Contamination of Zearalenone from China in 2019 by a Visual and Digitized Immunochromatographic Assay

Zearalenone (ZEN) is a prevalent mycotoxin that needs intensive monitoring. A semi-quantitative and quantitative immunochromatographic assay (ICA) was assembled for investigating ZEN contamination in 187 samples of cereal and their products from China in 2019. The semi-quantitative detection model had a limit of detection (LOD) of 0.50 ng/mL with visual judgment and could be completely inhibited within 5 min at 3.0 ng/mL ZEN. The quantitative detection model had a lower LOD of 0.25 ng/mL, and ZEN could be accurately and digitally detected from 0.25–4.0 ng/mL. The ICA method had a high sensitivity, specificity, and accuracy for on-site ZEN detection. For investigation of the authentic samples, the ZEN-positive rate was 62.6%, and the ZEN-positive levels ranged from 2.7 to 867.0 ng/g, with an average ZEN-positive level being 85.0 ng/g. Of the ZEN-positive samples, 6.0% exceeded the values of the limit levels. The ZEN-positive samples were confirmed to be highly correlated using LC-MS/MS (R2 = 0.9794). This study could provide an efficiency and accuracy approach for ZEN in order to achieve visual and digitized on-site investigation. This significant information about the ZEN contamination levels might contribute to monitoring mycotoxin occurrence and for ensuring food safety.

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Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading

Toxins ◽

10.3390/toxins13110781 ◽

2021 ◽

Vol 13 (11) ◽

pp. 781

Author(s):

Zhuolin Song ◽

Lin Feng ◽

Yuankui Leng ◽

Mingzhu Huang ◽

Hao Fang ◽

...

Keyword(s):

Ochratoxin A ◽

Limit Of Detection ◽

High Sensitivity ◽

Cross Reaction ◽

Molar Ratio ◽

Quantitative Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Enzyme Loading ◽

M13 Bacteriophage ◽

Mimic Peptide

Enzyme-linked immunosorbent assay (ELISA) is widely used in the routine screening of mycotoxin contamination in various agricultural and food products. Herein, a cascade-amplifying system was introduced to dramatically promote the sensitivity of an immunoassay for ochratoxin A (OTA) detection. Specifically, a biotinylated M13 bacteriophage was introduced as a biofunctional competing antigen, in which a seven-peptide OTA mimotope fused on the p3 protein of M13 was used to specifically recognize an anti-OTA monoclonal antibody, and the biotin molecules modified on capsid p8 proteins were used in loading numerous streptavidin-labeled polymeric horseradish peroxidases (HRPs). Owing to the abundance of biotinylated p8 proteins in M13 and the high molar ratio between HRP and streptavidin in streptavidin-polyHRP, the loading amount of HRP enzymes on the M13 bacteriophage were greatly boosted. Hence, the proposed method exhibited high sensitivity, with a limit of detection of 2.0 pg/mL for OTA detection, which was 250-fold lower than that of conventional ELISA. In addition, the proposed method showed a slight cross-reaction of 2.3% to OTB, a negligible cross-reaction for other common mycotoxins, and an acceptable accuracy for OTA quantitative detection in real corn samples. The practicability of the method was further confirmed with a traditional HRP-based ELISA method. In conclusion, the biotinylated bacteriophage and polyHRP structure showed potential as a cascade-amplifying enzyme loading system for ultra-trace OTA detemination, and its application can be extended to the detection of other analytes by altering specific mimic peptide sequences.

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Fluorescence Polarization Immunoassay for Determination of Enrofloxacin in Pork Liver and Chicken

Molecules ◽

10.3390/molecules24244462 ◽

2019 ◽

Vol 24 (24) ◽

pp. 4462

Author(s):

Xing Shen ◽

Jiahong Chen ◽

Shuwei Lv ◽

Xiulan Sun ◽

Boris B. Dzantiev ◽

...

Keyword(s):

Fluorescence Polarization ◽

Limit Of Detection ◽

Polyclonal Antibodies ◽

Screening Method ◽

Sample Pretreatment ◽

High Sensitivity ◽

Cross Reactivity ◽

Fluorescence Polarization Immunoassay ◽

Pork Liver ◽

Sensitivity Specificity

Enrofloxacin (ENR) is a widely used fluoroquinolone (FQ) antibiotic for antibacterial treatment of edible animal. In this study, a rapid and highly specific fluorescence polarization immunoassay (FPIA) was developed for monitoring ENR residues in animal foods. First, ENR was covalently coupled to bovine serum albumin (BSA) to produce specific polyclonal antibodies (pAbs). Three fluorescein-labeled ENR tracers (A, B, and C) with different spacers were synthesized and compared to obtain higher sensitivity. Tracer C with the longest arm showed the best sensitivity among the three tracers. The developed FPIA method showed an IC50 (50% inhibitory concentration) of 21.49 ng·mL−1 with a dynamic working range (IC20–IC80) of 4.30–107.46 ng·mL−1 and a limit of detection (LOD, IC10) of 1.68 ng·mL−1. The cross-reactivity (CR) of several structurally related compounds was less than 2%. The recoveries of spiked pork liver and chicken samples varied from 91.3% to 112.9%, and the average coefficients of variation were less than 3.83% and 5.13%, respectively. The immunoassay took only 8 min excluding sample pretreatment. This indicated that the established method had high sensitivity, specificity, and the advantages of simplicity. Therefore, the proposed FPIA provided a useful screening method for the rapid detection of ENR residues in pork liver and chicken.

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Validation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium parasites

PLoS ONE ◽

10.1371/journal.pone.0252887 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0252887

Author(s):

Renate Schneider ◽

Aline Lamien-Meda ◽

Herbert Auer ◽

Ursula Wiedermann-Schmidt ◽

Peter L. Chiodini ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Melting Curve ◽

Resonance Energy ◽

High Sensitivity ◽

Significant Rise ◽

Quantitative Detection ◽

Melting Curve Analysis ◽

Nucleotide Polymorphisms

Increasing numbers of travelers returning from endemic areas, migrants, and refugees have led to a significant rise in the number of imported malaria cases in non-endemic countries. Real- time PCR serves as an excellent diagnostic tool, especially in regions where experience in microscopy is limited. A novel fluorescence resonance energy transfer-based real-time PCR (FRET-qPCR) was developed and evaluated using 56 reference samples of the United Kingdom National External Quality Assessment Service (UK NEQAS) for molecular detection of malaria, including P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Species identification is based on single nucleotide polymorphisms (SNPs) within the genome where the MalLC640 probe binds, lowering the melting temperature in the melting curve analysis. The novel FRET-qPCR achieved 100% (n = 56) correct results, compared to 96.43% performing nested PCR. The high sensitivity, with a calculated limit of detection of 199.97 parasites/mL blood for P. falciparum, is a significant advantage, especially if low-level parasitemia has to be ruled out. Even mixed infections of P. falciparum with P. vivax or P. ovale, respectively, were detected. In contrast to many other real-time PCR protocols, this novel FRET-qPCR allows the quantitative and species-specific detection of Plasmodium spp. in one single run. Solely, P. knowlesi was detected but could not be differentiated from P. vivax. The turnaround time of this novel FRET-qPCR including DNA extraction is less than two hours, qualifying it for routine clinical applications, including treatment monitoring.

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Detection of EGFR deletion using unique RepSeq technology

10.1101/584045 ◽

2019 ◽

Author(s):

Thuraiayah Vinayagamoorthy ◽

Dahui Qin ◽

Fei Ye ◽

Minghao Zhong

Keyword(s):

Internal Control ◽

Sanger Sequencing ◽

Limit Of Detection ◽

High Sensitivity ◽

Wild Type ◽

Sequencing Technology ◽

Sequencing Method ◽

Exon 19 Deletion ◽

Sensitivity Specificity ◽

Generation Sequencing

AbstractWe are reporting a novel sequencing technology, RepSeq (Repetitive Sequence), that has high sensitivity, specificity and quick turn-around time. This new sequencing technology is developed by modifying traditional Sanger sequencing technology in several aspects. The first, a homopolymer tail is added to the PCR primer(s), which makes interpreting electropherograms a lot easier than that in traditional Sanger sequencing. The second, an indicator nucleotide is added at the 5’end of the homopolymer tail. In the presence of a deletion, the position of the indicator nucleotide in relation to the wild type confirms the deletion. At the same time, the indicator of the wild type serves as the internal control. Furthermore, the specific design of the PCR and/or sequencing primers will specifically enrich/select mutant alleles, which increases sensitivity and specificity significantly. Based on serial dilution studies, the analytical lower limit of detection was 1.47 copies. A total of 89 samples were tested for EGFR exon 19 deletion, of which 21 were normal blood samples and 68 were samples previously tested by either pyrosequencing or TruSeq Next Generation Sequencing Cancer Panel. There was 100 % concordance among all the samples tested. RepSeq technology has overcome the shortcomings of Sanger sequencing and offers an easy-to-use novel sequencing method for personalized precision medicine.

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A FRET-ICT Dual-Modulated Ratiometric Fluorescence Sensor for Monitoring and Bio-Imaging of Cellular Selenocysteine

Molecules ◽

10.3390/molecules25214999 ◽

2020 ◽

Vol 25 (21) ◽

pp. 4999

Author(s):

Zongcheng Wang ◽

Chenhong Hao ◽

Xiaofang Luo ◽

Qiyao Wu ◽

Chengliang Zhang ◽

...

Keyword(s):

Real Time ◽

Limit Of Detection ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

High Sensitivity ◽

Fluorescence Sensor ◽

Quantitative Detection ◽

Fluorescence Signal ◽

Ratiometric Fluorescence ◽

Sensitivity And Selectivity

Since the fluctuation of cellular selenocysteine (Sec) concentration plays an all-important role in the development of numerous human disorders, the real-time fluorescence detection of Sec in living systems has attracted plenty of interest during the past decade. In order to obtain a faster and more sensitive small organic molecule fluorescence sensor for the Sec detection, a new ratiometric fluorescence sensor Q7 was designed based on the fluorescence resonance energy transfer (FRET) strategy with coumarin fluorophore as energy donor and 4-hydroxy naphthalimide fluorophore (with 2,4-dinitrobenzene sulfonate as fluorescence signal quencher and Sec-selective recognition site) as an energy acceptor. The sensor Q7 exhibited only a blue fluorescence signal, and displayed two well distinguished emission bands (blue and green) in the presence of Sec with ∆λ of 68 nm. Moreover, concentrations ranging of quantitative detection of Sec of Q7 was from 0 to 45 μM (limit of detection = 6.9 nM), with rapid ratiometric response, high sensitivity and selectivity capability. Impressively, the results of the living cell imaging test demonstrated Q7 has the potentiality of being an ideal sensor for real-time Sec detection in biosystems.

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Combination of 16S rRNA and procalcitonin in diagnosis of neonatal clinically suspected sepsis

Journal of International Medical Research ◽

10.1177/0300060519892418 ◽

2019 ◽

Vol 48 (3) ◽

pp. 030006051989241

Author(s):

Renqiang Yu ◽

Qin Zhou ◽

Shanyu Jiang ◽

Yingzi Mei ◽

Min Wang

Keyword(s):

16S Rrna ◽

Sensitivity And Specificity ◽

Bacterial Culture ◽

High Sensitivity ◽

16S Rrna Sequencing ◽

Detection Methods ◽

Suspected Sepsis ◽

Rrna Sequencing ◽

Positive Rate ◽

Sensitivity Specificity

Objective To investigate the application of 16S rRNA in diagnosing patients with neonatal sepsis. Methods We studied 60 consecutive neonatal patients with clinically suspected sepsis and 20 non-infective cases as controls. All patients were diagnosed with sepsis by clinical and experimental criteria. Clinical characteristics were recorded and 16S rRNA sequencing was conducted for all patients. The sensitivity, specificity, and accuracy of the detection methods were analyzed. Results The detection limit of 16S rRNA sequencing was 1 × 102 CFU/mL. For suspected sepsis, the positive rate of 16S rRNA detection was 93.3%, which was similar to that of procalcitonin detection (85%), and was significantly higher than that of bacterial culture (51.7%). The specificity of procalcitonin detection (74.1%) was significantly lower than that of 16S rRNA detection (100%). Moreover, the combination of 16S rRNA and procalcitonin detection showed a sensitivity of 100%, specificity of 74.1%, and accuracy of 92.0%. For proven sepsis, the sensitivity and specificity of 16S rRNA detection were both 100.0%, and those for procalcitonin were 87.1% and 87.0%, respectively. Conclusion Detection of 16S rRNA has high sensitivity and specificity in diagnosing sepsis. The combination of 16S rRNA and procalcitonin has even better sensitivity with acceptable specificity.

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Sample Preincubation Strategy for Sensitive and Quantitative Detection of Clenbuterol in Swine Urine Using a Fluorescent Microsphere–Based Immunochromatographic Assay

Journal of Food Protection ◽

10.4315/0362-028x.jfp-14-086 ◽

2014 ◽

Vol 77 (11) ◽

pp. 1998-2003 ◽

Cited By ~ 18

Author(s):

SHENG L. DENG ◽

SHAN SHAN ◽

CHAO L. XU ◽

DAO F. LIU ◽

YONG H. XIONG ◽

...

Keyword(s):

Limit Of Detection ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Rapid Screening ◽

Immunochromatographic Assay ◽

Quantitative Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Fluorescent Microsphere ◽

Swine Urine ◽

Relative Standard

We describe an ultrasensitive and quantitative immunochromatographic assay to determine the amount of clenbuterol (CLB) in swine urine. In this study, fluorescein isothiocyanate polystyrene fluorescent microspheres were used as probes. A sample preincubation strategy was introduced to this immunochromatographic assay. Results showed that the strategy evidently improved the sensitivity and accuracy of lateral flow assay. The method was completed in 20 min, and a half-maximal inhibitory concentration of 0.13 μg liter−1 was obtained. The limit of detection of the proposed method to determine CLB in swine urine was 0.01 μg liter−1, which was lower than the limit of detection of immunochromatographic assays without preincubation. Intra-and interday recoveries of spiked swine urine ranged from 85.0 to 107.5%. The relative standard deviation values of the preincubated test strip ranged from 2.7 to 12.5%. Analysis of the CLB in swine urine samples showed that the result obtained from the lateral flow assay is consistent with that obtained from a commercial enzyme-linked immunosorbent assay kit. Our results suggest that the developed fluorescent microsphere–based immunochromatographic assay may be useful as a rapid screening method to detect CLB quantitatively.

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Polystyrene Microsphere-Based Immunochromatographic Assay for Detection of Aflatoxin B1 in Maize

Biosensors ◽

10.3390/bios11060200 ◽

2021 ◽

Vol 11 (6) ◽

pp. 200

Author(s):

Jin Wang ◽

Xiangmei Li ◽

Xing Shen ◽

Ang Zhang ◽

Jinxiu Liu ◽

...

Keyword(s):

Aflatoxin B1 ◽

Limit Of Detection ◽

Point Of Care ◽

Immunochromatographic Assay ◽

Quantitative Detection ◽

Point Of Care Testing ◽

Polystyrene Microsphere ◽

Accuracy And Precision ◽

Wide Range ◽

Liquid Chromatography Tandem Mass

Aflatoxin B1 (AFB1), a mycotoxin, is hepatotoxic, carcinogenic, and nephrotoxic in humans and animals, and contaminate a wide range of maize. In this study, an immunochromatographic assay (ICA) based on polystyrene microspheres (PMs) was developed for sensitive and quantitative detection of AFB1 in maize. The amounts of PMs, the condition for activating carboxyl groups of PMs, the amount of monoclonal antibody (mAb), and the volume of the immune probe were optimized to enhance the performance PMs-ICA for point-of-care testing of AFB1 in maize. The PMs-ICA showed the cut-off value of 1 ng/mL in phosphate buffer (PB) and 6 µg/kg in maize samples, respectively. The quantitative limit of detection (qLOD) was 0.27 and 1.43 µg/kg in PB and maize samples, respectively. The accuracy and precision of the PMs-ICA were evaluated by analysis of spiked maize samples with recoveries of 96.0% to 107.6% with coefficients of variation below 10%. In addition, the reliability of PMs-ICA was confirmed by the liquid chromatography-tandem mass spectrometry method. The results indicated that the PMs-ICA could be used as a sensitive, simple, rapid point-of-care testing of AFB1 in maize.

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Colorimetric detection of organophosphates with cysteamine capped gold nanoparticle sensors

10.3762/bxiv.2020.137.v1 ◽

2020 ◽

Author(s):

Muhammad Musaddiq Shah ◽

Wen Ren ◽

Bashir Ahmad ◽

Joseph Irudayaraj

Keyword(s):

Gold Nanoparticle ◽

Limit Of Detection ◽

Colorimetric Detection ◽

High Sensitivity ◽

Food Samples ◽

Quantitative Detection ◽

Enzyme Mimics ◽

Nanoparticle Probes ◽

Site Monitoring ◽

20 Nm

Nanozyme biosensors have the potential to provide high sensitivity, multiple functionality, and tunable activity. A facile colorimetric biosensor for the detection of organophosphates (OPs) using cysteamine capped gold nanoparticle probes (C-AuNPs) as enzyme mimics is proposed. Parathion ethyl (PE) a class of OPs is a potent insecticide that functions by inhibiting the acetylcholinesterase (AChE) in the nervous system of insects. The inhibition kinetics of AChE using PE enables the development of a PE sensor. C-AuNPs possess the ability to catalyze the oxidization of 3, 3’, 5, 5’-tetramethylbenzidine (TMB) to a blue-colored product without peroxidase. The detection of PE was monitored by the inability of AChE to generate choline. Choline causes the aggregation of C-AuNPs and the aggregated C-AuNPs has decreased ability to catalyze the oxidization of TMB. A calibration was developed in the 40-320 nM range for the quantitative detection of PE. The limit of detection observed was 20 nM and the method had excellent specificity. The proposed sensor provides an excellent platform for on-site monitoring of PE in environmental and food samples with high sensitivity and greater selectivity.

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Broad-range and Effective Detection of Human Noroviruses by Colloidal Gold Immunochromatographic Assay Based on the Shell Domain of the Major Capsid Protei

10.21203/rs.3.rs-55525/v1 ◽

2020 ◽

Author(s):

Meng Xu ◽

Feifeng Lu ◽

Qingping Wu ◽

Jumei Zhang ◽

Peng Tian ◽

...

Keyword(s):

Colloidal Gold ◽

High Sensitivity ◽

Relative Sensitivity ◽

Immunochromatographic Assay ◽

Clinical Samples ◽

Special Equipment ◽

Human Noroviruses ◽

Infected People ◽

Sensitivity Specificity

Abstract Background: Human noroviruses (HuNoVs) are a major cause of nonbacterial gastroenteritis in all ages worldwide. As the replication of HuNoVs in vitro is immature, the detection of HuNoVs is depended on molecular assays such as RT-PCR and RT-qPCR. However, these molecular-based techniques require special equipment, unique reagents, experienced operators to perform, and extended time to get results. In addition, the genotypes of HuNoVs are broad. Therefore, a method for rapidly, broad-range, and effective approach for HuNoVs would be more applicable for screening infected people when outbreaks occur.Results: In this study, a colloidal-gold-based immunochromatographic assay (ICA) was developed for highly effective detection of HuNoVs in clinical samples. Monoclonal antibodies (McAbs) against the shell (S) domain in the major capsid protein of HuNoV were used in the ICA. The limitations of detection for HuNoVs in clinical samples were 1.2×106 genomic copies per gram (gc/g) and 4.4×105 gc/g for genogroup I and II (GI and GII) HuNoVs, respectively. A total of 122 samples were tested for HuNoVs by ICA and compared against that by RT-qPCR. The relative sensitivity, specificity and agreement of the ICA was 84.2 % (95% CI: 83.6-84.8 %), 100.0 % (95 % CI: 98.5-100.0 %) and 87.7 % (95% CI: 85.6-89.8 %), respectively. No cross-reaction with other common enteric viruses or bacteria was observed. The ICA could detect a broad range of genotypes, including GI.1, GI.3, GI.4, GI.6, GI.14, GII.2, GII.3, GII.4, GII.6, GII.13, and GII.17 HuNoVs.Conclusions: Our results demonstrated that ICA targeting the S domain protein is a promising candidate for effectively improve identifying different genotypes of HuNoVs in clinical samples with high sensitivity and specificity.

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