Fluorescence Detection Method Research Articles

Repurposing expired metformin to fluorescent carbon quantum dots for ratiometric and color tonality visual detection of tetracycline with greenness evaluation

Repurposing expired drugs is highly significant as it prevents the accumulation of pharmaceutical wastes, optimizes resource usage, and reduces environmental harm. Metformin, used for type II diabetes, lowers blood sugar via decreasing liver glucose production and increasing muscle insulin sensitivity. However, the growing number of expired tablets annually necessitates a strategic approach for repurposing or reusing these tablets. Herein, we develop a simple hydrothermal approach for converting expired metformin to valuable fluorescent carbon quantum dots (m-CQDs) with sizes smaller than 10 nm. The m-CQDs displayed a blue emission at 420 nm when excited at 350 nm. Interestingly, when tetracycline (TC) was added, the blue emission diminished, and a greenish-yellow emission emerged at 530 nm. This emission tunning was the resulting of combination of inner filter effect and aggregation induced emission. Leveraging this dynamic emission tunning, we developed both ratiometric-based fluorescence and color tonality-based visual detection methods for detecting TC in pharmaceuticals and wastewater. More importantly, three assessment tools, ComplexMoGAPI, AGREE, and BAGI were used to evaluate the environmental sustainability of the proposed analytical method. The evaluation confirmed the sustainability of the method and the alignment with the greenness guidelines for analytical methods.

A clenbuterol detection method based on magnetic separation up-conversion fluorescent probe

In this study, a fluorescence detection method combining aptamer-modified up-conversion nanoparticles (UCNPs) and magnetic nanoparticles (MNPs) was developed for detection of Clenbuterol (CLB). The aptamer-modified magnetic NPs captured CLB, which reacted with the aptamer-modified UCNPs and generated a sandwich complex. The aptamer-modified UCNPs acted as a fluorescence source. The MNP-CLB-UCNP complex was retrieved from the solution using an magnetic field, and the fluorescence intensity was detected by fluorescence spectrophotometry with excitation and emission spectra at 980 nm and in the 400–800 nm region, respectively. The results showed that the fluorescence intensity gradually increased with increasing concentrations of CLB with a good specify. The method was highly sensitive for the quantification of CLB, with a limit of detection of 0.304 ng mL−1. The recovery rate of CLB from pork samples ranged from 84 % to 94.87 %. This fluorescence method enables the sensitive, precise, and accurate quantification of CLB residues in pork samples.

A portable optical sensor combining smartphone with phycocyanin-based fluorescent test paper for rapid, visual and on-site detection of CO32–

With the development of global industry, carbon dioxide emissions surged. The conversion of carbon dioxide from the air results in some CO32−, which can exacerbate environmental disasters like ocean acidification. Therefore, the content of CO32− in seawater is an important indicator of the degree of ocean acidification. In this study, natural fluorescent protein phycocyanin (PC) was used as a fluorescent probe, and a fluorescence detection method was established for quantitative monitoring of CO32− with quick response time (within 50 s), high sensitivity, and selectivity. The fluorescence quenching phenomenon between PC and CO32− was mainly attributed to static quenching. The limit of detection (LOD) was 0.42 μM and the method was successfully applied to monitor CO32− in tap water and seawater, acquiring satisfactory recovery between 99.28 % and 106.40 %. More importantly, paper-based test strips were easily fabricated using PC, enabling the rapid, visual, and on-site detection of CO32− with the aid of a smartphone. The visual detection integrated with the smartphone was converted to data information (RGB value) through a Color Picker APP and successfully used for quantitative identification of CO32−. By capturing fluorescent images and analyzing the corresponding RGB value via a smartphone, the linear calibration ranged from 0.5 μM to 500.0 μM with LOD of 0.11 μM was obtained. Satisfactory recoveries were acquired in tap water (98.00 %-107.50 %) and seawater (97.30 %-101.74 %), respectively. Therefore, integrating the PC fluorescent paper with a smartphone realizes the rapid, visual, and on-site detection of CO32− in the water environment, which is expected to broaden application prospects of monitoring ocean acidification degree.

In-capillary magnetic separation and enrichment coupled with laser-induced fluorescence for rapid determination of biomolecules

Biomolecules are indispensable in the life activities and metabolism of various organisms, and their highly sensitive detection is of great significance for disease diagnosis and food safety. Due to the complexity of the matrix and the low concentration of the target, separation and enrichment of biomolecules prior to detection are necessary and time-consuming. Herein, a novel approach combining in-capillary magnetic separation and enrichment with laser-induced fluorescence detection method was proposed for qualitative and quantitative analysis of biomolecules. Two different sequence aptamers modified with magnetic nanoparticles and labeled with fluorescence were used as capture probes and signal probes to form sandwich structures with target molecules for detection. Under the combined influence of magnetic field force and fluid drag force, the integration of separation, enrichment and on-line detection were finished within 5min. Under optimal conditions, the developed method provided a low limit of detection as 2.7 fmol/L, 0.57 nmol/L, 3 CFU/mL for DNA, thrombin and Staphylococcus aureus, respectively. This proposed method offers a promising platform for the sensitive and rapid determination of biomolecules. It holds significant potential for future applications in fields of environmental and biochemical analysis.

Polypod Carboxylic Acid-Rare Earth Complex with High Cyclic Stability for Nitrobenzene Compound Detection.

The 5',5''-bis(4-carboxyphenyl)-[1,1':3',1'':3'',1'''-quaterphenyl]-4,4'''-dicarboxylic (H4L1) ligand has a large conjugated rigid planar structure and good absorption of ultraviolet radiation, which can provide effective "antenna effect". However, rare earth complexes using H4L1 as the sole ligand have not been reported. In this paper, rare earth Eu was combined with H4L1 ligand to produce organic rare earth composite L1-Eu by solvothermal synthesis method. It was found through fluorescence spectroscopy that the emission of L1-Eu complex has a linear response to nitrobenzene compounds. The L1-Eu composite material has a low detection limit for nitrobenzene compounds, with detection limits of 0.910, 8.401, 24.510, and 8.171 µM for nitrobenzene, o-nitrophenol, m-nitrophenol, and p-nitrophenol, respectively. Further more the L1-Eu complex can sensitively respond to nitrobenzene compounds while resisting interference from common metal ions and organic solvents. In particular, L1-Eu composite material has good stability and recyclability. Therefore, L1-Eu composite material can serve as a fluorescent probe for specific detection of nitrobenzene compounds. We believe that the L1-Eu complex provides a new method for fluorescence detection of nitrobenzene compounds.

A ratiometric fluorescent biosensing platform based on CDs and AuNCs@CGO for patulin detection

BackgroundPatulin (PAT) is a mycotoxin, usually found in fruit and their products, that can potentially be harmful to human health. In order to achieve rapid detection of mycotoxins and ensure the safety of food. This study reported a novel ratiometric fluorescent aptasensor for PAT detection. In this study, we used aptamer as the recognition element, Hybrid double stranded modified with fluorescent substances as the fluorescent donor, and AuNCs@CGO as the fluorescent acceptor. After the addition of PAT, the ratiometric fluorescence “turn on” response was exhibited. ResultsThe AuNCs@CGO are obtained by amide reaction between BSA-AuNCs and carboxylated graphene oxide (CGO). The prepared AuNCs@CGO can shorten the time of FRET effect and exhibit highly efficient quenching ability by adsorption effects (π-π stacking and electrostatic gravity) on the Aptamer-modified CDs. When the target PAT bound specifically to the CDs-apt, the fluorescence of the CDs-apt would recover, while the fluorescence of ROX modified cDNA remained unchanged. This ratiometric fluorescence response improved the accuracy of PAT detection. In addition, the proposed had good linearity for PAT in the range of 0.1–50 ng/mL with a limit of detection 0.16 ng/mL. The recovery of standard addition in grapes were 95.9%–105.4 %. SignificanceAn effective fluorescent detection method for PAT was constructed based on aptamer and nanomaterials. This new fluorescent biosensor has the characteristics of simple synthesis, easy operation, high sensitivity, strong selectivity and a low LOD, which may be a promising idea and platform for the detection of food safety hazard factors.

Molybdenum disulfide quantum dots for rapid fluorescence detection of glutathione and ascorbic acid

A method for the specific detection of glutathione (GSH) and ascorbic acid (AA) using 3-aminophenylboronic acid functionalized molybdenum disulfide quantum dots (APBA-MoS2 QDs) was reported. The APBA-MoS2 QDs synthesized using the hydrothermal method showed the strongest fluorescence intensity at an emission wavelength of 375 nm with the optimum excitation wavelength of 320 nm. The detection method was constructed by the combination of the dynamic and static quenching and the inner filter effect (IFE) of APBA-MoS2 QDs by permanganate (MnO4−) and the rapid redox reaction (1 min) of MnO4− by GSH or AA. The concentrations of GSH and AA showed good linear ranges of 10–500 μM and 10–100 μM, respectively, with limits of detection (LOD) of 0.476 μM and 0.185 μM, respectively. The spiked recovery tests in human serum and fresh fruit samples showed that APBA-MoS2 QDs had significant potential in the construction of fluorescent biological detection methods.

Novel electrochemiluminescence platform utilizing AuNPs@Uio-66-NH2 bridged luminescent substrates and aptamers for the detection of pesticide residues in Chinese herbal medicines

A large number of Chinese herbal medicines (CHMs) are included in daily recipes, but their pesticide residues have aroused more and more concerns. In this paper, an electrochemiluminescence aptasensor was constructed for the trace detection of acetamiprid (ACE) in Angelica sinensis and Lycium barbarum. Possessing a large specific surface area, UiO-66 was modified with amino groups to improve biocompatibility, and the addition of AuNPs allowed UiO-66-NH2 to catalyze the formation of excited states of luminescent molecules (TPrA⁎; Ru(bpy)32+⁎), and AuNPs@UiO-66-NH2 was used to bridge the aptamer (Au–S) and luminescent substrate (peptide bond). The conventional luminescent reagent Ru(bpy)32+ was doped with multi-walled carbon nanotubes (MWCNTs) to obtain a more powerful and stable light signal. After optimizing the experimental parameters, the aptasensor could give results in 10 min with a detection range from 1×10−2-1×104 nM and a lower limit of detection (LOD) of 0.8 pM. The LOD of the study was at least one order of magnitude lower than that of the fluorescence detection method. Furthermore, the accuracy of the aptasensor was validated for spiked recovery experiments.

Development of a RPA-CRISPR/Cas12a based rapid visual detection assay for Porcine Parvovirus 7.

Porcine Parvovirus (PPV) is a significant pathogen in the pig industry, with eight genotypes, including PPV7, identified since its emergence in 2016. Co-infections with viruses such as Porcine Circovirus 2 (PCV2) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) pose serious risks to swine health. Thus, there is an urgent need for rapid, sensitive, and specific detection methods suitable for use in field settings or laboratories with limited resources. We developed a CRISPR/Cas12a-based assay combined with recombinase polymerase amplification (RPA) for the rapid detection of PPV7. Specific RPA primers and five CRISPR RNAs (crRNAs) were designed to target a highly conserved region within the NS1 gene of PPV7. Optimization of crRNA and single-stranded DNA (ssDNA) concentrations was performed to enhance the assay's performance. CrRNA optimization identified crRNA-05 as the optimal candidate for Cas12a-based detection of PPV7, as all synthesized crRNAs demonstrated similar performance. The optimal crRNA concentration was determined to be 200 nM, yielding consistent results across tested concentrations. For ssDNA optimization, the strongest fluorescence signal was achieved with 500 nM of the FAM-BHQ ssDNA receptor. The assay showed a minimal detection limit of 100copies/μl for PPV7, confirmed through fluorescence and lateral flow detection methods. Specificity testing indicated that only PPV7 DNA samples returned positive results, confirming the assay's accuracy. In tests of 50 lung tissue samples from diseased pigs, the RPA-Cas12a assay identified 29 positive samples (58%), surpassing the 22 positive samples (44%) detected by conventional PCR. This highlights the RPA-Cas12a method's enhanced detection capability and its potential utility in clinical surveillance and management of PPV7 in swine populations. The RPA-Cas12a assay effectively detects PPV7 in clinical samples, enhancing disease surveillance and control in pigs. Its adaptability to resource-limited settings significantly improves PPV7 management and prevention strategies, thereby supporting the overall health and development of the pig industry.

Facial detection of formaldehyde by using acidichromic carbon dots and the reaction between formaldehyde and ammonium chloride

Herein we report the facial detection of formaldehyde (FA) by using an interesting red acidichromic carbon dots (ACDs) which turns blue when pH gradually decreases. The color change was attributed to the conversion between the double bonds (C=N) and single bonds (C-N) on the surface of the ACDs. Inspired by the reaction between FA and ammonium chloride that produces H+ and methenamine and decrease the pH value of the solution, a fast and simple visual detection method for FA was found with a minimum discriminated concentration of 0.04 mol/L. A fluorescence detection method for FA was also found with LOD of 0.029 mol/L and FA in real sample, e.g., shredded squid was successfully analyzed. This work provides a new idea of developing fast visual detection method for daily monitor or in-site semiquantitative assessment on FA.

Visual fluorescence detection of Listeria monocytogenes with CRISPR-Cas12a aptasensor.

Listeria monocytogenes (L. monocytogenes) is a prevalent food-borne pathogen that can cause listeriosis, which manifests as meningitis and other symptoms, potentially leading to fatal outcomes in severe cases. In this study, we developed an aptasensor utilizing carboxylated magnetic beads and Cas12a to detect L. monocytogenes. In the absence of L. monocytogenes, the aptamer maintains its spatial configuration, keeping the double-stranded DNA attached and preventing the release of a startup template and activation of Cas12a's trans-cleavage capability. Conversely, in the presence of L. monocytogenes, the aptamer undergoes a conformational change, releasing the double-stranded DNA to serve as a startup template, thereby activating the trans-cleavage capability of Cas12a. Consequently, as the concentration of L. monocytogenes increases, the observable brightness in a blue light gel cutter intensifies, leading to a rise in fluorescence intensity difference compared to the control. This Cas12a aptasensor demonstrates excellent sensitivity towards L. monocytogenes, with a lowest detection limit (LOD) of 57.15CFU/mL and a linear range of 4×102 to 4×107 CFU/mL (R2=0.9858). Notably, the proposed Cas12a aptasensor exhibited outstanding selectivity and recovery in beef samples, and could be employed for precise monitoring. This Cas12a aptasensor not only provides a novel fluorescent and visual rapid detection method for L. monocytogenes but also offers simplicity, speed, and stability compared to previous detection methods. Furthermore, it is suitable for on-site detection of beef samples.

Ghost cells as a two-phase blood analog fluid-high-volume and high-concentration production.

Hemolysis in mechanical circulatory support systems is currently determined quantitatively. To also locally resolve hemolysis, we are developing a fluorescent hemolysis detection method. This requires a translucent two-phase blood analog fluid combined with particle image velocimetry, an optical flow field measurement. The blood analog fluid is composed of red blood cell surrogates. However, producing surrogates in sufficient volume is a challenge. We therefore present a high-volume and high-concentration production for our surrogates: ghost cells, hemoglobin-depleted erythrocytes. In the ghost cell production, the hemoglobin is removed by a repeated controlled osmolar lysis. We have varied the solution mixture, centrifugation time, and centrifugation force in order to increase production efficiency. The production is characterized by measurements of output volume, hematocrit, transparency, and rheology of the blood analog fluid. The volume of produced ghost cells was significantly increased, and reproducibility was improved. An average production of 389 mL of ghost cells were achieved per day. Those ghost cells diluted in plasma have a rheology similar to blood while being permeable to light. The volume of ghost cells produced is sufficient for optical measurements as particle image velocimetry in mechanical circulatory support systems. This makes further work on experimental measurements for a locally resolved hemolysis detection possible.

Ultrasensitive and on-site diagnosis of rice bakanae disease based on CRISPR-LbCas12a coupled with LAMP.

Rice bakanae disease (RBD) has longstanding challenges impacted rice production, which is predominantly induced by Fusarium fujikuroi Nirenberg. Early diagnosis of F. fujikuroi is important to control RBD and improve quality and quantity of rice production. This study presents a novel on-site diagnosis platform combined with CRISPR/LbCas12a and LAMP to detect F. fujikuroi. LAMP amplification of TEF1-α, a characteristic gene of F. fujikuroi were performed, followed with trans-cleavage reaction of LbCas12a, cleaving the single-stranded DNA reporter, which is modified by the terminal fluorophore and quencher groups, producing fluorescence signal. The platform was confirmed with high specificity and sensitivity (LOD <1 aM). Furthermore, we designed a lateral flow strip experiment based on the trans-cleavage activity of LbCas12a, which was identified with similar sensitivity and specificity to the fluorescence detection method. In summary, this study achieved a platform with remarkable sensitivity and specificity for F. fujikuroi detection and provide potential for on-site and ultrasensitive diagnostic tools for RBD. © 2024 Society of Chemical Industry.

Sensitive detection of pymetrozine residues based on β-lactoglobulin as a fluorescence probe

A method was developed for the detection of pymetrozine (PYM) using β-lactoglobulin (β-LG) as a probe. The interaction between PYM and β-LG was studied by fluorescence spectra, circular dichroism spectra (CD), synchronous fluorescence spectra, ultraviolet visible spectra (UV–vis), fourier transform infrared (FT-IR) and molecular docking. The fluorescence quenching type between PYM and β-LG was demonstrated to be static by Stern-Volmer analysis and time-resolved fluorescence. The binding constant (Ka) was 2.95 × 104 L mol−1 at 293 K. The electrostatic attraction was confirmed to be the primary binding force by Ross theory, and the binding distance was 2.02 nm by fluorescence Resonance Energy Transfer (FRET) theory. Based on the interaction between β-LG and PYM, a fluorescence detection method for PYM was established. Under optimal experimental conditions, the standard equations were established in the range of 0.43 – 10.68 μg mL−1. The limit of detection (LOD) for PYM was 0.03 μg mL−1. The method was successfully applied to apple and pear fruit samples. Recoveries of PYM in these samples were 97.44 % – 102.29 %, the relative standard deviation (RSD) of these samples were 0.24 % – 0.88 % (n = 3).

Fluorescence detection of valence speciation of Cr(III) based on the catechol oxidase mimic enzyme activity of CuSeNP nanozymes.

Copper selenide nanoparticles (CuSeNP) weresynthesized using histidine, ethylenediamine, and sodium selenate as precursors by one-step microwave digestion methods. The as-prepared CuSeNPs exhibit excellent catechol oxidase mimic enzyme and catalase (CAT)-like activities. Dopamine (DA) can be oxidized to aminochrome with H2O2 by CuSeNPs, and the intermediate product aminochrome can further react with α-naphthol to yield a highly fluorescent derivative. It was confirmed that Cr(III) could adsorb on the surface of CuSeNPs and inhibit the production of semiquinone radicals in the reaction system, and the catalytic activity of CuSeNPs was inhibited. The detection mechanisms, kinetics, and catalytic properties of CuSeNPs were systematically investigated. As a result, a novel fluorescence method for the assay of Cr(III) was established. The feasibility of CuSeNP nanozyme in detecting speciation Cr(III) in food samples was explored with satisfactory results. It showed the obvious potential for developing effective and dependable fluorescent detection method for protecting food safety.

Eco-friendly fluorescent detection method for Cu2+ ions combined with smartphone-integrated paper strip sensors based on highly fluorescent 2-aminoterephthalic acid in milk samples

The creation of smart, real-time sensing devices is crucial in food monitoring, though it continues to be a major challenge. Here, we report an eco-friendly fluorescent detection method for Cu2+ ions using 2-aminoterephthalic acid (NH2-H2BDC). Fluorescence (FL) quenching occurs at 425 nm under excitation at 330 nm, with detection limits (LOD) as low as 16.25 nM for Cu2+ ions. Additionally, a smartphone can visually and quantitatively detect Cu2+ ions by analyzing the RGB values on test paper. The results show high sensitivity, with a LOD of 11.07 μM. This work not only reveals fluorescent detection methods but also develops a convenient, low-cost, and portable smartphone-integrated paper strip for Cu2+ ion sensing. Two metrics, AGREEprep and BAGI, were employed to assess the method's environmental friendliness. The evaluation confirmed its exceptional eco-friendliness. This method marks a significant advancement for Cu2+ ion monitoring in diverse fields, especially food monitoring.

An Eu (III)-functionalized covalent organic framework fluorescent probe for specific detection of Flumequine based on pore restriction and antenna effect

The overuse of quinolone antibiotics has led to a series of health and environmental issues. Herein, we combine the distinct luminescence properties of Eu3+ with the unique structure of covalent organic frameworks (COFs) to develop a precise and sensitive fluorescent probe for detecting Flumequine (Flu) in water. Eu3+ is thoroughly anchored into the channels of COFs as recognition sites, while the synthesized probe material still maintains its intact framework structure. The unique structure of COFs provides excellent support and protection for Eu3+. Therefore, COF-Eu can rapidly bind with Flu which can transfer the absorbed energy to Eu3+ through an “antenna effect”, resulting in red fluorescence. Moreover, there is a good linear relationship between Flu concentration in the range of 0–30 µM, with a detection limit of 41 nM. Simultaneously, the material maintains remarkable reproducibility, with its performance remaining almost unchanged after five cycles of use. Remarkably, the probe demonstrates excellent Flu recovery rates in real samples. This study provides a viable approach for the recognition of flumequine in the environment through a customized fluorescence detection method.

Detection and quantification of C-terminally tagged proteins by in-gel fluorescence

The analysis of recombinant proteins in complex solutions is often accomplished with tag-specific antibodies in western blots. Recently, I introduced an antibody-free alternative wherein tagged proteins are visualized directly within polyacrylamide gels. For this, I used the protein ligase Connectase to selectively attach fluorophores to target proteins possessing an N-terminal recognition sequence. In this study, I extend this methodology to encompass the detection and quantification of C-terminally tagged proteins. Similar to the N-terminal labeling method, this adapted procedure offers increased speed, heightened sensitivity, and an improved signal-to-noise ratio when compared to western blots. It also eliminates the need for sample-specific optimization, enables more consistent and precise quantifications, and uses freely available reagents. This study broadens the applicability of in-gel fluorescence detection methods and thereby facilitates research on recombinant proteins.

Analytical Determination of Aflatoxin in Ground Corn Check Samples Completed by Multiple Laboratories over Several Years.

Mycotoxins are toxic molecules produced by multiple fungal species, including Aspergillus and Fusarium. Fungal infection of crops can result in mycotoxins entering the animal and human food supply. Enzyme-linked immunosorbent assays and other immunological assays have been developed to detect mycotoxins in foods. To calibrate the response of those methods, reference materials with known amounts of homogeneously dispersed mycotoxins are often utilized, where the mycotoxin concentrations have been determined using high-performance liquid chromatography coupled with absorbance or fluorescence detection methods, or high-performance liquid chromatography coupled with mass spectrometry detection methods. Therefore, it is important that the analytical methods provide accurate and precise quantitation of mycotoxins. The reference materials must also contain homogeneously dispersed known quantities of mycotoxin. To evaluate the accuracy and precision of mycotoxin reference materials and the analytical methods, quantitative results from multiple laboratories were completed each year for several years on ground corn check samples containing known levels of mycotoxins. Results for the quantitation of aflatoxin-containing corn reference samples are presented in this article.

Development of a CRISPR/Cas12a-based fluorescent detection method of Senecavirus A

BackgroundSenecavirus A (SVA), identified in 2002, is known to cause porcine idiopathic vesicular disease (PIVD), which presents with symptoms resembling other vesicular diseases. This similarity complicates field diagnosis. Conventional molecular diagnostic techniques are limited by their cost, sensitivity, and requirement for complicated instrumentation. Therefore, developing an effective and accurate diagnostic method is crucial for timely identification and isolation of affected pigs, thereby preventing further disease spread.MethodsIn this study, we developed a highly-specific and ultra-sensitive SVA detection method powered by CRISPR/Cas12a. To enhance the availability in laboratories with varied equipment conditions, microplate reader and ultraviolet light transilluminator were introduced. Moreover, PCR amplification has also been incorporated into this method to improve sensitivity. The specificity and sensitivity of this method were determined following the preparation of the recombinant Cas12a protein and optimization of the CRISPR/Cas12a-based trans-cleavage system.ResultsThe method demonstrated no cross-reactivity with ten kinds of viruses of swine. The minimum template concentration required to activate substantial trans-cleavage activity was determined to be 106 copies/µL of SVA templates. However, when PCR amplification was incorporated, the method achieved a detection limit of one copy of SVA templates per reaction. It also exhibited 100% accuracy in simulated sample testing. The complete testing process does not exceed three hours.ConclusionsImportantly, this method utilizes standard laboratory equipment, making it accessible for use in resource-limited settings and facilitating widespread and ultra-sensitive screening during epidemics. Overall, the development of this method not only broadens the array of tools available for detecting SVA but also holds significant promise for controlling the spread of PIVD.