Lateral Flow Strip Assay Research Articles

Wheat Germ Agglutinin-Modified "Three-in-One" Multifunctional Probe Driven Broad-Spectrum and Flexible Immunochromatographic Diagnosis of viruses With High Sensitivity.

The conventional lateral flow assay (LFA) fails to the demands for the accurate screening of viruses as a result of its low sensitivity of colorimetric signal output and poor universality limited by antibody pairs. Here, a magnetically assisted dual-signal output LFA platform is developed for the ultrasensitive, universal, and flexible detection of viruses. A "three-in-one" multifunctional probe (MAuDQD) is prepared using a 180nm Fe3O4 core to load numerous Au nanoparticles (NPs) and two layers of QDs, which can substantially improve the sensitivity of LFA through coupling with the effects of magnetic enrichment and colorimetric/fluorescent enhancement. Wheat germ agglutinin (WGA)-modified MAuDQD attained the broad-spectrum capture viral membrane proteins and the colorimetric/fluorescent dual-mode detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and monkeypox virus (MPXV) on the LFA strip. In the colorimetric mode, the target viruses detected directly, with the visual sensitivity reaching 0.1-0.5ngmL-1 and the fluorescent mode supported quantitative analysis of SARS-CoV-2/MPXV with limits of detection decreasing to pgmL-1 level. Practicability of the MAuDQD@WGA-LFA is verified through the detection of 33 real clinical samples, showing the proposed assay has a great potential to become a sensitive, accurate, and universal tool for on-site monitoring of viral infections.

A polydiacetylene (PDA)-based colorimetric sensor for on-site cyanide anion monitoring integrated with a lateral flow assay platform

An advanced sensing platform for the on-site and real-time monitoring of cyanide (CN−) pollution was developed, addressing the key challenges of enhancing the selectivity and sensitivity, whilst simplifying the sensing process. This platform features a novel colorimetric sensor (PDA-BMN) created by incorporating a receptor for CN− recognition within a polydiacetylene (PDA) conjugated polymer system. The interaction of CN− with the receptor distorted the π-conjugated backbone, causing a distinct color change from blue to orange, with a limit of detection (LOD) of 0.55 µM, thereby highlighting its high sensitivity. The PDA-BMN system was integrated into a lateral flow assay (LFA) strip to facilitate on-site monitoring. The LFA strip was strategically designed with a partially pressed zone to reduce the fluid flow rate, enhance the reaction time, and increase the probability of interaction with the PDA-BMN sensor. This innovation resulted in a significant colorimetric transition, enabling the rapid and sensitive detection of CN− without the requirement for analytical instruments. This study demonstrates the effectiveness of combining PDA and LFA systems for real-time environmental monitoring, offering a robust, efficient, and user-friendly solution for detecting CN− pollution.

Fluorescent lateral flow assay strip for Mycobacterium tuberculosis and Mycobacterium smegmatis based on mycobacteriophage tail protein and aptamer

Timely and facile monitoring of Mycobacterium tuberculosis (M. tuberculosis) plays an important role for preventing and controlling tuberculosis infection. Mycobacterium smegmatis (M. smegmatis) has long been employed as a safe surrogate for the investigation of M. tuberculosis. In this work, an aqueous soluble tail protein derived from our previously isolated mycobacteriophage was prepared with a recombinant expression technique and noted as GP89, which shows noticeable binding capacity to Mycobacterium genus. GP89 was sprayed as a capture agent onto a nitrocellulose membrane for forming the test line of a lateral flow assay (LFA) strip. Moreover, an aptamer binding M. tuberculosis and M. smegmatis was labeled with fluorescent microspheres to act as the signal tracer of the LFA method. With the GP89 based LFA, M. tuberculosis and M. smegmatis can be detected with the aid of a handheld UV flashlight or a portable fluorescent strip reader within 10 min. The concentration range for quantitating M. tuberculosis and M. smegmatis are both 1.0 × 102 CFU mL−1 - 1.0 × 106 CFU mL−1, and the detection limits for the two mycobacteria are 2.0 and 24 CFU mL−1 (S/N = 3), respectively. The test strip was applied to detect M. tuberculosis and M. smegmatis in different samples such as physiological salt solution, urine, and saliva. This study offers a promising screening tool for diagnosing M. tuberculosis infection in resource-limited institutes.

Achieving precise dual detection: One-tube reverse transcription-recombinase aided amplification (RT-RAA) combined with lateral flow strip (LFS) assay for RNA and DNA target genes from pepper mild mottle virus and Colletotrichum species in crude plant samples

Ensuring the detection sensitivity of both RNA-derived and DNA-derived target genes in a single reaction has posed a significant challenge for on-site detection of plant pathogens. This challenge was addressed by developing a one-tube dual RT-RAA assay combined with LFS for the rapid on-site detection of pepper mild mottle virus (PMMoV) and four Colletotrichum species causing anthracnose in Solanaceous crops. By testing four different combinations of primer groups, two combinations were precisely adjusted within the dual RT-RAA system to balance amplification efficiency and maintain consistent levels of amplification in crude plant samples. Utilizing commercially accessible small-scale equipment and following a streamlined optimization strategy, the assay achieved a limit of detection of 0.32 copies/μL of target genes in the reaction. Importantly, it demonstrated no cross-reactivity with other plant pathogens, thereby affirming the high sensitivity and specificity of the developed dual RT-RAA-LFS detection assay. Moreover, the entire process took only 25 min from sample collection to the visible presentation of results. The assay was validated with 60 field samples and 10 seed samples, producing results consistent with reverse transcription quantitative polymerase chain reaction (RT-qPCR). Notably, it successfully detected PMMoV in systemic leaves without visible symptoms three days post-inoculation, underscoring its effectiveness in early disease detection. This streamlined strategy offers a valuable approach for rapid, low-cost, and highly sensitive on-site simultaneous detection of RNA genome-contained PMMoV and DNA genome-contained Colletotrichum species.

Integration of Group A Streptococcus Rapid Tests with the Open Fluidic CandyCollect Device.

The CandyCollect device is a lollipop-inspired open fluidic oral sampling device designed to provide a comfortable user sampling experience. We demonstrate that the CandyCollect device can be coupled with a rapid antigen detection test (RADT) kit designed for Group A Streptococcus (GAS). Through in vitro experiments with pooled saliva spiked with Streptococcus pyogenes we tested various reagents and elution volumes to optimize the RADT readout from CandyCollect device samples. The resulting optimized protocol uses the kit-provided reagents and lateral flow assay (LFA) while replacing the kit's pharyngeal swab with the CandyCollect device, reducing the elution solution volume, and substituting the tube used for elution to accommodate the CandyCollect device. Positive test results were detected by eye with bacterial concentrations as low as the manufacturer's "minimal detection limit" - 1.5×105 CFU/mL. LFA strips were also scanned and quantified with image analysis software to determine the signal-to-baseline ratio (SBR) and categorize positive test results without human bias. We tested our optimized protocol for integrating CandyCollect and RADT using CandyCollect clinical samples from pediatric patients (n=6) who were previously diagnosed with GAS pharyngitis via pharyngeal swabs tested with RADT as part of their clinical care. The LFA results of these CandyCollect devices and interspersed negative controls were determined by independent observers, with positive results obtained in four of the six participants on at least one LFA replicate. Taken together, our results show that CandyCollect devices from children with GAS pharyngitis can be tested using LFA rapid tests.

Centrifugated lateral flow assay strips based on dual-emission carbon dots modified with europium ions for ratiometric determination and on-site discrimination of tetracyclines in environment

Due to the serious detrimental impact on human health, antibiotic pollution particularly tetracyclines residues has become a serious problem. Herein, a multiple response fluorescent probe consisted of dual-emission carbon dots and Eu3+ (D-CDs@Eu3+) is designed for the determination and discrimination of tetracyclines (TCs). Specifically, the carboxyl and amidogen group of dual-emission carbon dots (D-CDs) can coordinate with Eu3+ to form the D-CDs@Eu3+. Upon adding TCs, the fluorescence intensities of D-CDs at 405 nm and 495 nm are quenched due to inner filter effect (IFE) and the localization of fluorescence resonance energy transfer (L-FRET) between the D-CDs@Eu3+ and TC. Simultaneously, the D-CDs@Eu3+ may chelate with TCs to enhance the occurrence of antenna effect, while the characteristic peaks of Eu3+ at 590 nm and 615 nm are enhanced. On these bases, the TCs detection is achieved with low detection limits from 46.7 to 72.0 nM. Additionally, through the distinct efficiencies of L-FRET, the discrimination of TCs is achieved. Moreover, a novel centrifugated lateral flow assay strips (CLFASs) device is developed by integrating the D-CDs@Eu3+, lateral flow assay strips and smartphone using RGB variations for TCs detection, achieving remarkable recoveries (98.6–103.7 %) in real samples. Therefore, this CLFASs device provides a reliable approach for the TCs detection, demonstrating potential applications.

Fast, portable and sensitive detection of group B streptococcus DNA using one-pot MIRA-CRISPR system with suboptimal PAM

The group B Streptococcus (GBS) can generate vertical transmission to infants during delivery, has been seriously threatening the health of infants. Rapid and accurate prenatal GBS diagnosis for pregnant women is a deterministic blueprint to avoid infant viruses. Here, we developed an extraction-free nucleic acid isothermal amplification/CRISPR-Cas12a cutting one-pot system for GBS diagnostic assay by using suboptimal protospacer adjacent motifs, effectively avoiding multiple handling steps and uncapping contamination. The GBS diagnosis assay based on a one-pot system was validated by using fluorescent technique and lateral flow assay strips, exhibited fantastic specificity, accuracy and sensitivity with a limit of detection of 32 copies per reaction (0.64 copies/μL). Moreover, a portable device was constructed and integrated with the one-pot system to realize the GBS detection without professional and scene restrictions, it showed excellent performance in clinical sample detection, which achieved optical and portable GBS detection for point-of-care testing or home-self testing.

Fluorescent Quantum Dots Based Lateral Flow Assay for Rapid Quantitative Detection of Ciliary Neurotrophic Factor in Glaucoma

AbstractEarly and accurate diagnosis of glaucoma is crucial to prevent the progressive deterioration that leads to irreversible vision loss. It is imperative to develop an effective screening tool for glaucoma. Ciliary neurotrophic factor (CNTF) is a tear biomarker implicated in glaucoma pathogenesis. Lateral flow assay (LFA) provides an ideal platform for detection of glaucoma in tear fluid. A quantum dot‐based fluorescence LFA, integrated with a 3D printed readout box, is developed for fast (30 min), sensitive, and quantitative CNTF detection in tears. A standard curve is firstly generated for the quantitative detection of CNTF. The limit of detection (LOD) of the obtained LFA strip at 6.45 pg·mL−1 is comparable to that of the enzyme‐linked immunosorbent assay (ELISA) at 6.42 pg·mL−1. This enables the identification of low CNTF levels (25.7 ± 14.9 pg·mL−1) reported in tear fluid from glaucoma patients. This LFA is found to be highly selective for CNTF and maintained consistent results in different pH condition. The strip remaines stablewhen stored in the darkat room temperature. A smartphone app is developed to simplify analysis and enable prompt and easily obtainable results. This method shows great potential to be a powerful tool for point‐of‐care glaucoma screening.

Photoactivatable CRISPR/Cas12a Sensors for Biomarkers Imaging and Point-of-Care Diagnostics.

In recent years, the CRISPR/Cas12a-based sensing strategy has shown significant potential for specific target detection due to its rapid and sensitive characteristics. However, the "always active" biosensors are often insufficient to manipulate nucleic acid sensing with high spatiotemporal control. It remains crucial to develop nucleic acid sensing devices that can be activated at the desired time and space by a remotely applied stimulus. Here, we integrated photoactivation with the CRISPR/Cas12a system for DNA and RNA detection, aiming to provide high spatiotemporal control for nucleic acid sensing. By rationally designing the target recognition sequence, this photoactivation CRISPR/Cas12a system could recognize HPV16 and survivin, respectively. We combined the lateral flow assay strip test with the CRISPR/Cas12a system to realize the visualization of nucleic acid cleavage signals, displaying potential instant test application capabilities. Additionally, we also successfully realized the temporary control of its fluorescent sensing activity for survivin by photoactivation in vivo, allowing rapid detection of target nucleic acids and avoiding the risk of contamination from premature leaks during storage. Our strategy suggests that the CRISPR/Cas12a platform can be triggered by photoactivation to sense various targets, expanding the technical toolbox for precise biological and medical analysis. This study represents a significant advancement in nucleic acid sensing and has potential applications in disease diagnosis and treatment.

A nanogap-enhanced SERS nanotag-based lateral flow assay for ultrasensitive and simultaneous monitoring of SARS-CoV-2 S and NP antigens.

A lateral flow assay (LFA) strip based on dual 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB)-encoded satellite Fe3O4@Au (Mag@Au) SERS tags with nanogap is reported for ultrasensitive and simultaneous diagnosis of two SARS-CoV-2 functional proteins. Composed of Fe3O4 core, satellite gold shell with nanogaps, and double-layer DTNB, the Mag@Au nanoparticles with an average size of 238nm were designed as multifunctional tags to efficiently enrich the target SARS-CoV-2 protein from complex samples, significantly enhancing theSERS signal of the LFA strip and provide quantitative SERS detection of analyte on test lines. The developed dual DTNB-encoded satellite Mag@Au-based LFA allowed simultaneous quantification of spike (S) protein and nucleocapsid (NP) protein with detection limits of 23pgmL-1 and 2pgmL-1, respectively, lower than commercial ELISA kits and reported SERS-LFA detection system-based Au NPs and Fe3O4@3nm Au MNPs. This magnetic SERS-LFA also showed high performance of multi-variant strain detection and further distinguished clinical samples of Omicron variant infection, demonstrating the potential of in situ detection of respiratory virus diseases.

Dual Detection of Hepatitis B and C Viruses Using CRISPR‐Cas Systems and Lateral Flow Assay

The development of sensitive and specific diagnostic tools for hepatitis B virus (HBV) and hepatitis C virus (HCV) remains crucial for effective disease management and control. In this study, we utilized CRISPR‐Cas12 and CRISPR‐Cas13 systems for the detection of HBV (DNA virus) and HCV (RNA virus), respectively. We designed and tested multiple guide RNAs (gRNAs) targeting both viruses, confirming successful cleavage of target sequences through gel electrophoresis and a fluorescent reporter assay. Using optimized gRNAs, we developed a lateral flow assay (LFA) for sensitive detection of HBV and HCV, demonstrating a concentration‐dependent signal increase. Importantly, no cross‐reactivity was observed with other viral targets. To further enhance sensitivity, we employed a dual‐enzyme approach, combining Cas12 and Cas13 in a single reaction, which significantly improved detection limits for both viruses. Finally, we developed a dual antigen detection LFA strip capable of simultaneously detecting both HBV and HCV in a single sample. This approach holds promise for point‐of‐care (POC) diagnostics where the specific viral infection is unknown. This study addresses the current limitations in CRISPR‐Cas based diagnostics, namely, the need for ultrasensitive detection methods and the ability to detect multiple antigens using a single test strip. Our findings demonstrate the feasibility of using CRISPR‐Cas systems for highly sensitive and specific detection of HBV and HCV, paving the way for potential POC application.

Lateral flow strip assay of a gene segment in the COVID-19 virus with combined dual readout mode and preliminary multisite hybrid chain reaction amplification.

The past and present scenario of COVID-19 has revealed the necessity of simple point-of-care tests. When combined with the great advantages of amplification, lateral flow assay nucleic acid analysis represents a more sensitive molecular diagnostic technique compared to universal protein analysis. Room temperature operation, an enzyme-free nature, and in situ elongation make hybrid chain reaction amplification (HCR) a good candidate for amplified combined lateral flow assays (LFAs). Since dual modes of detection can not only satisfy different application scenarios, but also reduce the false-negative rate, in this paper, visual and fluorescent detection based on labelling with colloidal gold nanoparticles and fluorescence labelling were incorporated into a HCR integrated with a LFA. The detection assay was finished in 30 minutes. The linear relationship between the signal and the concentration of the characteristic segment in the COVID-19 ORF gene was demonstrated. The obtained detection limits of as low as 10 fM (6.02 × 103 copies per mL) and 1 fM (6.02 × 102 copies per mL), respectively, were comparable with those in the literature. The multi-site HCR amplification integrated with LFA of a 1053 bp nucleic acid chain was also preliminarily studied, and tri-site amplification was found to exhibit higher signal intensity than single-site amplification. This study provides a promising strategy for simple, sensitive, and wide-ranging detection of pathogenic bacteria.

A lateral flow assay strip for simultaneous detection of miRNA and exosomes in liver cancer.

By employing an aptamer as the bridge and combining catalytic hairpin assembly with the Au aggregation amplification effect, a lateral flow assay (LFA) is designed for simultaneous detection of liver cancer-associated miRNA and exosomes. The LFA can differentiate between liver cancer patients and healthy individuals with simple operation and high accuracy.

Devising a colorimetric aptasensor for detection of basal stem rot-associated RNA marker during early Ganoderma boninense infection in oil palm

Basal stem rot (BSR) is a detrimental disease in oil palm (Elaeis guineensis) due to the host's lack of symptoms during the early phase of infection. Early detection of BSR at non-symptomatic phase is feasible via molecular-based approaches and has become the major interest among researchers. Our previous study had identified a differentially expressed gene encoding auxin efflux carrier component 5-like (EgPIN5) at 11 day-post-inculation of Ganoderma boninense (symptomless early stage of infection) on oil palm seedlings. We further evaluated the gene expression on samples collected from an oil palm plantation at different BSR stages (symptomless, mild and severe). Our findings revealed that EgPIN5 was expressed in symptomless group but was not expressed in either mild or severe BSR groups. Next, we developed a sandwich-type aptamer-based lateral flow assay (LFA) against EgPIN5 RNA by adapting the versatile DNA-functionalized gold nanoparticles for colorimetric detection. The LFA strip demonstrated visible colorimetric signal of EgPIN5 RNA at 12.5 ng/μL with limit of detection at 2.45 ng/μL using a scanning device. Based on our consistent results, we presented EgPIN5 as a diagnostic biomarker for symptomless BSR infection in oil palm. Utilization of LFA assay strip for practical EgPIN5 RNA detection is hoped to encourage establishment of a fast, cheap, and sensitive point-of-care detection system in plant biology.

Development of highly sensitive plasmonic biosensors encoded with gold nanoparticles on M13 bacteriophage networks

In this study, a novel nanoplasmonic substrate is developed for biomedical applications. In nanoplasmonic biosensors, a substrate that can induce strong hot spots by forming uniform nanogaps smaller than 10 nm is required to implement a device capable of detecting biomarkers at low concentrations. Herein, gold nanoparticles-encoded M13 bacteriophage (AuNPs@M13) substrate was developed by immobilizing 60 nm AuNPs onto the surface of an M13 bacteriophage scaffold with a network assembly structure. The plasmonic characteristics of the proposed AuNPs-M13 were evaluated by assessing the nanogap distribution, electromagnetic enhancement effects, and reproducibility of the fabricated substrate. Additionally, an immunoassay for severe acute respiratory syndrome coronavirus 2 was performed to confirm the clinical applicability of the proposed AuNPs@M13 substrate. When the AuNPs@M13 was used as a nanoplasmonic substrate, it was possible to achieve better sensitivity (limit of detection [LoD] = 1.21 PFU/mL) than that with the commercially available lateral flow assay strip (LoD = 300–500 PFU/mL) or enzyme-linked immunoassay (LoD = 48 PFU/mL). The proposed AuNPs@M13 in this study is expected to be used as an innovative biomedical device for the high-sensitivity detection of various biomarkers.

CRISPR/Cas13a-Assisted accurate and portable hepatitis D virus RNA detection

ABSTRACT Background & Aims Hepatitis delta virus (HDV) infection accelerates the progression of chronic hepatitis B virus (HBV) infection, posing a large economic and health burden to patients. At present, there remains a lack of accurate and portable detection methods for HDV RNA. Here, we aim to establish a convenient, rapid, highly sensitive and specific method to detect HDV RNA using CRISPR–Cas13a technology. Methods We established fluorescence (F) and lateral flow strip (L) assays based on CRISPR–Cas13a combined with RT–PCR and RT-RAA for HDV RNA detection, respectively. we conducted a cohort study of 144 patients with HDV-IgG positive to evaluate the CRISPR–Cas13a diagnostic performance for identifying HDV in clinical samples, compared to RT–qPCR and RT-ddPCR. Results For synthetic HDV RNA plasmids, the sensitivity of RT–PCR-CRISPR-based fluorescence assays was 1 copy/μL, higher than that of RT–qPCR (10 copies/μL) and RT-ddPCR (10 copies/μL); for HDV RNA-positive samples, the sensitivity of RT-RAA-CRISPR-based fluorescence and lateral flow strip assays was 10 copies/μL, as low as that of RT–qPCR and RT-ddPCR, and the assay took only approximately 85 min. Additionally, the positivity rates of anti-HDV IgG-positive samples detected by the RT–qPCR, RT-ddPCR, RT–PCR-CRISPR fluorescence and RT-RAA-CRISPR lateral flow strip methods were 66.7% (96/144), 76.4% (110/144), 81.9% (118/144), and 72.2% (104/144), respectively. Conclusions We developed a highly sensitive and specific, as well as a portable and easy CRISPR-based assay for the detection of HDV RNA, which could be a prospective measure for monitoring the development of HDV infection and evaluating the therapeutic effect.

Synchronous detection of Burkholderia pseudomallei and its ceftazidime resistance mutation based on RNase-HII hydrolysis combined with lateral flow strip assay.

Melioidosis is a severe and often fatal infectious disease caused by Burkholderia pseudomallei, which poses significant challenges due to its high mortality rate and frequent misdiagnosis. To effectively treat infected patients, especially in epidemic regions like Hainan with a high resistance rate to ceftazidime (CAZ), early diagnosis and resistance testing are critical. Here, we designed a strategy that combines RNase-HII-dependent PCR with lateral flow strip assay (LFSA) for simultaneous detection of B. pseudomallei and its CAZ resistance mutation. In this strategy, we utilized rhPCR technology to design specific primers with a C3-spacer modification. These primers target a 115-base pair region within the ORF2 of the B. pseudomallei type III secretion system gene cluster, as well as the P174L mutation in the penA gene, which is a major mutation associated with CAZ resistance in Hainan. The primers contain complementary RNA bases that can be hydrolyzed only in the presence of thermostable RNase-HII enzymes, facilitating subsequent PCR reactions. Additionally, we introduced two pairs of universal probes carrying fluorescein isothiocyanate (FITC) and biotin, as well as digoxin and biotin. The sequences of these universal probes align with specific regions of the primers, resulting in target products labeled with different tags. The resulting amplification products are then subjected to LFSA, where the presence of specific markers is visually interpreted based on color development. These markers flow to test line 1 (T1) and are captured by a fixed anti-FITC antibody, generating a red line that indicates the presence of B. pseudomallei. Similarly, the occurrence of a red line on test line 2 (T2), which is fixed with anti-digoxin antibodies, indicates the presence of the P174L mutation in B. pseudomallei. This protocol serves as an excellent tool for the rapid diagnosis of melioidosis and provides a basis for selecting appropriate antibiotic medication. It has the potential to revolutionize the diagnosis of melioidosis, particularly in resource-limited settings. Furthermore, this technique holds promise as a versatile tool for disease diagnosis in general. IMPORTANCE This study focused on the development of a reaction system using rhPCR to amplify a specific gene, ORF2, of B. pseudomallei and to identify the P174L mutation associated with increased drug resistance to ceftazidime (CAZ). The system incorporated universal primer probes and a simple temperature cycle reaction. The amplified products were then analyzed using lateral flow strip assay (LFSA) for strain identification and mutation interpretation. The developed system provides a reliable basis for diagnosing melioidosis and selecting appropriate drugs. Its potential impact is particularly significant in resource-limited settings where access to advanced diagnostic techniques is limited. This platform stands out for its simplicity, convenience, sensitivity, specificity, and portability. It shows promise as a point-of-care testing method for detecting single nucleotide polymorphism in genes associated with other diseases. By leveraging the advantages of this platform, researchers and healthcare professionals can potentially expand its use beyond melioidosis and apply it to the rapid detection of genetic variations in other disease-related genes.

Identification of horse, donkey and pig ingredients by species-specific ERA-based methods to assess the authenticity of meat products

Ensuring the authenticity of horse-, donkey- and pig-related meat products is of great religious and economic significance. In this study, with the mitochondrial genes of ATPase 6 or ND2 as targets, the species-specific real-time enzymatic recombinase amplification (real-time ERA) and ERA combined with lateral flow strip (LFS ERA) assays for horse, donkey and pig ingredients were developed. The optimization results showed that both ERA assays had the same optimal temperature for the detection of horse, donkey and pig ingredients, i.e., 39 °C for real-time ERA and 37 °C for LFS ERA, respectively. The assays could be performed simultaneously and the results could obtained within 25 min. Both developed assays showed good specificity and no cross-reactions with other common meat species were observed. The limit of detection of the developed ERA assays for horse, donkey and pig ingredients was 10 pg genomic DNA/per reaction and 0.1% target meat (w/w). Their applicability were verified with 45 donkey meat products (both raw and marinated minced meat), and the results showed that the detection rate of horse or pig ingredient was as high as 53.33%, including a complete substitution or partial substitution of donkey meat. The results were consistent with those of the standard real-time PCR in China, but the whole detection time was significantly reduced from 2 h to 35 min. It is concluded that the developed real-time ERA and LFS ERA assays are feasible for the rapid authentication of horse, donkey and pig meats and identification of the adulteration of donkey meat-related products.

Toward rapid and sensitive point‐of‐care diagnosis with surface‐enhanced Raman scattering‐based optofluidic systems

AbstractWith the recent global outbreaks of infectious diseases such as coronavirus disease 2019, developing a detection system capable of quickly and accurately diagnosing diseases on‐site has become a pressing need. The ability to diagnose patients in the field is crucial for the prompt isolation and treatment of infected individuals and the prevention of the spread of the disease. Our research group has recently developed a surface‐enhanced Raman scattering optofluidic system that enables rapid and accurate point‐of‐care diagnostics. This account will introduce the principle and configuration of the fluidic devices, such as lateral flow assay strips or microfluidic channels, and the portable Raman spectrometer. We will also highlight the challenges that must be addressed for using this system in clinical settings. Rapid and accurate diagnosis is critical for effective disease management and control, and developing this system can significantly improve our ability to respond to outbreaks of infectious diseases.

Two-color lateral flow nucleic acid assay combined with double-tailed recombinase polymerase amplification for simultaneous detection of chicken and duck adulteration in mutton

We propose a visual strategy for simultaneous detection of chicken and duck ingredients in mutton by integration of a double tailed multiplex recombinase polymerase amplification (mRPA) assay with a multicolor nucleic acid hybridization lateral flow strip (mNAH-LFS) assay. Two colored gold nanoparticles (AuNPs), including gold nanospheres (red) and gold nanoflowers (blue), were synthesized and functionalized with the corresponding nucleic acid probe to target each analyte. The RPA product was analyzed by this two-colored mNAH-LFS assay which includes a red T1 line for chicken adulteration and a blue T2 line for duck adulteration. In this RPA reaction, special Spacer9 and tail nucleotide sequence-tagged primers (Tag+Spacer9+F-primer, Tag+Spacer9+R-primer) were used to obtain double-stranded amplicons with specific wobbled sequences that can directly bind with AuNPs-labeled probes (AuNSs-cT1 hybridized with T1, AuNFs-cT4 hybridized with T4) and solid-phase probes (biotin-cT2 hybridized with T2, biotin-cT3 hybridized with T3) on the mNAH-LFS producing a red or/and cyan color on the T lines. The detection limits of this mRPA-mNAH-LFS assay for chicken and duck were 0.01% and 0.01%, respectively. Finally, this mRPA-mNAH-LFS assay was successfully used to authenticate 59 commercial meat products. In conclusion, this mRPA-mNAH-LFS is a sensitive, accurate, and cost-effective approach for meat quality assurance and traceability.