Assay For Quantitative Detection Research Articles

Copper-based nanozyme-linked immunosorbent assay for quantitative detection of fumonisin B1

Fumonisin B1 (FB1) is a Group 2B carcinogen. It is vital to develop an ultra-sensitive, low-cost, and rapid screening method for FB1 for food safety risk control. In this study, a novel copper-based nanozyme was synthesized. The anti-FB1 monoclonal antibody (mAb) was conjugated with copper-based nanozyme through physical adsorption to probe construction. The probe could compete with the limited antigen and trigger a color change when employing the 3,3’,5,5’-tetramethylbenzidine (TMB) as chromogenic substrate in the presence of H2O2. Several experimental conditions were optimized, including the concentration of coating antigen, the concentration of mAb coupled to the nanozyme, incubation time, and methanol concentration. Under optimal conditions, a direct competitive nanozyme-linked immunosorbent assay (NLISA) has been established with a limit of detection (LOD) of 6.52 pg/mL. The developed method presented good accuracy and precision. The spiked recoveries ranged from 97.368 % to 120.916 %, with coefficient of variation (CV) below 10 %. In addition, there was a high consistency (R2=0.9820 for corn; R2=0.9936 for wheat) between the developed detection method and the reference method, liquid chromatography/mass spectrometry (LC-MS/MS). This research can expand the application areas of copper-based nanozymes and improve the sensitivity and efficiency of FB1 monitoring.

Quantitative immunochromatographic assay for rapid detection of rosemary herb extract in ham sausage and dairy products

The important constituents of rosemary herb extract are carnosic acid (CarA), carnosol (Car) and rosmanol (Ros), which are burdensome to the human body when consumed in excessive amounts. Using a computer-assisted method, we prepared 3B10-antibody with both broad-spectrum and high affinity for CarA, Car and Ros with median inhibitory concentration of 0.41 ng/mL, 0.45 ng/mL, and 0.53 ng/mL, respectively. Subsequently, a quantitative lateral flow immunoassay (LFIA) was developed to identify CarA, Car and Ros in ham sausage and dairy within 30 min, and the respectively calculated limit of detection values of 0.044 μg/kg, 0.148 μg/kg and 0.169 μg/kg in sample, which could meet maximum additive limits. The quantitative results of LFIA method in positive samples were consistent with that of the instrumental method.These results also show that the high recognition performance and reliability of the LFIA strips validate the field test method.

Quantitative MODS-Wayne assay for rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis from sputum samples.

PZA susceptibility testing continues to be a challenge, particularly in countries with high TB incidence. In response to this pressing need, we have developed a quantitative MODS-Wayne (MODS-WQ) assay. This approach offers a direct and cost-effective solution representing a significant advancement in TB diagnostics, particularly benefiting resource-limited laboratories, primarily in developing regions. The MODS-WQ assay stands out for its ability to quantify pyrazinoic acid (POA) production, as a reliable indicator of PZA resistance. Unlike traditional qualitative assays, MODS-WQ eliminates the inherent subjectivity in interpretation, providing more accurate and actionable results. Moreover, the MODS-WQ approach accounts for critical factors influencing PZA resistance, including enzymatic efficiency and efflux pump activity. By integrating these factors into the detection process, our methodology offers a comprehensive understanding of PZA resistance levels, enabling tailored treatment strategies for patients.

A highly sensitive and specific real-time quantitative polymerase chain reaction assay for Perkinsus marinus detection in oysters

Oyster aquaculture is one of the fastest-growing food production industries worldwide; however, it faces a significant challenge from the protist Perkinsus marinus, particularly in the USA. Although several quantitative molecular diagnostic methodologies are available for identifying diseases caused by P. marinus, the primer pairs used therein led to non-specific identification of other Perkinsus spp. Hence, a quantitative real-time PCR (Pm-qPCR) assay specific for P. marinus was developed using a TaqMan-based probe with the internal quencher in this study. A primer pair and probe specific to P. marinus were designed from a hypothetical protein of P. marinus collected from the whole-genome shotgun sequence database of the National Center for Biotechnology Information (NCBI). In silico analysis using homologous sequences of P. olseni and P. chesapeaki confirmed the high specificity of primers designed in this study. The Pm-qPCR assay was performed using seven different strains of P. marinus, P. olseni, and P. chesapeaki, revealing high specificity and sensitivity for detecting only P. marinus strains. In conclusion, it was demonstrated that Pm-qPCR can effectively and accurately diagnose P. marinus with high specificity and sensitivity. This assay is promising for monitoring oyster health and disease management in ecosystems and aquaculture.

Development and Evaluation of Real-Time Quantitative PCR Assays for Detection of Phellinus noxius Causing Brown Root Rot Disease.

Brown root rot disease (BRRD) is a highly destructive tree disease. Early diagnosis of BRRD has been challenging because the first symptoms and signs are often observed after extensive tissue colonization. Existing molecular detection methods, all based on the internal transcribed spacer (ITS) region, were developed without testing against global Phellinus noxius isolates, other wood-decay fungi, or host plant tissues. This study aimed to develop SYBR Green real-time quantitative PCR (qPCR) assays for P. noxius. The primer pair Pn_ITS_F/Pn_ITS_R targets the ITS, and the primer pair Pn_NLR_F/Pn_NLR_R targets a P. noxius-unique group of homologous genes identified through a comparative genomics analysis. The homologous genes belong to the nucleotide-binding-oligomerization-domain-like receptor (NLR) superfamily. The new primer pairs and a previous primer pair G1F/G1R were optimized for qPCR conditions and tested for specificity using 61 global P. noxius isolates, 5 other Phellinus species, and 22 non-Phellinus wood-decay fungal species. Although all three primer pairs could detect as little as 100 fg (approximately 2.99 copies) of P. noxius genomic DNA, G1F/G1R had the highest specificity and Pn_NLR_F/Pn_NLR_R had the highest efficiency. To avoid false positives, the cutoff quantification cycle values were determined as 34 for G1F/G1R, 29 for Pn_ITS_F/Pn_ITS_R, and 32 for Pn_NLR_F/Pn_NLR_R. We further validated these qPCR assays using Ficus benjamina seedlings artificially inoculated with P. noxius, six tree species naturally infected by P. noxius, rhizosphere soil, and bulk soil. The newly developed qPCR assays provide sensitive detection and quantification of P. noxius, which is useful for long-term monitoring of BRRD status.

ImageJ and smartphone app chemistry analyzer using image-based colorimetric assay for quantitative detection of melamine

Abstract A smartphone-assisted microchemistry analyzer with an image-based colorimetric assay using ImageJ and a smartphone app detection method was successfully developed for the quantitative detection of melamine (MEL). The color changes of the MEL in the colloidal gold lateral flow immunoassay strip were captured and analyzed using a smartphone-controlled analyzer with an LED light source and a smartphone camera. The smartphone camera and light source were used to read the colorimetric signal from the strip, and a smartphone app was written and installed onto the smartphone. The quantitative analysis was validated with ImageJ and smartphone app colorimetric analysis. The limits of detection (LODs) for ImageJ and the smartphone app were calculated as 0.30 and 0.07 mg/L, respectively. The highly quantitative relationships between the MEL concentrations and the optical density and gray value of the ImageJ and smartphone app detection method. The designed image-based biosensor is successfully applied to detect MEL in solution of standard MEL and commercial milk samples.

Quantitative real-time PCR assays for species-specific detection and quantification of Baltic Sea spring bloom dinoflagellates.

In the Baltic Sea, the dinoflagellates Apocalathium malmogiense, Biecheleria baltica, and Gymnodinium corollarium are important contributors to the spring bloom. However, their relative contribution to the bloom community cannot be unambiguously determined by conventional light microscopy due to a lack of resolution of distinctive morphological features of the three species. Here, we describe a molecular approach based on a quantitative real-time polymerase chain reaction (qPCR) primer and probe system, targeting the ITS1 and ITS2 regions of the rRNA gene for all three species and enabling their quantification. The specificity of the method was demonstrated using monocultures of A. malmogiense, B. baltica, G. corollarium as well as three other dinoflagellate species co-occurring in the Baltic Sea during spring and validated using field-collected phytoplankton samples.

Development and validation of a quantitative PCR assay for detection of Sulawesi tortoise adenovirus

In 2007, a mortality event involving over 100 Sulawesi tortoises (Indotestudo forsteni), two Impressed tortoises (Manouria impress) and a critically endangered Burmese star tortoise (Geochelone platynota) was attributed to Sulawesi tortoise adenovirus (STADV; genus Siadenovirus). We developed a TaqMan quantitative PCR assay targeting the DNA polymerase gene of STADV for use in clinical diagnosis and epidemiologic surveillance. This assay failed to amplify five closely-related chelonian adenoviruses, indicating high analytical specificity. The assay performed with high efficiency (slope = −3.337; R2 = 0.999) and high inter- and intra-assay repeatability (coefficient of variation <1.36 % at all standard curve dilutions). Dynamic range included 1.00 × 107 to 1.00 × 101 target copies per reaction and limit of detection was 101 target copies per reaction, though 100 target copies per reaction were intermittently detected. This qPCR assay provides a valuable diagnostic tool for characterization of STADV epidemiology, including potential identification of the North American reservoir host.

Evaluation of a modified quantitative polymerase chain reaction assay for genus Schistosoma detection using stool and urine samples from schistosomiasis endemic areas in Kenya

Evaluation of a modified quantitative polymerase chain reaction assay for genus Schistosoma detection using stool and urine samples from schistosomiasis endemic areas in Kenya

Homemade isothermal amplification-initiated Cas14a assay for rapid quantitative detection of aquatic RNA virus gene with no PAM

The red-spotted grouper nervous necrosis virus (RGNNV) is the most widely distributed nervous necrosis virus (NNV) and causes a significant risk to aquatic food safety. Though a few CRISPR-based aquatic pathogen detection methods have been reported recently, the absence of the PAM site of RGNNV specific gene RNA2 impedes its CRISPR-based diagnostic. Herein, we developed a rapid and accurate homemade isothermal amplification-initiated Cas14a assay (HiaCas14a) for RGNNV detection. The introduction of the PAM site into RT-LAMP specific amplicons of RGNNV RNA2 unlocked its PAM restriction. Furthermore, fish genomic RNA purified by magnetic beads and polymerases used in RT-LAMP prepared by ourselves obviously gave the system an enhanced convenience and a reduced cost. HiaCas14a detected 63.4 aM RNA with excellent specificity and sensitivity quantitatively, and it demonstrated a high accuracy of field sample validation results compared with RT-qPCR. The method provided an accurate, low-cost, and sensitive tool for aquatic pathogen detection in resource-poor areas.

A quantitative real-time PCR assay for rapid detection and quantification of Amyloodinium ocellatum parasites in seawater samples

Amyloodinium ocellatum is one of the most important ectoparasites of marine fish, causing amyloodiniosis and mass mortality in aquaculture. To date, no diagnostic method has been developed to detect and quantify parasite abundance in seawater. In this study, a quantitative real-time PCR (qPCR) assay using the ITS-F3 and ITS-R3 primer pair based on the internal transcribed spacers of nuclear ribosomal DNA (ITS rDNA) of A. ocellatum was successfully established for the detection and quantification of dinospores in seawater. The dinospores were collected by suction filtration of seawater using a vacuum pump, and examination by light microscopy and scanning electron microscopy (SEM) showed that the dinospores were adsorbed on the surface of the cellulose acetate membrane. The linear relationship between seawater parasite abundance and Ct values followed the model y = −3.0607 x + 29.919 with a coefficient of determination (R2) of 0.985. The results of the assay showed high reproducibility and no cross-reactivity with other aquatic pathogens. The assay successfully detected dinospores (100 % success rate) at concentrations of 8 and 10 cell per 300 mL of seawater. The protective fluid buffer effectively protected the dinospores DNA from degradation for over 96 h. The method was used to investigate the life span of dinospores in seawater and the incubation patterns of tomonts in the tank, the results showed that a small fraction of dinospores can survive for more than 7 days in seawater and the process of dinospores incubation from the tomonts persisted up to 84 h. This study provides a valuable quantitative diagnostic tool to assist in the early monitoring and understanding of disease progression in amyloodinosis.

Inter-laboratory comparison of eleven quantitative or digital PCR assays for detection of proviral bovine leukemia virus in blood samples

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis and causes a persistent infection that can leave cattle with no symptoms. Many countries have been able to successfully eradicate BLV through improved detection and management methods. However, with the increasing novel molecular detection methods there have been few efforts to standardize these results at global scale. This study aimed to determine the interlaboratory accuracy and agreement of 11 molecular tests in detecting BLV. Each qPCR/ddPCR method varied by target gene, primer design, DNA input and chemistries. DNA samples were extracted from blood of BLV-seropositive cattle and lyophilized to grant a better preservation during shipping to all participants around the globe. Twenty nine out of 44 samples were correctly identified by the 11 labs and all methods exhibited a diagnostic sensitivity between 74 and 100%. Agreement amongst different assays was linked to BLV copy numbers present in samples and the characteristics of each assay (i.e., BLV target sequence). Finally, the mean correlation value for all assays was within the range of strong correlation. This study highlights the importance of continuous need for standardization and harmonization amongst assays and the different participants. The results underscore the need of an international calibrator to estimate the efficiency (standard curve) of the different assays and improve quantitation accuracy. Additionally, this will inform future participants about the variability associated with emerging chemistries, methods, and technologies used to study BLV. Altogether, by improving tests performance worldwide it will positively aid in the eradication efforts.

Multiplex methylation detection assays using a blocking FRET probe with machine learning-assisted quantitative melting curve method targeting early-stage breast cancer

Breast cancer (BC) is the second most prevalent form of cancer, and poses a significant threat to public health. DNA methylation is an ideal marker for the early detection of BC. Fluorescence quantitative polymerase chain reaction (PCR)-based DNA methylation detection is simpler and faster but is constrained by its multiplexing capability and specificity. To address this, we developed a multiplex quantitative methylation PCR assay for the simultaneous analysis of methylation status at multiple sites specific to BC (cg11754974, cg13828440, cg18637238, and cg16652347). The machine learning model was trained using 1200 cases of multipeak data to enhance the melting curve resolution. Performance testing demonstrated the method’s ability to selectively amplify methylated genes at a DNA concentration of 1 × 105 copies μL−1, with high replicability (coefficient of variation <5 %) and mutation detection capabilities as low as 10 %. When applied to 80 clinical BC samples, the assay effectively distinguished patients with early-stage BC from normal controls, achieving an area under the curve of 0.8938, sensitivity of 83.33 %, and specificity of 84.62 %. Our essay exhibits superior clinical performance when compared to the quantitative methylation-specific PCR assays for noninvasive detection of early-stage BC, which is poised to become a favorable clinical diagnostic method for early-stage BC owing to its simplicity, speed, and capacity to improve diagnostic accuracy.

Establishment and performance analysis of a new multiplex detection method for influenza an and B virus antigen.

Influenza A and B virus detection is pivotal in epidemiological surveillance and disease management. Rapid and accurate diagnostic techniques are crucial for timely clinical intervention and outbreak prevention. Quantum dot-encoded microspheres have been widely used in immunodetection. The integration of quantum dot-encoded microspheres with flow cytometry is a well-established technique that enables rapid analysis. Thus, establishing a multiplex detection method for influenza A and B virus antigens based on flow cytometry quantum dot microspheres will help in disease diagnosis. To establish a codetection method of influenza A and B virus antigens based on flow cytometry quantum dot-encoded microsphere technology, which forms the foundation for the assays of multiple respiratory virus biomarkers. Different quantum dot-encoded microspheres were used to couple the monoclonal antibodies against influenza A and B. The known influenza A and B antigens were detected both separately and simultaneously on a flow cytometer, and the detection conditions were optimized to establish the influenza A and B antigen codetection method, which was utilized for their detection in clinical samples. The results were compared with the fluorescence quantitative polymerase chain reaction (PCR) method to validate the clinical performance of this method. The limits of detection of this method were 26.1 and 10.7 pg/mL for influenza A and B antigens, respectively, which both ranged from 15.6 to 250000 pg/mL. In the clinical sample evaluation, the proposed method well correlated with the fluorescent quantitative PCR method, with positive, negative, and overall compliance rates of 57.4%, 100%, and 71.6%, respectively. A multiplex assay for quantitative detection of influenza A and B virus antigens has been established, which is characterized by high sensitivity, good specificity, and a wide detection range and is promising for clinical applications.

Hydrogen peroxide positively regulates ABA signaling via oxidative modification of the C2H2-type zinc finger protein ZFP36 in rice

The rice zinc finger protein ZFP36 serves as a pivotal regulator of the hydrogen peroxide (H2O2) signaling pathway in response to abscisic acid (ABA). Its role is crucial for integrating H2O2 signals with the plant defense mechanisms against water deficit and oxidative stress. However, it remains unclear whether ZFP36 directly modulates ABA-induced H2O2 signaling. This study explored the effects of oxidative post-translational modifications (OxiPTMs) on ZFP36 in rice, with an emphasis on the H2O2-induced oxidation through its cysteine (Cys) residues. We found that ZFP36 undergoes oxidative modification as a target of H2O2 in the presence of ABA, specifically at Cys32. Employing quantitative detection and fluorescence assays, we observed that ZFP36 oxidation enhances the expression and activity of genes encoding protective antioxidant enzymes. Moreover, our investigation into the thioredoxin (Trx) and glutaredoxin (Grx) families revealed that OsTrxh1 facilitates the reduction of oxidized ZFP36. Genetic evidence indicates that ZFP36 positively influences rice resilience to oxidative and water stress, while OsTrxh1 exerts an opposing effect. These insights reveal a distinctive pathway for plant cells to perceive ABA-induced H2O2 signaling, advance our comprehension of H2O2 signaling dynamics, and ABA-related plant responses, and lay a vital groundwork for enhancing crop stress tolerance.

Novel strategy to quantify the viability of oocysts of Cryptosporidium parvum and C. hominis, a risk factor of the waterborne protozoan pathogens of public health concern

While waters might be contaminated by oocysts from >40 Cryptosporidium species, only viable oocysts of C. parvum and C. hominis truly pose the main health risk to the immunocompetent population. Oocyst viability is also an important but often neglected risk factor in monitoring waterborne parasites. However, commonly used methods in water monitoring and surveys cannot distinguish species (microscopic observation) or oocyst viability (PCR), as dead oocysts in water could retain gross structure and DNA content for weeks to months. Here, we report new TaqMan qRT-PCR/qPCR assays for quantitative detection of viable C. parvum and C. hominis oocysts. By targeting a hypothetical protein-encoding gene cgd6_3920 that is highly expressed in oocysts and variable between species, the qRT-PCR/qPCR assays achieve excellent analytical specificity and sensitivity (limit of quantification [LOQ] = 0.25 and 1.0 oocyst/reaction). Using calibration curves, the number and ratio of viable oocysts in specimens could be calculated. Additionally, we also establish a TaqMan-18S qPCR for cost-effective screening of pan-Cryptosporidium-positive specimens (LOQ = 0.1 oocyst/reaction). The assay feasibility is validated using field water (N = 43) and soil (79) specimens from 17 locations in Changchun, China, which detects four Cryptosporidium species from seven locations, including three gp60-subtypes (i.e., IIdA19G1, IIdA17G1 and IIdA24G2) of C. parvum oocysts showing varied viability ratios. These new TaqMan q(RT)-PCR assays supplement current methods in the survey of waters and other samples (e.g., surfaces, foods and beverages), and are applicable to assessing the efficiency of oocyst deactivation protocols.

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.

Development and evaluation of sandwich ELISA for detection and quantification of staphylococcal enterotoxin‐A in food

AbstractStaphylococcus aureus is an opportunistic zoonotic pathogen which secretes 24 different types of enterotoxins (SEs) and enterotoxin‐like (SEls) proteins. Classical enterotoxins (SEA–SEE) are responsible for &gt;95% of food poisoning outbreaks, of which SEA alone is responsible for &gt;75% of them. This study was undertaken to develop a sandwich enzyme‐linked immunosorbent assay (ELISA) for sensitive, specific, and quantitative detection of staphylococcal enterotoxins‐A in food. Optimization of sandwich ELISA was attempted in two ways: rabbit polyclonal anti‐SEA as a capture antibody and mouse monoclonal anti‐SEA as a detector antibody, and vice versa. In the optimization of sandwich ELISA, mouse monoclonal anti‐SEA as a capture antibody and rabbit polyclonal anti‐SEA as a detector antibody yielded the highest sensitivity of 0.5–0.75 ng mL−1. The developed assay was found to be highly specific and exhibited equivalent sensitivity to a commercial kit. The developed sandwich ELISA may be utilized for the detection of staphylococcal enterotoxin‐A in food as a cheap alternative to available commercial kits. The developed sandwich ELISA may be useful for microbiological quality assurance of foods, especially in resource‐limited developing countries.

Quantitative immunochromatographic assay for rapid and cost-effective on-site detection of benzo[a]pyrene in oilfield chemicals

Contamination of oilfield chemicals (OFCs) by benzo[a]pyrene (B[a]P) is increasingly becoming a severe environmental security issue. There is an urgent need to develop a rapid and accurate method for B[a]P detection in OFCs. In this study, B[a]P hapten was designed using computer aided molecular design. A high-affinity, specific, and matrix-insensitive monoclonal antibody (mAb) with IC50 values of 6.77 ng/mL was obtained. Based on this mAb, we developed a rapid gold nanoparticle-based immunochromatographic strip assay (GICA) with double T-line mode for on-site detection of B[a]P in OFCs samples. The GICA exhibited excellent detection performance in OFCs samples with strong acidity, strong alkalinity, and deep color. Under optimal conditions, the proposed method detected B[a]P in OFCs at 0.42–300 mg/kg, and limit of detection was 0.23–1.07 mg/kg. The recovery rate was 88–106% with a coefficient of variation of 1.46–6.35%. Confirmed by natural positive OFCs samples and high-performance liquid chromatography, this GICA is accurate and reliable, with great potential for rapid and cost-effective on-site detection.

Development of real-time PCR assay for quantitative detection of Clostridium septicum

Cellulitis is an important disease in commercial turkey farms associated with significant economic loss. Although the etiology of cellulitis is not fully elucidated, Clostridium septicum (C. septicum) is one of the main causes of this infectious disease. In this study, we report the development of a quantitative real-time PCR (qRT PCR) assay targeting the alpha-toxin gene (csa), which involves a prior 15-cyle PCR using a nested pair of primers to increase the detection sensitivity. Additionally, the TaqMan probe was employed to increase the target-specificity of the assay. The performance of our nested qRT-PCR assay was evaluated using Clostridium isolates from turkey farms, representing both septicum and non-septicum species, as well as sponge swab samples from turkey farms. Our step-by-step development of the assay showed that the csa gene is a suitable target for specific detection of C. septicum strains and that the inclusion of nested PCR step significantly increased the detection sensitivity of the final qRT PCR assay. The performance of the assay was also validated by a high correlation of the threshold cycle numbers of the qRT PCR assay with the relative abundance of C. septicum read counts in 16S rRNA gene microbiota profiles of the C. septicum-containing samples from turkey farms.