Resistivity detection of perfluoroalkyl substances with fluorous polyaniline in an electrical lateral flow sensor.

Perfluoroalkyl substances (PFAS), known as "forever chemicals," are a growing concern in the sphere of human and environmental health. In response, rapid, reproducible, and inexpensive methods for PFAS detection in the environment and home water supplies are needed. We have developed a simple and inexpensive perfluoroalkyl acid detection method based on an electrically read lateral flow assay (e-LFA). Our method employs a fluorous surfactant formulation with undoped polyaniline (F-PANI) fabricated to create test lines for the lateral flow assay. In perfluoroalkyl acid sensing studies, an increase in conductivity of the F-PANI film is caused by acidification and doping of PANI. A conductivity enhancement by 104-fold can be produced by this method, and we demonstrate a limit of detection for perfluorooctanoic acid (PFOA) of 400 ppt and perfluorobutanoic acid of 200 ppt. This method for PFOA detection can be expanded for wide-scale environmental and at-home water testing.

Lateral Flow Assay Biotesting by Utilizing Plasmonic Nanoparticles Made of Inexpensive Metals─Replacing Colloidal Gold.

Nanoparticles (NPs) can be conjugated with diverse biomolecules and employed in biosensing to detect target analytes in biological samples. This proven concept was primarily used during the COVID-19 pandemic with gold-NP-based lateral flow assays (LFAs). Considering the gold price and its worldwide depletion, here we show that novel plasmonic NPs based on inexpensive metals, titanium nitride (TiN) and copper covered with a gold shell (Cu@Au), perform comparable to or even better than gold nanoparticles. After conjugation, these novel nanoparticles provided high figures of merit for LFA testing, such as high signals and specificity and robust naked-eye signal recognition. Since the main cost of Au NPs in commercial testing kits is the colloidal synthesis, our development with the Cu@Au and the laser-ablation-fabricated TiN NPs is exciting, offering potentially inexpensive plasmonic nanomaterials for various bioapplications. Moreover, our machine learning study showed that biodetection with TiN is more accurate than that with Au.

"Cave Effect" Induces Self-Assembled Bimetallic Hollow Structure for Three-in-One Lateral Flow Immunoassay.

Bimetallic hollow structures have attracted much attention due to their unique properties, but they still face the problems of nonuniform alloys and excessive etching leading to structural collapse. Here, uniform bimetallic hollow nanospheres are constructed by pore engineering and then highly loaded with hemin (Hemin@MOF). Interestingly, in the presence of polydopamine (PDA), the competitive coordination between anionic polymer (γ-PGA) and dimethylimidazole does not lead to the collapse of the external framework but self-assembly into a hollow structure. By constructing the Hemin@MOF immune platform and using E. coli O157:H7 as the detection object, we find that the visual detection limits can reach 10, 3, and 3 CFU/mL in colorimetric, photothermal, and catalytic modes, which is 4 orders of magnitude lower than the traditional gold standard. This study provides a new idea for the morphological modification of the metal-organic skeleton and multifunctional immunochromatography detection.

Bimetallic Nanozyme: A Credible Tag for In Situ-Catalyzed Reporter Deposition in the Lateral Flow Immunoassay for Ultrasensitive Cancer Diagnosis.

The lateral flow immunoassay (LFIA) is a sought-after point-of-care testing platform, yet the insufficient sensitivity of the LFIA limits its application in the detection of tumor biomarkers. Here, a colorimetric signal amplification method, bimetallic nanozyme-mediated in situ-catalyzed reporter deposition (BN-ISCRD), was designed for ultrasensitive cancer diagnosis. The bimetallic nanozyme used, palladium@iridium core-shell nanoparticles (Pd@Ir NPs), had ultrahigh enzyme-like activity, which was further explained by the electron transfer of Pd@Ir NPs and the change in the Gibbs free energy during catalysis through density functional theory calculations. With gastric cancer biomarkers pepsinogen I and pepsinogen II as model targets, this assay could achieve a cutoff value of 10 pg/mL, which was 200-fold lower than that without signal enhancement. The assay was applied to correctly identify 8 positive and 28 negative clinical samples. Overall, this BN-ISCRD-based LFIA showed great merits and potential in the application of ultrasensitive disease diagnosis.

Signal Amplification by Spatial Concentration for Immunoassay on Cellulose Media.

Immunoassay is one of the most common bioanalytical techniques from lab-based to point-of-care settings. Over time, various approaches have been developed to amplify signals for greater sensitivity. However, the need for effective, versatile, and simple signal amplification methods persists yet. This paper presents a novel signal amplification method for immunoassay that utilizes spatial concentration of a cellulose-based plate possessing sensor transducers, specifically gold nanoparticles. By modifying the dimensions of the plate, the density of nanoparticles increased, resulting in intensified color signals. The coating material, polydopamine, which is utilized to protect the gold nanoparticles. Chemical changes in nanocomposites are characterized using scanning electron microscopy, Raman spectroscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and scanning electron microscopy. The application of this method to colorimetric quantification demonstrated great consistency across various concentrations of nanoparticles, with better reliability at lower concentration ranges. A model immunoassay is designed to evaluate the analytical performance. As a result, this method successfully corrected a false-negative result with a lowered Kd of 0.509 pmol per zone. This method shows strong signal enhancement capability that can correct false-negative signals in the immunoassays, with potential benefits including versatility, simplicity, low cost, and the ability to operate multiple plates simultaneously.

Microstructure-Driven Self-Transport and Convection of Water on Membrane Surface for Ultra-Fast, Highly Sensitive, Low-Cost Lateral-Flow Assays.

Lateral-flow assay (LFA) is one of the most commonly used detection technologies, in which the chromatographic membranes are currently used as the lateral-flow membrane (e.g., nitrocellulose membrane, NC Mem). However, several disadvantages of existing chromatographic membranes limit the performance of LFA, including relatively low flow velocity of sample solution and relatively more residuals of sample on membrane, which increase detection time and detection noise. Herein, a surface structure membrane (SS Mem) is proposed, which enables fast self-transport of water with a convection manner and realizes low residuals of sample on membrane surface after the flow. On SS Mem, the flow velocity of water is 7.1-fold higher, and the residuals of sample are decreased by 60-67%, comparing those in NC Mem. SS Mem is used as lateral-flow membrane to prepare lateral-flow strips of nanogold LFA and fluorescence LFA for rapid detection of SARS CoV-2 nucleocapsid protein. These LFAs require 210 s per detection, with limits of detection of 3.98 pg mL-1 and 53.3 fg mL-1, sensitivity of 96.5%, and specificity of 90%. The results suggest that SS Mem enables ultrafast, highly sensitive lateral-flow immunoassays and shows great potential as a new type of lateral-flow membrane to broaden the application of LFA.

"Four-In-One" Multifunctional Dandelion-Like Gold@platinum Nanoparticles-Driven Multimodal Lateral Flow Immunoassay.

The traditional lateral flow immunoassay (LFIA) with a single signal output mode may encounter challenges such as low sensitivity, poor detection range, and susceptibility to external interferences. These limitations hinder its ability to meet the growing demand for advanced LFIA. To address these issues, the rational development of multifunctional labels for multimodal LFIA emerges as a promising strategy. Herein, this study reports a multimodal LFIA using "four-in-one" multifunctional dandelion-like gold@platinum nanoparticles (MDGP). The inherent properties of MDGP, such as the broad absorption spectrum, porous dandelion-like nanostructure, and bimetallic composition with gold and platinum, endow them with capacities in dual spectral-overlapped fluorescence quenching, optical readout, catalytic activity, and photothermal effect. Benefiting from their multifunctional properties, the MDGP-LFIA enables multimodal outputs including fluorescent, colorimetric, and photothermal signals. This multimodal MDGP-LFIA allows for the detection of acetamiprid at a range of 0.01-50 ng mL-1, with the lowest qualitative and quantitative detection results of 0.5 and 0.008 ng mL-1, respectively, significantly better than the traditional gold nanoparticles-based LFIA. The diversity, complementarity, and synergistic effect of integrated output signals in this multimodal MDGP-LFIA improve the flexibility, practicability, and accuracy of detection, holding great promise as a point-of-care testing platform in versatile application scenarios.

Kiwi-Inspired Rational Nanoarchitecture with Intensified and Discrete Magneto-Fluorescent Functionalities for Ultrasensitive Point-of-Care Immunoassay.

Fluorescent lateral flow immunoassays (FLFIA) is a well-established rapid detection technique for quantitative analysis. However, achieving accurate analysis of biomarkers at the pg mL-1 level using FLFIA still poses challenges. Herein, an ultrasensitive FLFIA platform is reported utilizing a kiwi-type magneto-fluorescent silica nanohybrid (designated as MFS) that serves as both a target-enrichment substrate and an optical signal enhancement label. The spatially-layered architecture comprises a Fe3O4 core, an endocarp-fibers like dendritic mesoporous silica, seed-like quantum dots, and a kiwi-flesh like silica matrix. The MFS demonstrates heightened fluorescence brightness, swift magnetic response, excellent size uniformity, and dispersibility in water. Through liquid-phase capturing and fluorescence-enhanced signal amplification, as well as magnetic-enrichment sample amplification and magnetic-separation noise reduction, the MFS-based FLFIA is successfully applied to the detection of cardiac troponin I that achieved a limit of detection at 8.4 pg mL-1, tens of times lower than those of previously published fluorescent and colorimetric lateral flow immunoassays. This work offers insights into the strategic design of magneto-fluorescent synergetic signal amplification on LFIA platform and underscores their prospects in high-sensitive rapid and on-site diagnosis of biomarkers.

Efficient Photothermal Sensor Based on Coral-Like Hollow Gold Nanospheres for the Sensitive Detection of Sulfonamides.

Gold nanoparticles (AuNPs), universally regarded as colorimetric signal reporters, are widely employed in lateral flow immunoassays (LFIAs). However, it is difficult for AuNPs-LFIA to achieve a wide range and sensitive detection. Herein, novel coral-like hollow gold nanospheres (CHGNPs) are synthesized. The growth of gold nanospheres can be regulated to obtain a multibranched and hollow construction. The obtained CHGNPs possess intense broadband absorption across the visible to near-infrared region, exhibiting a high molar extinction coefficient of 14.65 × 1011 M-1 cm-1 and a photothermal conversion efficiency of 79.75%. Thus, the photothermal/colorimetric dual-readout LFIA is developed based on CHGNPs (CHGNPs-PT-LFIA and CHGNPs-CM-LFIA) to effectively improve the detection sensitivity and broaden the detection range in regard to sulfonamides (SAs). The limits of detection of the CHGNPs-PT-LFIA and CHGNPs-CM-LFIA reached 1.9 and 2.8 pg mL-1 for the quantitative detection of sulfaquinoxaline, respectively, which are 6.3-fold and 4.3-fold lower than that of the AuNPs-LFIA. Meanwhile, the CHGNPs-PT-LFIA broadened the detection range to three orders of magnitude, which ranged from 2.5 to 5000 pg mL-1. The synthesized photothermal CHGNPs have been proven effective in improving the performance of the LFIA and provide a potential option for the construction of sensing platforms.

Rapid and sensitive detection of chikungunya virus using one-tube, reverse transcription, semi-nested multi-enzyme isothermal rapid amplification, and lateral flow dipstick assays.

Chikungunya fever is an acute infectious disease caused by chikungunya virus (CHIKV), which is transmitted by Aedes mosquitoes. Simple, rapid, and sensitive detection of CHIKV is critical for its prevention and spread. To address this issue, we combined one-tube, reverse transcription semi-nested, multi-enzyme isothermal rapid amplification, and lateral flow dipstick strips assay to detect CHIKV RNA. The study used a 318-bp gene fragment of CHIKV NSP4 as the target of the assay. This method of amplification takes 30 min for two-step amplification at 39°C. The dilution of amplification products was added to the LFD strip with results visible to the naked eye after 10 min. The method has a sensitivity of 1 copy/µL for the detection of CHIKV RNA, which is 100-fold higher than the conventional reverse transcription-multi-enzyme isothermal rapid amplification and 10-fold higher than the reverse transcription quantitative PCR (RT-qPCR) method. In addition, the method demonstrated good specificity and a better detection rate (85.7%, 18 of 21) than RT-qPCR (80.9%, 17 of 21) in clinically confirmed patient plasma samples. Thus, the rapid CHIKV RNA assay developed in this study will be an important tool for the rapid and accurate screening of patients for chikungunya fever. IMPORTANCE: This study presents a new one-tube, reverse transcription semi-nested, multi-enzyme isothermal rapid amplification assay combined with lateral flow dipstick strips for the detection of CHIKV. This technique significantly improves sensitivity and outperforms RT-qPCR for the detection of CHIKV, especially in samples with low viral loads. It is also significantly faster than conventional RT-qPCR and does not require special equipment or a standard PCR laboratory. The combination of the isothermal amplification technology developed in this study with point-of-care molecular testing offers the potential for rapid, on-site, low-cost molecular diagnosis of CHIKV.

A Lateral Flow Assay Based on Streptavidin-biotin Amplification System with Recombinase Polymerase Amplification for Rapid and Quantitative Detection of Salmonella enteritidis.

Salmonella constitutes a prevalent alimentary pathogen, instigating zoonotic afflictions. Consequently, the prompt discernment of Salmonella in sustenance is of cardinal significance. Lateral flow assays utilizing colorimetric methodologies adequately fulfill the prerequisites of point-of-care diagnostics, however, their detection threshold remains elevated, generally permitting only qualitative discernment, an impediment to the preliminary screening of nascent pathogens. In response to this conundrum, we propose a lateral flow diagnostic predicated upon a streptavidin-biotin amplification system with recombinase polymerase amplification engineered for the expeditious and quantitative discernment of Salmonella enteritidis. Trace nucleic acids within a sample undergo exponential amplification via recombinase polymerase amplification to a level discernable, constituting the initial signal amplification. Subsequently, along the test line (T-line) of the lateral flow strip, the chromatic signal undergoes augmentation by securing a greater quantity of AuNPs through the magnification capacity of the streptavidin-biotin mechanism, affecting the second signal amplification. Quantitative results are procured via smartphone capture and transferred to computer software for precise calculation of the targeted quantity. The lateral flow strip exhibits a LOD at 19.41 CFU/mL for cultured S. enteritidis. The RSD of three varying concentrations were respectively 3.74 %, 5.96 %, and 4.25 %.

Development of a multiple cross displacement amplification combined with nanoparticles-based biosensor assay for rapid and sensitive detection of Streptococcus pyogenes.

BACKGROUND: S. pyogenes, is a primary pathogen that leads to pharyngitis and can also trigger severe conditions like necrotizing fasciitis and streptococcal toxic shock syndrome (STSS), often resulting in high mortality rates. Therefore, prompt identification and appropriate treatment of S. pyogenes infections are crucial in preventing the worsening of symptoms and alleviating the disease's impact. RESULTS: In this study, a newly developed technique called multiple cross displacement amplification (MCDA) was employed to detect S. pyogenes,specifically targeting the speB gene, at a temperature of 63°C within 30 min. Then, an easily portable and user-friendly nanoparticles-based lateral flow biosensor (LFB) assay was introduced for the rapid analysis of MCDA products in just 2 min. The results indicated that the LFB offers greater objectivity compared to Malachite Green and is simpler than electrophoresis. The MCDA-LFB assay boasts a low detection limit of 200 fg and exhibits no cross-reaction with non-S. pyogenes strains. Among 230 clinical swab throat samples, the MCDA-LFB method identified 27 specimens as positive, demonstrating higher sensitivity compared to 23 samples detected positive by qPCR assay and 18 samples by culture. The only equipment needed for this assay is a portable dry block heater. Moreover, each MCDA-LFB test is cost-effective, priced at approximately $US 5.5. CONCLUSION: The MCDA-LFB assay emerges as a straightforward, specific, sensitive, portable, and user-friendly method for the rapid diagnosis of S. pyogenes in clinical samples.

Rapid and sensitive lateral flow biosensor for the detection of GII human norovirus based on immunofluorescent nanomagnetic microspheres.

Human norovirus (HuNoV) is the most predominant viral agents of acute gastroenteritis. Point-of-care testing (POCT) based on lateral flow immunochromatography (LIFC) has become an important tool for rapid diagnosis of HuNoVs. However, low sensitivity and lack of quantitation are the bottlenecks of traditional LIFC. Thus, we established a rapid and accurate technique that combined immunomagnetic enrichment (IM) with LFIC to identify GII HuNoVs in fecal specimens. Before preparing immunofluorescent nanomagnetic microspheres and achieving the effect of HuNoV enrichment in IM and fluorescent signal in LFIC, amino-functionalized magnetic beads (MBs) and carboxylated quantum dots (QDs) were coupled at a mass ratio of 4:10. Anti-HuNoV monoclonal antibody was then conjugated with QDs-MB. The limit of detection was 1.56 × 104 copies/mL, and the quantitative detection range was 1.56 × 104 copies/mL-1 × 106 copies/mL under optimal circumstances. The common HuNoV genotypes GII.2, GII.3, GII.4, and GII.17 can be detected, there was no cross-reaction with various enteric viruses, including rotavirus, astrovirus, enterovirus, and sapovirus. A comparison between IM-LFIC and RT-qPCR for the detection of 87 fecal specimens showed a high level of agreement (kappa = 0.799). This suggested that the method is rapid and sensitive, making it a promising option for point-of-care testing in the future.

A field diagnostic method for rapid and sensitive detection of mpox virus.

The mpox outbreak has subdued with fewer reported cases at the present in high-income countries. It is known that mpox virus (MPXV) infection has been epidemic for more than 50 years in African countries. The ancestral MPXV strain has changed into multiple clades, indicating the ongoing evolution of MPXV, which reflects the historical neglect of mpox in Africa, especially after smallpox eradication, and bestows the danger of more severe mpox epidemics in the future. It is thus imperative to continue the development of mpox diagnostics and treatments so we can be prepared in the event of a new mpox epidemic. In this study, we have developed an MPXV detection tool that leverages the recombinase-aid amplification assay by integrating lateral flow strips (RAA-LF) and one-step sample DNA preparation, with visible readout, no need of laboratory instrument, and ready for field deployment. The detection limit reaches 10 copies per reaction. The performance of our RAA-FL assay in diagnosing mpox clinical samples is on par with that of the quantitative polymerase chain reaction (PCR) assay. Taken together, we have developed a point-of-care RAA-LF method of high accuracy and sensitivity, readily deployable for field detection of MPXV. This diagnostic tool is expected to improve and accelerate field- and self-diagnosis, allow timely isolation and treatment, reduce the spread of MPXV, thus effectively mitigate MPXV outbreak in the future.

Advanced technologies towards improved HPV diagnostics.

Persistent infection with high-risk types of human papillomaviruses (HPV) is a major cause of cervical cancer, and an important factor in other malignancies, for example, head and neck cancer. Despite recent progress in screening and vaccination, the incidence and mortality are still relatively high, especially in low-income countries. The mortality and financial burden associated with the treatment could be decreased if a simple, rapid, and inexpensive technology for HPV testing becomes available, targeting individuals for further monitoring with increased risk of developing cancer. Commercial HPV tests available in the market are often relatively expensive, time-consuming, and require sophisticated instrumentation, which limits their more widespread utilization. To address these challenges, novel technologies are being implemented also for HPV diagnostics that include for example, isothermal amplification techniques, lateral flow assays, CRISPR-Cas-based systems, as well as microfluidics, paperfluidics and lab-on-a-chip devices, ideal for point-of-care testing in decentralized settings. In this review, we first evaluate current commercial HPV tests, followed by a description of advanced technologies, explanation of their principles, critical evaluation of their strengths and weaknesses, and suggestions for their possible implementation into medical diagnostics.

Single-tube, single-strip lateral flow assays utilizing loop-mediated isothermal amplification for simultaneous hepatitis B and C viral detection.

Globally, hepatitis B virus (HBV) affects over 250 million people, whereas hepatitis C virus (HCV) affects approximately 70 million people, posing major public health challenges. Despite the availability of vaccines and treatments, a lack of comprehensive diagnostic coverage has left many cases undiagnosed and untreated. To address the need for sensitive, specific, and accessible diagnostics, this study introduced a multiplex loop-mediated isothermal amplification assay with lateral flow detection for simultaneous HBV and HCV testing. This assay achieved exceptional sensitivity and was capable of detecting HBV and HCV concurrently in a single tube and on a single strip within 25 min, achieving the required clinical sensitivity (10 and 103 genomic copies/reaction for HBV and HCV, respectively). The method was validated in clinical samples of various viral genotypes, achieving an equivalent limit of detection. Additionally, a custom portable heating device was developed for field use. The assay developed here, capable of direct viral detection on the strip, shows promise in supplanting current methods that solely identify antibodies and necessitate additional qPCR for viral activity assessment. This economical and rapid assay aligns with point-of-care testing needs, offering significant advancements in enhancing viral hepatitis diagnostics in settings with limited resources.

The development of RT-RPA and CRISPR-Cas12a based assay for sensitive detection of Hirame novirhabdovirus.

Hirame novirhabdovirus (HIRRV) is a highly pathogenic fish virus that poses a significant threat to the farming of a variety of economic fish. Due to no commercial vaccines and effective drugs available, sensitive and rapid detection of HIRRV at latent and early stages is important and critical for the control of disease outbreaks. However, most of the current methods for HIRRV detection have a large dependence on instruments and operations. For better detection of HIRRV, we have established a detection technology based on the reverse transcription and recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a to detect the N gene of HIRRV in two steps. Following the screening of primer pairs, the reaction temperature and time for RPA were optimized to be 40 °C and 32min, respectively, and the CRISPR/Cas12a reaction was performed at 37 °C for 15min. The whole detection procedure including can be accomplished within 1 h, with a detection sensitivity of about 8.7 copies/µl. The detection method exhibited high specificity with no cross-reaction to the other Novirhabdoviruses IHNV and VHSV, allowing naked-eye color-based interpretation of the detection results through lateral flow (LF) strip or fluorescence under violet light. Furthermore, the proliferation dynamic of HIRRV in the spleen of flounder were comparatively detected by LF- and fluorescence-based RPA-CRISPR/Cas12a assay in comparison to qRT-PCR at the early infection stage, and the results showed that the viral positive signal could be firstly detected by the two RPA-CRISPR/Cas12a based methods at 6 hpi, and then by qRT-PCR at 12 hpi. Overall, our results demonstrated that the developed RPA-CRISPR/Cas12a method is a stable, specific, sensitive and more suitable in the field, which has a significant effect on the prevention of HIRRV. RT-RPA-Cas12a-mediated assay is a rapid, specific and sensitive detection method for visual and on-site detection of HIRRV, which shows a great application promise for the prevention of HIRRV infections.

Development of a triplex RT-RAA-LFA assay for the rapid differential diagnosis of porcine epidemic diarrhea virus, porcine deltacoronavirus and transmissible gastroenteritis virus.

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) are three clinically common coronaviruses causing diarrhea in pigs, with indistinguishable clinical signs and pathological changes. Rapid, portable and reliable differential diagnosis of these three pathogens is crucial for the prompt implementation of appropriate control measures. In this study, we developed a triplex nucleic acid assay that combines reverse transcription recombinase-aided amplification (RT-RAA) with lateral flow assay (LFA) by targeting the most conserved genomic region in the ORF1b genes of PEDV, PDCoV and TGEV. The entire detection process of the triplex RT-RAA-LFA assay included 10-min nucleic acid amplification at 42 °C and 5-min visual LFA readout at room temperature. The assay could specifically differentiate PEDV, PDCoV and TGEV without cross-reaction with any other major swine pathogens. Sensitivity analysis showed that the triplex RT-RAA-LFA assay was able to detect the viral RNA extracted from the spiked fecal samples with the minimum of 1 × 100 TCID50 PEDV, 1 × 104 TCID50 PDCoV, and 1 × 102 TCID50 TGEV per reaction, respectively. Further analysis showed that the 95 % detection limit (LOD) of triplex RT-RAA-LFA for PEDV, PDCoV, and TGEV were 22, 478, and 205 copies of recombinant plasmids per reaction, respectively. The diagnostic performance of triplex RT-RAA-LFA was compared with that of PEDV, PDCoV and TGEV respective commercial real-time RT-PCR kits by testing 114 clinical rectal swab samples in parallel. The total diagnostic coincidence rates of triplex RT-RAA-LFA with real-time RT-PCR kits of PEDV, PDCoV and TGEV were 100 %, 99.1 % and 99.1 %, respectively, and their Kappa values were 1.00, 0.958 and 0.936, respectively. Collectively, the RT-RAA-LFA assay is a powerful tool for the rapid, portable, visual, and synchronous differential diagnosis of PEDV, PDCoV, and TGEV.

Single-tube four-target lateral flow assay detects human papillomavirus types associated with majority of cervical cancers.

Isothermal nucleic acid amplification methods have many advantages for use at the point of care. However, there is a lack of multiplexed isothermal amplification tests to detect multiple targets in a single reaction, which would be valuable for many diseases, such as infection with high-risk human papillomavirus (hrHPV). In this study, we developed a multiplexed loop-mediated isothermal amplification (LAMP) reaction to detect the three most common hrHPV types that cause cervical cancer (HPV16, HPV18, and HPV45) and a cellular control for sample adequacy. First, we characterized the assay limit of detection (LOD) in a real-time reaction with fluorescence readout; after 30 min of amplification the LOD was 100, 10, and 10 copies/reaction of HPV16, HPV18, and HPV45, respectively, and 0.1 ng/reaction of human genomic DNA (gDNA). Next, we implemented the assay on lateral flow strips, and the LOD was maintained for HPV16 and HPV18, but increased to 100 copies/reaction for HPV45 and to 1 ng/reaction for gDNA. Lastly, we used the LAMP test to evaluate total nucleic acid extracted from 38 clinical samples; compared to qPCR, the LAMP test had 89% sensitivity and 95% specificity. When integrated with sample preparation, this multiplexed LAMP assay could be useful for point-of-care testing.

A method for Porphyromonas gingivalis based on recombinase polymerase amplification and lateral flow strip technology.

OBJECTIVE: A practical visual detection method was established to detect Porphyromonas gingivalis (P. gingivalis) by employing a combination of recombinase polymerase amplification and lateral flow strips (RPA-LF) assay, designed for conducting point-of-care testing in clinical settings. METHODS: Primers and probes targeting the P. gingivalis pepO gene were designed. The RPA-LF assay was established by optimising reaction temperature and time, determining the limit of detection (LOD). The specificity of the method was determined by assessing its cross-reactivity with deoxyribonucleic acid from 23 pathogenic bacteria. Finally, the clinical samples from healthy controls (n = 30) and individuals with periodontitis (n = 31) were analysed. The results were compared with those obtained using real-time polymerase chain reaction (PCR). RESULTS: The optimal reaction temperature and time were 39 °C and 12 min. The method exhibited a LOD at 6.40 × 10-4 µg/mL and demonstrated high specificity and sensitivity during cross-reactivity assessment. The RPA-LF assay achieved a P. gingivalis detection rate of 84 % in individuals with periodontitis and 3 % in healthy controls. The results were consistent with those obtained through real-time PCR. CONCLUSION: An RPA-LF assay was developed for detecting P. gingivalis, characterised by its high sensitivity, high specificity, simple operational procedure, and rapid reaction time.