Flow Assay For Rapid Detection Research Articles

Isothermal Amplification and Hypersensitive Fluorescence Dual-Enhancement Nucleic Acid Lateral Flow Assay for Rapid Detection of Acinetobacter baumannii and Its Drug Resistance.

Acinetobacter baumannii (A. baumannii) is among the main pathogens that cause nosocomial infections. The ability to rapidly and accurately detect A. baumannii and its drug resistance is essential for blocking secondary infections and guiding treatments. In this study, we reported a nucleic acid fluorescent lateral flow assay (NFLFA) to identify A. baumannii and carbapenem-resistant A. baumannii (CRAB) in a rapid and quantitative manner by integrating loop-mediated isothermal amplification (LAMP) and silica-based multilayered quantum dot nanobead tag (Si@MQB). First, a rapid LAMP system was established and optimised to support the effective amplification of two bacterial genes in 35 min. Then, the antibody-modified Si@MQB was introduced to capture the two kinds of amplified DNA sequences and simultaneously detect them on two test lines of a LFA strip, which greatly improved the detection sensitivity and stability of the commonly used AuNP-based nucleic acid LFA. With these strategies, the established LAMP-NFLFA achieved detection limits of 199 CFU/mL and 287 CFU/mL for the RecA (house-keeping gene) and blaOXA-23 (drug resistance gene) genes, respectively, within 43 min. Furthermore, the assay exhibited good repeatability and specificity for detecting target pathogens in real complex specimens and environments; thus, the proposed assay undoubtedly provides a promising and low-cost tool for the on-site monitoring of nosocomial infections.

Utilization of an immunochromatographic lateral flow assay for rapid detection of carbapenemase production in gram negative bacilli.

Rapid detection of carbapenemase production in gram negative bacilli has important treatment considerations. We evaluated a lateral flow assay (LFA) for carbapenemase production compared with molecular detection of 5 (blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48) carbapenemase genes. A total of 218 carbapenem nonsusceptible strains, including species of Enterobacterales, Pseudomonas aeruginosa isolated from clinical cultures were tested using the Cepheid Xpert Carba-R assay and the NG Biotech Carba-5 lateral flow immunoassay. Overall agreement with LFA was 98.2% with accuracy for each target >99% compared with polymerase chain reaction. Results were available within 15 minutes compared with 1 hour for molecular detection. The use of accurate, rapid diagnostics compliments antimicrobial stewardship programs.

Bio-SCAN V2: A CRISPR/dCas9-based lateral flow assay for rapid detection of theophylline.

Rapid, specific, and robust diagnostic strategies are needed to develop sensitive biosensors for small molecule detection, which could aid in controlling contamination and disease transmission. Recently, the target-induced collateral activity of Cas nucleases [clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases] was exploited to develop high-throughput diagnostic modules for detecting nucleic acids and small molecules. Here, we have expanded the diagnostic ability of the CRISPR-Cas system by developing Bio-SCAN V2, a ligand-responsive CRISPR-Cas platform for detecting non-nucleic acid small molecule targets. The Bio-SCAN V2 consists of an engineered ligand-responsive sgRNA (ligRNA), biotinylated dead Cas9 (dCas9-biotin), 6-carboxyfluorescein (FAM)-labeled amplicons, and lateral flow assay (LFA) strips. LigRNA interacts with dCas9-biotin only in the presence of sgRNA-specific ligand molecules to make a ribonucleoprotein (RNP). Next, the ligand-induced ribonucleoprotein is exposed to FAM-labeled amplicons for binding, and the presence of the ligand (small molecule) is detected as a visual signal [(dCas9-biotin)-ligRNA-FAM labeled DNA-AuNP complex] at the test line of the lateral flow assay strip. With the Bio-SCAN V2 platform, we are able to detect the model molecule theophylline with a limit of detection (LOD) up to 2μM in a short time, requiring only 15min from sample application to visual readout. Taken together, Bio-SCAN V2 assay provides a rapid, specific, and ultrasensitive detection platform for theophylline.

Development and application of a recombinase-aided amplification and lateral flow assay for rapid detection of pseudorabies virus from clinical crude samples

Pseudorabies virus (PRV) is an economically important viral agent affecting the swine industry in China. Accurate, rapid and simple detection is critical to PRV control and eradication. In the present study, a visible and low equipment-dependent recombinase-aid amplification assay integrated with lateral flow assay (RAA-LFA) was successfully developed to detect the PRV against the gE gene. The RAA-LFA did not react with the other swine pathogens, indicating the method has a good specificity. The limit of detection (LOD) for this RAA-LFA method was 21 copies per reaction against standard plasmids containing gE gene. Notably, the RAA-LFA can detect as low as 6.0 × 100 50 % tissue culture infective dose (TCID50) viral titer per reaction under nucleic-acid-extraction free condition. Clinical detection showed that the results detected by RAA-LFA were completely consistent with that of the qPCR assay. Taken together, the developed PRV RAA-LFA method provides approachability, comparable accuracy and sensitivity tool for PRV point-of-care testing (POCT), which is valuable to PRV control in areas where equipment and personnel resources are scarce.

Non-thiolated nucleic acid functionalized gold nanoparticle-based aptamer lateral flow assay for rapid detection of kanamycin.

Anovel Apt-LFA has been established for kanamycin based on non-thiolated nucleic acid-modified colloidal gold nanoprobe (AuNPs@polyA-DNA). The improvement in nucleic acid hybridization speed and efficiency was verified by modifying AuNPs with polyA-DNA strands instead of thiolated oligonucleotides (SH-DNA) strands. Moreover, the AuNPs@polyA-DNA was explored to apply in an Apt-LFA. Theexperimental factors including the concentration of the aptamer, the concentration of SA-DNAT conjugate, the incubation time, and temperature were carefully investigated. In addition, the kanamycin aptamer was modified by extending several bases at its end to modulate the hybridization complementary strand, which was found to significantly improve the performance of Apt-LFA. Under optimal experimental conditions, the Apt-LFA can detect kanamycin in honey with a LOD of 250ngmL-1 by the naked eyes. A linear range of 50-1250ngmL-1 was obtained with a LOD of 15ngmL-1 in honey by a portable reader. The Apt-LFA was successfully applied to the detection of kanamycin in honey with recoveries of 95.1-105.2%.

Development and evaluation of an isothermal recombinase polymerase amplification–lateral flow assay for rapid detection of strawberry vein banding virus in the field

The disease caused by strawberry vein banding virus (SVBV) is one of the main virus diseases affecting the growth and development of strawberries, and the development of an on-site method for rapid detection of SVBV would be useful for breeding virus-free strawberry seedlings. In this study, we used isothermal recombinant polymerase amplification (RPA) combined with lateral flow strip (LF strip) testing to achieve rapid diagnosis of SVBV following on-site rapid DNA extraction. The DNA target for the SVBV-RPA amplification was the specific nucleotide region, which is conserved between different SVBV isolates in China. This assay allowed detection of SVBV within 30 min, comprising rapid N3 DNA extraction for 10 min, DNA amplification with RPA for 15 min at 40 °C, and visualization of the amplicons by LF strip for 5 min. The conservative detection limit was as low as 10 pg DNA under optimized conditions. Furthermore, the diagnostic results obtained by SVBV-LF-RPA on-site for detection of suspected samples were consistent with those obtained by polymerase chain reaction (PCR), achieving the aim of highly sensitive on-site rapid diagnosis of most isolated SVBV strains. The system was successfully applied and validated for on-site detection of SVBV in the production of strawberry virus-free seedlings in Shanghai, demonstrating its suitability for on-site diagnosis of SVBV and high potential for application to SVBV detection nationwide.

Evaluation of a Lateral Flow Assay for Rapid Detection of African Swine Fever Virus in Multiple Sample Types.

Antibody-based lateral flow assay (LFA) is a quick and inexpensive tool used to detect pathogens in field samples, especially in hard-to-reach remote areas that may have limited access to central laboratories during an outbreak or surveillance. In this study, we investigated the ability of a commercially available LFA, PenCheck®, to detect African swine fever virus (ASFV) in clinical samples derived from pigs infected with highly virulent ASFV strains. The assay was specific and positively identified the majority of pigs showing high fever during the early stages (between 3 and 5 days) of infection. PenCheck® LFA also detected ASFV in serum and tissue samples collected from pigs that succumbed to experimental ASFV infection and whole blood, plasma, and tissue samples from the field. The limit of detection of the assay was ASFV titer 107.80 TCID50/mL, corresponding to ASFV real-time PCR values below 23 Ct. Although the sensitivity of the assay is less than that of the laboratory-based real-time PCR assays, the results obtained with the PenCheck® LFA in this study suggest that it can be used as a herd-level, field-deployable, and easy-to-use diagnostic tool to identify ASF-affected farms when access to portable molecular assays or central laboratories is not possible.

Development of a Multiplex Lateral Flow Assay for Rapid Detection of Waterborne Pathogens

Development of a Multiplex Lateral Flow Assay for Rapid Detection of Waterborne Pathogens

A Time-Resolved-Fluorescence Lateral Flow Assay for Rapid Detection of Cholyglycine Acid for the Diagnosis of Liver Diseases

Liver disease is a great danger to human health. The determination of blood level of Cholyglycine acid (CG) is a vital biomarker for the assessment of liver function in clinic, which is contribute to the diagnosis of liver diseases. Thus, establishing accuracy, rapid and convenient method for the detection of glycolic acid is of great significance. In this study, a time-resolved-fluorescence (TRF) lateral flow assay for rapid detection of CG was development. The analytical detection limit (mean of zero-2 SD) was 0.06 μg/mL The method showed good linearity in the range of 0.2–40 g/mL and was not affected by biomolecules with similar structure to CG. The analytical mean recovery of control was between 90–110% and the imprecision of intra- and inter-assay of CVs was less than 10%. No significant matrix effect was observed in saline, serum, plasma or whole blood. A good correlation was found with the homogeneous enzyme immunoassay (HEIA) assay (slopes 1.0463, y-intercepts 0.2721 μg/mL, R = 0.989, n = 50, P < 0.001). The CG TRF analysis could provide reproducible and quantitative information about the state of liver in a few minutes, which is suitable for the detection of liver diseases in point-of-care-testing (POCT) conditions.

Development of a PCR-lateral flow assay for rapid detection of Yersinia pestis, the causative agent of plague

Plague is a zoonotic disease caused by Yersinia pestis, a Gram-negative, rod shaped coccobacillus, which is primarily found in rodents and can be transmitted to humans through flea bite. The disease has three major clinical forms bubonic (by flea bite), pneumonic (by respiratory droplets) and septicemic plague. Y. pestis is classified as a category ‘A’ agent by NIAID, USA due to its high mortality and easy person to person dissemination. The conventional diagnostic methods available for Y. pestis show cross-reactivity with other enteropathogenic bacteria making its detection difficult. There is a need to develop sensitive and specific molecular assay for accurate detection of Y. pestis. PCR is well suited molecular biology tool for rapid diagnosis of plague but after completion of thermal cycling steps, it requires additional time to analyze amplified product using agarose gel electrophoresis. In the present study, PCR assay coupled with lateral flow strips has been developed for rapid detection of Y. pestis. Lateral flow strips give an alternative to gel electrophoresis and permit easy and rapid detection of PCR products. The PCR was performed with 5′ 6-FAM and biotin tagged primers specific for Y. pestis, targeting yihN gene located on chromosome. The PCR product was analyzed using lateral flow strips which yielded result within 2-3 minutes. The analytical sensitivity of PCR-lateral flow (PCR-LF) assay was 1 pg genomic DNA of Y. pestis and 500 copies of target DNA sequence harboured in a recombinant plasmid. The assay could detect Y. pestis DNA extracted from spiked human blood samples containing ≥104 CFU per mL of bacteria. The assay was found to be specific and did not cross react with other closely related bacterial species. The developed assay was highly specific, sensitive and also did not require agarose gel electrophoresis for post amplification analysis.

An up-converting phosphor technology-based lateral flow assay for rapid detection of major mycotoxins in feed: Comparison with enzyme-linked immunosorbent assay and high-performance liquid chromatography-tandem mass spectrometry.

Current methods for detection of mycotoxin in feed are time-consuming and tedious. An up-converting phosphor technology-based lateral flow (UPT-LF) assay system is a new emerging technique for analytes detection. The aim of this study was to compare the performance of UPT-LF, an enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for detecting aflatoxin B1 (AFB1), zearalenone (ZEN) and deoxynivalenol (DON) in feed. The results showed that the use of UPT-LF for AFB1, ZEN and DON detection exhibited the following: limits of detection of 3, 50 and 200 μg/kg; average recoveries of 104.39%, 102.94% and 103.65%; and precision of 13.96%, 13.71% and 12.56%; respectively. UPT-LF required 45 min to determine one mycotoxin and 1.5 h to determine three mycotoxins in a sample, which took the shortest time. Besides, there were positive correlations between the UPT-LF, ELISA and HPLC/MS/MS methods. In conclusion, UPT-LF can be used to detect and quantify AFB1, ZEN and DON in feed samples. Though the sensitivity, accuracy and precision of UPT-LF are inferior to those of HPLC-MS/MS and ELISA, the UPT-LF assay is the most convenient and rapid technique for on-site detection among the three methods.

Development of a reverse-transcription recombinase polymerase amplification assay with a lateral flow assay for rapid detection of avian orthoavulavirus 1.

Newcastle disease is an avian infectious disease caused by avian orthoavulavirus 1, also known as Newcastle disease virus (NDV). This disease has caused significant economic losses to the poultry industry worldwide. The rapid and simple detection of NDV infection is crucial to inform the appropriate control measures. We developed a reverse-transcription recombinase polymerase amplification (RT-RPA) assay combined with a lateral flow assay (LFA) for NDV detection. The RPA assay can be completed at 37°C within 20 min, and the RPA result can be visualized by the LFA within 5 min. The NDV RT-RPA-LFA detected NDV specifically with no cross-reactivity with other pathogens. The detection limit of NDV cDNA with our RT-RPA-LFA was 3.34 × 10-3 ng/μL. Consequently, the RT-RPA-LFA showed good potential for the detection of NDV infection in the field, especially in resource-limited settings.

Aptamer-gold nanozyme based competitive lateral flow assay for rapid detection of CA125 in human serum

For several decades, point-of-care technology (POCT) has proven its potential regarding swift and cost-efficient detection of analytes. Lateral flow assay is a highly popular POC technology that needs improvisation to increase its sensitivity, cost effectiveness and quantification so that it becomes more user friendly and affordable technology. In this context, the present study has investigated the use of aptamers and nanozymes together for the first time in developing an Aptamer-nanozyme lateral flow assay (ALFA). The present study uses a specific aptamer for CA125 as capture reagent and peroxidase mimetic gold nanoparticles as label for detection of CA125 in human serum through developed competitive ALFA. The assay was specific and has a limit of detection of 5.21 U/mL. The ALFA test was in house validated for its precision, recovery and showed a significant correlation with established CA125 chemiluminiscent ELISA with P-value<0.0001. In summary, this assay quantitatively detects an analyte by using an aptamer and peroxidase mimetic gold nanoparticles that ensures circumventing the use of antibodies and incorporating enzyme mimetic activity in assay systems.

Characterization of the highly immunogenic VP2 protrusion domain as a diagnostic antigen for members of Birnaviridae family.

Birnaviridae is a family of viruses (birnaviruses) which consists of four genera, members of which cause diseases in fish, birds, mollusks, and insects. The genome of birnaviruses encodes the highly immunogenic VP2 capsid protein. In order to demonstrate that the VP2 protein can be exploited as a diagnostic antigen for birnaviruses, we developed a lateral flow assay based on the surface-exposed VP2 protrusion domain of a representative birnavirus, infectious bursal disease virus (IBDV) of serotype 1 which causes the highly devastating infectious bursal disease in chickens. The biophysical characterization of the purified domain reveals that the domain predominantly consists of β-sheets, exists in a trimeric form, and remains folded at high temperatures, making it suitable for diagnostic purposes. Owing to its highly immunogenic nature and excellent biophysical properties, we employed the VP2 protrusion domain in a gold nanoparticle-based lateral flow assay for rapid detection of anti-IBDV antibodies in serum samples of infected chickens. Our results indicate that the domain binds anti-IBDV antibodies with high specificity during laboratory testing and on-site testing. The lateral flow assay reported here yields comparable results in a qualitative manner as obtained through a commercial enzyme-linked immunosorbent assay (ELISA). As VP2 is a common capsid protein of birnaviruses, the lateral flow assay can be generalized for other birnaviruses, and members of Tetraviridae and Nodaviridae families which contain homologous VP2 capsid proteins.Electronic supplementary materialThe online version of this article (10.1007/s00253-020-10458-6) contains supplementary material, which is available to authorized users.

A novel carbon nanoparticle probe-based ultrasensitive lateral flow assay for rapid detection of Ebola virus

Ebola virus (EBOV) is an extremely contagious pathogen first discovered in Africa associated with severe hemorrhagic disease in humans and nonhuman primates, which has resulted in at least 28 500 suspected cases and 11 300 confirmed deaths in 2014-2016 Ebola epidemic in West Africa. Rapid and sensitive detection of EBOV is the key to increasing the probability of survival and reducing infection rates in pandemic regions. Here, we report an ultrasensitive and instrument-free EBOV detection assay based on colloidal carbon immunochromatography. Carbon nanoparticle-labeled rabbit anti-EBOV-VP40 IgG were concentrated in the conjugate pad, monoclonal antibody (McAb, 4B7F9) against EBOV-VP40 and goat anti-rabbit IgG were immobilized on the nitrocellulose membrane with 2 μL/cm at a concentration of 1 mg/mL as test and control lines, respectively. Then the sample application pad, conjugate release pad, nitrocellulose membrane and absorbent pad were assembled into a lateral flow test strip. The test strip shows strong specificity against related viruses that share similar clinical symptoms and geographic range with EBOV, including marburg virus, influenza virus, yellow fever virus and dengue virus. In addition, 1 500 negative serums were tested with false-positive rate of 1.3‰ which significantly lower than that of ReEBOV™ colloidal gold test kit recommended by World Health Organization (WHO). The sensitivity of this strip was analyzed using inactivated EBOV with detection limit of 100 ng/mL (10⁶ copies/mL) which clearly higher than that of ReEBOV™ dipstick (10⁸ copies/mL). Furthermore, the strip showed excellent thermal stability characteristics in room temperature and could be as a point-of-care (POC), ultra-sensitive and specific promising candidate for EBOV serological screening in rural Africa or entry/exit ports.

Lateral flow assay for rapid detection of Staphylococcus aureus enterotoxin A in milk

Staphylococcus aureus is closely associated with food poisoning incidences, more, because of intoxication due to contamination of food with its enterotoxins. Staphylococcus enterotoxin A (SEA) is highly resistant to heat and other food processing conditions and has been implicated in food borne diseases from dairy products. Here we describe a rapid, gold nanoparticle based lateral flow immunoassay (LFIA) for detecting SEA in milk. Assay is in sandwich format and uses two specific monoclonal antibodies against SEA, one of which is labeled with gold nanoparticles and other is immobilized at control line. We also demonstrate the effect of using a combination of additives in capture antibody in order to enhance assay sensitivity. The developed LFIA is specific and has a lower detection limit of 0.5μg/ml. LFIA is tested both on spiked buffer and milk samples and display good reproducibility. The assembled strips stable for a month at 4°C. The method may be suitable for testing SEA in milk samples as it is quick, easy to perform and reproducible.

A sensitive and specific lateral flow assay for rapid detection of antibodies against glycoprotein B of Aujeszky's disease virus

A direct double antibody lateral flow assay (DDA-gB-LFA) for the detection of antibodies against the glycoprotein B (gB) of Aujeszky's disease virus (ADV) in swine sera was developed. A native ADV gB was used for the preparation of a conjugate with colloidal gold particles and the immobilization on the strip membrane. The gB purified from ADV virions by immunoaffinity chromatography retained its native epitope structure after adsorption on the nitrocellulose membrane and the surface of colloidal gold particles. The diagnostic specificity and sensitivity of the DDA-gB-LFA were evaluated using 236 field swine sera. The diagnostic specificity and sensitivity of the DDA-gB-LFA compared to a commercially available gB-based ELISA were 98.0% and 98.6%, respectively, when determined with the use of the reader-detection mode, and 98.0% and 93.5%, respectively, when determined using visual detection. The DDA-gB-LFA provides a rapid, sensitive, and specific determination of ADV gB-directed antibodies in sera and can be used for the detection of ADV-exposed swine.

Lateral flow assay for rapid detection of white spot syndrome virus (WSSV) using a phage-displayed peptide as bio-recognition probe.

White spot disease caused by the white spot syndrome virus (WSSV) has a major socio-economic impact on shrimp farming in India. It has been realized that a field-usable diagnostic capable of rapid detection of WSSV can prevent huge economic losses in disease outbreaks. In this work, we explored the possibility of using a peptide as bio-recognition probe in a field-usable device for the detection of WSSV from infected shrimps and prawns. A commercially available random phage-display library was screened against rVP28 (a major structural protein of WSSV, expressed as a recombinant protein in Escherichia coli). A bacteriophage clone VP28-4L was obtained, and its binding to purified rVP28 protein as well as WSSV from infected shrimp Litopaeneus vannamei tissue was confirmed by ELISA and western blot. The apparent equilibrium dissociation constant (Kd,app) was calculated to be 810nM. VP28-4L did not show cross-reactivity with any other shrimp viruses. A 12-mer peptide (pep28, with the sequence 'TFQAFDLSPFPS') displayed on the VP28-4L was synthesized, and its diagnostic potential was evaluated in a lateral flow assay (LFA). Visual detection of WSSV could be achieved using biotinylated-pep28 and streptavidin-conjugated gold nanoparticles. In LFA, 12.5μg/mL of the virus could be detected from L. vannamei gill tissue homogenate within 20min. Pep28 thus becomes an attractive candidate in bio-recognition of WSSV in field-usable diagnostic platforms benefitting the aquaculture sector.

Optimizations needed for lateral flow assay for rapid detection of pathogenic E. coli.

Lateral flow assay (LFA), or the immunochromatographic strip test, is popular to use for rapid and sensitive immunoassays. Gold nanoparticles (GNPs), due to tunable optical characteristics and easy manipulation of size or shape, represent an attractive approach for LFA technology. Since most enterohemorrhagic infections result from water and food contaminations of Escherichia coli O157:H7, selective and rapid detection of this organism in environmental and biological complexes is necessary. In this study, optimized parameters of antibody (Ab)-based LFA for rapid detection of pathogenic E. coli O157:H7 are described. GNPs were used as visualizing agents. The measuring parameters include the Ab concentration on the capture lines, the concentration of gold conjugate, and flow rate. M180 and 36 nm were the ideal membrane and GNP size, respectively, for bacterial detection of LFA. The target, E. coli O157:H7, could be detected with a visual limit of detection of 105 cfu/mL in 3-5 min. Selectivity of the system was very high and the target was recognized by developed strips, regardless of its presence singly or in mixed bacterial samples.

Development and evaluation of an up-converting phosphor technology-based lateral flow assay for rapid detection of Francisella tularensis.

Francisella tularensis is a potential biowarfare/bioterrorism agent and zoonotic pathogen that causes tularemia; thus, surveillance of F. tularensis and first-level emergency response using point-of-care testing (POCT) are essential. The UPT-LF POCT assay was established to quantitatively detect F. tularensis within 15 min, and the sensitivity of the assay was 104 CFU · mL−1 (100 CFU/test). The linear quantitative range covered five orders of magnitude, and the coefficients of variation were less than 10%. Except Shigella dysenteriae, UPT-LF showed excellent specificity to four strains that are also potential biowarfare/bioterrorism agents and 13 food-borne pathogenic strains. Samples with pH 2–13, high ion strengths (≥2 mol · L−1 solution of KCl and NaCl), high viscosities (≤50 mg · mL−1 PEG20000 or ≥20% glycerol), and high concentrations of biomacromolecules (≥400 mg · mL−1 bovine serum albumin or ≥80 mg · mL−1 casein) showed little influence on the assay. For practical utilization, the tolerance limits for seven powders and eight viscera were determined, and operation errors of liquid measurement demonstrated a minor influence on the strip. Ftu-UPT-LF is a candidate POCT method because of its excellent sensitivity, specificity, and stability in complex samples, as well as low operation error.