This TFO-LAMP technique was applied for the detection of Group B Streptococcus (GBS). The TFO probe is designed to recognize a specific DNA sequence, termed the TFO targeting sequence (TTS), within the amplified product, facilitating detection via fluorescent instrumentation or lateral flow biosensors. A screening method was developed to identify TFO sequences with high affinity to integrate TFO into LAMP, subsequently incorporating a selected TTS into an LAMP primer. In the TFO-LAMP assay, a FAM-labeled TFO is added to target the TTS. This TFO can be captured by an anti-FAM antibody on lateral flow test strips, thus creating a nucleic acid testing biosensor. The efficacy of the TFO-LAMP assay was confirmed through experiments with specimens spiked with varying concentrations of GBS, demonstrating 85% sensitivity at 300 copies and 100% sensitivity at 30,000 copies. In conclusion, this study has successfully developed a TFO-LAMP technology that offers applicability in lateral flow biosensors and potentially other biosensor platforms. Full article
(This article belongs to the Special Issue Design and Application of Novel Nucleic Acid Probe)
Show Figures

Figure 1

15 pages, 3315 KiB  
Article
Point-of-Care Diagnostic System for Viable Salmonella Species via Improved Propidium Monoazide and Recombinase Polymerase Amplification Based Nucleic Acid Lateral Flow
by So-Young Lee and Se-Wook Oh
Diagnostics 2024, 14(8), 831; https://doi.org/10.3390/diagnostics14080831 - 17 Apr 2024
Cited by 2 | Viewed by 1780
Abstract
Salmonella species are prominent foodborne microbial pathogens transmitted through contaminated food or water and pose a significant threat to human health. Accurate and rapid point-of-care (POC) diagnosis is gaining attention in effectively preventing outbreaks of foodborne disease. However, the presence of dead bacteria [...] Read more.
Salmonella species are prominent foodborne microbial pathogens transmitted through contaminated food or water and pose a significant threat to human health. Accurate and rapid point-of-care (POC) diagnosis is gaining attention in effectively preventing outbreaks of foodborne disease. However, the presence of dead bacteria can interfere with an accurate diagnosis, necessitating the development of methods for the rapid, simple, and efficient detection of viable bacteria only. Herein, we used an improved propidium monoazide (PMAxx) to develop a nucleic acid lateral flow (NALF) assay based on recombinase polymerase amplification (RPA) to differentiate viable Salmonella Typhimurium. We selected an RPA primer set targeting the invA gene and designed a probe for NALF. RPA-based NALF was optimized for temperature (30–43 °C), time (1–25 min), and endonuclease IV concentration (0.025–0.15 unit/µL). PMAxx successfully eliminated false-positive results from dead S. Typhimurium, enabling the accurate detection of viable S. Typhimurium with a detection limit of 1.11 × 102 CFU/mL in pure culture. The developed method was evaluated with spiked raw chicken breast and milk with analysis completed within 25 min at 39 °C. This study has potential as a tool for the POC diagnostics of viable foodborne pathogens with high specificity, sensitivity, rapidity, and cost-effectiveness. Full article
(This article belongs to the Special Issue Visualization Technology in Point-of-Care Diagnostics)
Show Figures

Figure 1

16 pages, 1263 KiB  
Review
Advances and Challenges in SARS-CoV-2 Detection: A Review of Molecular and Serological Technologies
by Mai M. El-Daly
Diagnostics 2024, 14(5), 519; https://doi.org/10.3390/diagnostics14050519 - 29 Feb 2024
Cited by 15 | Viewed by 4448
Abstract
The urgent need for accurate COVID-19 diagnostics has led to the development of various SARS-CoV-2 detection technologies. Real-time reverse transcriptase polymerase chain reaction (RT-qPCR) remains a reliable viral gene detection technique, while other molecular methods, including nucleic acid amplification techniques (NAATs) and isothermal [...] Read more.
The urgent need for accurate COVID-19 diagnostics has led to the development of various SARS-CoV-2 detection technologies. Real-time reverse transcriptase polymerase chain reaction (RT-qPCR) remains a reliable viral gene detection technique, while other molecular methods, including nucleic acid amplification techniques (NAATs) and isothermal amplification techniques, provide diverse and effective approaches. Serological assays, detecting antibodies in response to viral infection, are crucial for disease surveillance. Saliva-based immunoassays show promise for surveillance purposes. The efficiency of SARS-CoV-2 antibody detection varies, with IgM indicating recent exposure and IgG offering prolonged detectability. Various rapid tests, including lateral-flow immunoassays, present opportunities for quick diagnosis, but their clinical significance requires validation through further studies. Challenges include variations in specificity and sensitivity among testing platforms and evolving assay sensitivities over time. SARS-CoV-2 antigens, particularly the N and S proteins, play a crucial role in diagnostic methods. Innovative approaches, such as nanozyme-based assays and specific nucleotide aptamers, offer enhanced sensitivity and flexibility. In conclusion, ongoing advancements in SARS-CoV-2 detection methods contribute to the global effort in combating the COVID-19 pandemic. Full article
(This article belongs to the Special Issue Review Research on SARS-CoV-2 Detection)
Show Figures

Figure 1

13 pages, 6602 KiB  
Article
Lateral Flow Biosensor for On-Site Multiplex Detection of Viruses Based on One-Step Reverse Transcription and Strand Displacement Amplification
by Xuewen Lu, Kangning Ding, Zhiyuan Fang, Yilei Liu, Tianxing Ji, Jian Sun, Zhenling Zeng and Limin He
Biosensors 2024, 14(2), 103; https://doi.org/10.3390/bios14020103 - 17 Feb 2024
Cited by 3 | Viewed by 2702
Abstract
Respiratory pathogens pose a huge threat to public health, especially the highly mutant RNA viruses. Therefore, reliable, on-site, rapid diagnosis of such pathogens is an urgent need. Traditional assays such as nucleic acid amplification tests (NAATs) have good sensitivity and specificity, but these [...] Read more.
Respiratory pathogens pose a huge threat to public health, especially the highly mutant RNA viruses. Therefore, reliable, on-site, rapid diagnosis of such pathogens is an urgent need. Traditional assays such as nucleic acid amplification tests (NAATs) have good sensitivity and specificity, but these assays require complex sample pre-treatment and a long test time. Herein, we present an on-site biosensor for rapid and multiplex detection of RNA pathogens. Samples with viruses are first lysed in a lysis buffer containing carrier RNA to release the target RNAs. Then, the lysate is used for amplification by one-step reverse transcription and single-direction isothermal strand displacement amplification (SDA). The yield single-strand DNAs (ssDNAs) are visually detected by a lateral flow biosensor. With a secondary signal amplification system, as low as 20 copies/μL of virus can be detected in this study. This assay avoids the process of nucleic acid purification, making it equipment-independent and easier to operate, so it is more suitable for on-site molecular diagnostic applications. Full article
(This article belongs to the Special Issue Biosensors for the Analysis and Detection of Drug, Food or Disease)
Show Figures

Figure 1

13 pages, 1275 KiB  
Article
Detection of Nucleic Acids of the Fish Pathogen Yersinia ruckeri from Planktonic and Biofilm Samples with a CRISPR/Cas13a-Based Assay
by Iván L. Calderón, M. José Barros, Nicolás Fernández-Navarro and Lillian G. Acuña
Microorganisms 2024, 12(2), 283; https://doi.org/10.3390/microorganisms12020283 - 29 Jan 2024
Cited by 5 | Viewed by 2458
Abstract
Yersinia ruckeri is the cause of hemorrhagic septicemia, known as enteric redmouth disease, in salmonid fish species. This bacterial pathogen can form biofilms on abiotic surfaces of aquaculture settings or even on the surfaces of the fish themselves, contributing to their persistence in [...] Read more.
Yersinia ruckeri is the cause of hemorrhagic septicemia, known as enteric redmouth disease, in salmonid fish species. This bacterial pathogen can form biofilms on abiotic surfaces of aquaculture settings or even on the surfaces of the fish themselves, contributing to their persistence in the aquatic environment. Detection methods for this and other fish pathogens can be time-consuming and lack specificity and sensitivity, limiting timely monitoring, the treatment of microbial infections, and effective control of their transmission in aquaculture settings. Rapid and sensitive detection methods for nucleic acids can be crucial for an appropriate surveillance of bacterial pathogens, and the CRISPR/Cas-based assays have emerged as a good alternative since it has been proven to be a useful tool for the rapid, specific, and sensitive detection of viruses and some bacteria. In this study, we explored the capability of the CRISPR/Cas13a system (SHERLOCK) to specifically detect both DNA and RNA (gene transcripts) from planktonic and biofilm samples of the bacterial fish pathogen Y. ruckeri. The assay was designed to detect the gyrA gene and the small noncoding RNAs (sRNAs) MicA and RprA from planktonic cultures and biofilm samples prepared in marine broth. The specific crRNA designed for these gene targets included a 28 nt specific gene sequence, and a scaffold sequence necessary for Cas13-binding. For all the assays, the nucleic acids obtained from samples were previously subjected to isothermal amplification with the recombinase polymerase amplification (RPA) method and the subsequent T7 transcription of the RPA amplicons. Finally, the detection of nucleic acids of Y. ruckeri was by means of a reporter signal released by the Cas13a collateral RNA cleavage triggered upon target recognition, measured by fluorescence- or lateral-flow-based readouts. This CRISPR/Cas13a-based assay was able to specifically detect both DNA and sRNAs from the Y. ruckeri samples, and the sensitivity was comparable to that obtained with qPCR analysis, highlighting the potential applicability of this CRISPR/Cas13a-based assay for fish pathogen surveillance. Full article
(This article belongs to the Special Issue Microorganisms and Diseases Associated with Aquatic Animals 2.0)
Show Figures

Figure 1

13 pages, 267 KiB  
Article
Comparison of the Diagnostic Performances of Five Different Tests in Diagnosing Visceral Leishmaniasis in an Endemic Region of Ethiopia
by Dawit Gebreegziabiher Hagos, Yazezew Kebede Kiros, Mahmud Abdulkader, Henk D. F. H. Schallig and Dawit Wolday
Diagnostics 2024, 14(2), 163; https://doi.org/10.3390/diagnostics14020163 - 11 Jan 2024
Cited by 5 | Viewed by 2403
Abstract
The lack of accurate and feasible diagnostic tests poses a significant challenge to visceral leishmaniasis (VL) healthcare services in endemic areas. To date, various VL diagnostic tests have been or are being developed, and their diagnostic performances need to be assessed. In the [...] Read more.
The lack of accurate and feasible diagnostic tests poses a significant challenge to visceral leishmaniasis (VL) healthcare services in endemic areas. To date, various VL diagnostic tests have been or are being developed, and their diagnostic performances need to be assessed. In the present study, the diagnostic performances of rk39 RDT, the direct agglutination test (DAT), microscopy, loop-mediated isothermal amplification (LAMP), and miniature direct-on-blood polymerase chain reaction–nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) were assessed using quantitative polymerase chain reaction (qPCR) as the reference test in an endemic region of Ethiopia. In this study, 235 suspected VL cases and 104 non-endemic healthy controls (NEHCs) were recruited. Among the suspected VL cases, 144 (61.28%) tested positive with qPCR. The sensitivities for rk39 RDT, DAT, microscopy, LAMP assay, and mini-dbPCR-NALFIA were 88.11%, 96.50%, 76.58%, 94.33%, and 95.80%, respectively. The specificities were 83.33%, 97.96%, 100%, 97.38%, and 98.92% for rk39 RDT, DAT, microscopy, LAMP assay, and mini-dbPCR-NALFIA, respectively. In conclusion, rk39 RDT and microscopy exhibited lower sensitivities, while DAT demonstrated excellent performance. LAMP and mini-dbPCR-NALFIA showed excellent performances with feasibility for implementation in remote endemic areas, although the latter requires further evaluation in such regions. Full article
(This article belongs to the Section Diagnostic Microbiology and Infectious Disease)
15 pages, 3694 KiB  
Article
Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow Assay
by Saloni Agarwal, Mojdeh Hamidizadeh and Frank F. Bier
Biosensors 2023, 13(11), 988; https://doi.org/10.3390/bios13110988 - 17 Nov 2023
Cited by 2 | Viewed by 2810
Abstract
This study focuses on three key aspects: (a) crude throat swab samples in a viral transport medium (VTM) as templates for RT-LAMP reactions; (b) a biotinylated DNA probe with enhanced specificity for LFA readouts; and (c) a digital semi-quantification of LFA readouts. Throat [...] Read more.
This study focuses on three key aspects: (a) crude throat swab samples in a viral transport medium (VTM) as templates for RT-LAMP reactions; (b) a biotinylated DNA probe with enhanced specificity for LFA readouts; and (c) a digital semi-quantification of LFA readouts. Throat swab samples from SARS-CoV-2 positive and negative patients were used in their crude (no cleaning or pre-treatment) forms for the RT-LAMP reaction. The samples were heat-inactivated but not treated for any kind of nucleic acid extraction or purification. The RT-LAMP (20 min processing time) product was read out by an LFA approach using two labels: FITC and biotin. FITC was enzymatically incorporated into the RT-LAMP amplicon with the LF-LAMP primer, and biotin was introduced using biotinylated DNA probes, specifically for the amplicon region after RT-LAMP amplification. This assay setup with biotinylated DNA probe-based LFA readouts of the RT-LAMP amplicon was 98.11% sensitive and 96.15% specific. The LFA result was further analysed by a smartphone-based IVD device, wherein the T-line intensity was recorded. The LFA T-line intensity was then correlated with the qRT-PCR Ct value of the positive swab samples. A digital semi-quantification of RT-LAMP-LFA was reported with a correlation coefficient of R2 = 0.702. The overall RT-LAMP-LFA assay time was recorded to be 35 min with a LoD of three RNA copies/µL (Ct-33). With these three advancements, the nucleic acid testing-point of care technique (NAT-POCT) is exemplified as a versatile biosensor platform with great potential and applicability for the detection of pathogens without the need for sample storage, transportation, or pre-processing. Full article
Show Figures

Figure 1

Back to TopTop