Quantitative and Sensitive Detection of Chloramphenicol by Surface-Enhanced Raman Scattering

PubMed Central

Ding, Yufeng; Yin, Hongjun; Meng, Qingyun; Zhao, Yongmei; Liu, Luo; Wu, Zhenglong; Xu, Haijun

2017-01-01

We used surface-enhanced Raman scattering (SERS) for the quantitative and sensitive detection of chloramphenicol (CAP). Using 30 nm colloidal Au nanoparticles (NPs), a low detection limit for CAP of 10−8 M was obtained. The characteristic Raman peak of CAP centered at 1344 cm−1 was used for the rapid quantitative detection of CAP in three different types of CAP eye drops, and the accuracy of the measurement result was verified by high-performance liquid chromatography (HPLC). The experimental results reveal that the SERS technique based on colloidal Au NPs is accurate and sensitive, and can be used for the rapid detection of various antibiotics. PMID:29261161

Loop-mediated isothermal amplification assay for rapid and sensitive detection of sheep pox and goat pox viruses in clinical samples.

PubMed

Venkatesan, G; Balamurugan, V; Bhanuprakash, V; Singh, R K; Pandey, A B

2016-06-01

A Loop-mediated isothermal amplification (LAMP) assay targeting the highly conserved DNA polymerase gene of capripox virus genome was developed and evaluated for rapid detection of sheep pox and goat pox viruses. The optimized LAMP assay is found specific and sensitive for amplification of target DNA with a diagnostic sensitivity and specificity of 96.6% and 100% respectively compared to quantitative PCR. The detection rate of LAMP, PCR and Q-PCR assays is found to be 81.5%, 67% and 83% respectively. This LAMP assay has the potential for rapid clinical diagnosis and surveillance of sheep pox and goat pox in field diagnostic laboratories. Copyright © 2016 Elsevier Ltd. All rights reserved.

Field Demonstration of a Multiplexed Point-of-Care Diagnostic Platform for Plant Pathogens.

PubMed

Lau, Han Yih; Wang, Yuling; Wee, Eugene J H; Botella, Jose R; Trau, Matt

2016-08-16

Effective disease management strategies to prevent catastrophic crop losses require rapid, sensitive, and multiplexed detection methods for timely decision making. To address this need, a rapid, highly specific and sensitive point-of-care method for multiplex detection of plant pathogens was developed by taking advantage of surface-enhanced Raman scattering (SERS) labeled nanotags and recombinase polymerase amplification (RPA), which is a rapid isothermal amplification method with high specificity. In this study, three agriculturally important plant pathogens (Botrytis cinerea, Pseudomonas syringae, and Fusarium oxysporum) were used to demonstrate potential translation into the field. The RPA-SERS method was faster, more sensitive than polymerase chain reaction, and could detect as little as 2 copies of B. cinerea DNA. Furthermore, multiplex detection of the three pathogens was demonstrated for complex systems such as the Arabidopsis thaliana plant and commercial tomato crops. To demonstrate the potential for on-site field applications, a rapid single-tube RPA/SERS assay was further developed and successfully performed for a specific target outside of a laboratory setting.

Study and evaluation of Wondfo rapid diagnostic kit based on nano-gold immunochromatography assay for diagnosis of Plasmodium falciparum.

PubMed

Peng, Yunping; Wu, Junlin; Wang, Jihua; Li, Wenmei; Yu, Shujuan

2012-04-01

Malaria has been recognized as a human disease for thousands of years and remains one of the most common diseases affecting humans worldwide. Therefore, a method for rapidly detecting Plasmodium falciparum is necessary and useful. We have developed Wondfo rapid diagnostic kit based on nano-gold immunochromatography assay for the detection of P. falciparum in patient specimen. In the present study, we demonstrated the sensitivity and specificity of the rapid diagnostic kit in which nano-gold labeling techniques and the monoclonal antibodies against histidine-rich protein-2 of P. falciparum were used to establish two-antibody sandwich immunochromatographic assay for detecting P. falciparum. By using microscopic examination of blood smears as control, the sensitivity, specificity, and feasibility of Wondfo rapid diagnostic kit was determined in the prompt and accurate diagnosis of malaria. In this study, 1,558 blood samples were collected from outpatient clinics in China and detected by both Wondfo kit and microscopic examination. The Wondfo kit did not show cross-reaction with microfilaria, Toxoplasma gondii, and other parasites in the blood. The patient samples positive for rheumatoid factor, HIV, tuberculosis, and syphilis did not show false positivity when testing with Wondfo kit. The detection sensitivity and specificity of Wondfo rapid diagnostic kit were 95.49% and 99.53%, respectively. These results indicate that our rapid diagnostic assay may be useful for detecting P. falciparum in patient specimen.

Nucleophile sensitivity of Drosophila TRPA1 underlies light-induced feeding deterrence

PubMed Central

Du, Eun Jo; Ahn, Tae Jung; Wen, Xianlan; Seo, Dae-Won; Na, Duk L; Kwon, Jae Young; Choi, Myunghwan; Kim, Hyung-Wook; Cho, Hana; Kang, KyeongJin

2016-01-01

Solar irradiation including ultraviolet (UV) light causes tissue damage by generating reactive free radicals that can be electrophilic or nucleophilic due to unpaired electrons. Little is known about how free radicals induced by natural sunlight are rapidly detected and avoided by animals. We discover that Drosophila Transient Receptor Potential Ankyrin 1 (TRPA1), previously known only as an electrophile receptor, sensitively detects photochemically active sunlight through nucleophile sensitivity. Rapid light-dependent feeding deterrence in Drosophila was mediated only by the TRPA1(A) isoform, despite the TRPA1(A) and TRPA1(B) isoforms having similar electrophile sensitivities. Such isoform dependence re-emerges in the detection of structurally varied nucleophilic compounds and nucleophilicity-accompanying hydrogen peroxide (H2O2). Furthermore, these isoform-dependent mechanisms require a common set of TRPA1(A)-specific residues dispensable for electrophile detection. Collectively, TRPA1(A) rapidly responds to natural sunlight intensities through its nucleophile sensitivity as a receptor of photochemically generated radicals, leading to an acute light-induced behavioral shift in Drosophila. DOI: http://dx.doi.org/10.7554/eLife.18425.001 PMID:27656903

A simple, rapid, cost-effective and sensitive method for detection of Salmonella in environmental and pecan samples.

PubMed

Dobhal, S; Zhang, G; Rohla, C; Smith, M W; Ma, L M

2014-10-01

PCR is widely used in the routine detection of foodborne human pathogens; however, challenges remain in overcoming PCR inhibitors present in some sample matrices. The objective of this study was to develop a simple, sensitive, cost-effective and rapid method for processing large numbers of environmental and pecan samples for Salmonella detection. This study was also aimed at validation of a new protocol for the detection of Salmonella from in-shell pecans. Different DNA template preparation methods, including direct boiling, prespin, multiple washing and commercial DNA extraction kits, were evaluated with pure cultures of Salmonella Typhimurium and with enriched soil, cattle feces and in-shell pecan each spiked individually with Salmonella Typhimurium. PCR detection of Salmonella was conducted using invA and 16S rRNA gene (internal amplification control) specific primers. The effect of amplification facilitators, including bovine serum albumin (BSA), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG) and gelatin on PCR sensitivity, was also evaluated. Conducting a prespin of sample matrices in combination with the addition of 0·4% (w/v) BSA and 1% (w/v) PVP in PCR mix was the simplest, most rapid, cost-effective and sensitive method for PCR detection of Salmonella, with up to 40 CFU Salmonella per reaction detectable in the presence of over 10(9 ) CFU ml(-1) of background micro-organisms from enriched feces soil or pecan samples. The developed method is rapid, cost-effective and sensitive for detection of Salmonella from different matrices. This study provides a method with broad applicability for PCR detection of Salmonella in complex sample matrices. This method has a potential for its application in different research arenas and diagnostic laboratories. © 2014 The Society for Applied Microbiology.

Rapid detection of bacteria in foods and biological fluids

NASA Technical Reports Server (NTRS)

Fealey, R. D.; Renner, W.

1973-01-01

Simple and inexpensive apparatus, called "redox monitoring cell," rapidly detects presence of bacteria. Bacteria is detected by measuring drop in oxygen content in test solution. Apparatus consists of vial with two specially designed electrodes connected to sensitive voltmeter.

Rapid and sensitive detection of mink circovirus by recombinase polymerase amplification.

PubMed

Ge, Junwei; Shi, Yunjia; Cui, Xingyang; Gu, Shanshan; Zhao, Lili; Chen, Hongyan

2018-06-01

To date, the pathogenic role of mink circovirus (MiCV) remains unclear, and its prevalence and economic importance are unknown. Therefore, a rapid and sensitive molecular diagnosis is necessary for disease management and epidemiological surveillance. However, only PCR methods can identify MiCV infection at present. In this study, we developed a nested PCR and established a novel recombinase polymerase amplification (RPA) assay for MiCV detection. Sensitivity analysis showed that the detection limit of nested PCR and RPA assay was 10 1 copies/reaction, and these methods were more sensitive than conventional PCR, which has a detection limit of 10 5 copies/reaction. The RPA assay had no cross-reactivity with other related viral pathogens, and amplification was completed in less than 20 min with a simple device. Further assessment of clinical samples showed that the two assays were accurate in identifying positive and negative conventional PCR samples. The detection rate of MiCV by the RPA assay in clinical samples was 38.09%, which was 97% consistent with that by the nested PCR. The developed nested PCR is a highly sensitive tool for practical use, and the RPA assay is a simple, sensitive, and potential alternative method for rapid and accurate MiCV diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

[Comparison of detection sensitivity in rapid-diagnosis influenza virus kits].

PubMed

Tokuno, Osamu; Fujiwara, Miki; Nakajoh, Yoshimi; Yamanouchi, Sumika; Adachi, Masayo; Ikeda, Akiko; Kitayama, Shigeo; Takahashi, Toshio; Kase, Tetsuo; Kinoshita, Shouhiro; Kumagai, Shunichi

2009-09-01

Rapid-diagnosis kits able to detect influenza A and B virus by immunochromatography developed by different manufacturers, while useful in early diagnosis, may vary widely in detection sensitivity. We compared sensitivity results for eight virus-detection kits in current use--Quick Chaser FluA, B (Mizuho Medy), Espline Influenza A & B-N (Fujirebio), Capilia Flu A + B (Nippon Beckton Dickinson & Alfesa Pharma), Poctem Influenza A/B (Otsuka Pharma & Sysmex), BD Flu Examan (Nippon Beckton Dickinson), Quick Ex-Flu "Seiken" (Denka Seiken), Quick Vue Rapid SP Influ (DP Pharma Biomedical), and Rapid Testa FLU stick (Daiichi Pure Chemicals)--against influenza virus stocks, contained five vaccination strains (one A/H1N1, two A/H3N2, and two B) and six clinical strains (two A/H1N1, two A/H3N2, and two B). Minimum detection concentrations giving immunologically positive signals in serial dilution and RNA copies in positive dilution in real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were assayed for all kits and virus stock combinations. RNA log10 copy numbers/mL in dilutions within detection limits yielded 5.68-7.02, 6.37-7,17, and 6.5-8.13 for A/H1N1, A/H3N2, and B. Statistically significant differences in sensitivity were observed between some kit combinations. Detection sensitivity tended to be relatively higher for influenza A than B virus. This is assumed due to different principles in kit methods, such as monoclonal antibodies, specimen-extraction conditions, and other unknown factors.

A rapid and simple method of detection of Blepharisma japonicum using PCR and immobilisation on FTA paper

PubMed Central

Hide, Geoff; Hughes, Jacqueline M; McNuff, Robert

2003-01-01

Background The rapid expansion in the availability of genome and DNA sequence information has opened up new possibilities for the development of methods for detecting free-living protozoa in environmental samples. The protozoan Blepharisma japonicum was used to investigate a rapid and simple detection system based on polymerase chain reaction amplification (PCR) from organisms immobilised on FTA paper. Results Using primers designed from the α-tubulin genes of Blepharisma, specific and sensitive detection to the equivalent of a single Blepharisma cell could be achieved. Similar detection levels were found using water samples, containing Blepharisma, which were dried onto Whatman FTA paper. Conclusion This system has potential as a sensitive convenient detection system for Blepharisma and could be applied to other protozoan organisms. PMID:14516472

A highly sensitive and specific capacitive aptasensor for rapid and label-free trace analysis of Bisphenol A (BPA) in canned foods.

PubMed

Mirzajani, Hadi; Cheng, Cheng; Wu, Jayne; Chen, Jiangang; Eda, Shigotoshi; Najafi Aghdam, Esmaeil; Badri Ghavifekr, Habib

2017-03-15

A rapid, highly sensitive, specific and low-cost capacitive affinity biosensor is presented here for label-free and single step detection of Bisphenol A (BPA). The sensor design allows rapid prototyping at low-cost using printed circuit board material by benchtop equipment. High sensitivity detection is achieved through the use of a BPA-specific aptamer as probe molecule and large electrodes to enhance AC-electroelectrothermal effect for long-range transport of BPA molecules toward electrode surface. Capacitive sensing technique is used to determine the bounded BPA level by measuring the sample/electrode interfacial capacitance of the sensor. The developed biosensor can detect BPA level in 20s and exhibits a large linear range from 1 fM to 10 pM, with a limit of detection (LOD) of 152.93 aM. This biosensor was applied to test BPA in canned food samples and could successfully recover the levels of spiked BPA. This sensor technology is demonstrated to be highly promising and reliable for rapid, sensitive and on-site monitoring of BPA in food samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantum Dot-Fullerene Based Molecular Beacon Nanosensors for Rapid, Highly Sensitive Nucleic Acid Detection.

PubMed

Liu, Ye; Kannegulla, Akash; Wu, Bo; Cheng, Li-Jing

2018-05-15

Spherical fullerene (C 60 ) can quench the fluorescence of a quantum dot (QD) through energy transfer and charge transfer processes, with the quenching efficiency regulated by the number of proximate C 60 on each QD. With the quenching property and its small size compared with other nanoparticle-based quenchers, it is advantageous to group a QD reporter and multiple C 60 -labeled oligonucleotide probes to construct a molecular beacon (MB) probe for sensitive, robust nucleic acid detection. We demonstrated a rapid, high-sensitivity DNA detection method using the nanosensors composed of QD-C 60 based MBs carried by magnetic nanoparticles (MNPs). The assay was accelerated by first dispersing the nanosensors in analytes for highly efficient DNA capture resulting from short-distance 3-dimensional diffusion of targets to the sensor surface and then concentrating the nanosensors to a substrate by magnetic force to amplify the fluorescence signal for target quantification. The enhanced mass transport enabled a rapid detection (< 10 min) with a small sample volume (1-10 µl). The high signal-to-noise ratio produced by the QD-C 60 pairs and magnetic concentration yielded a detection limit of 100 fM (~106 target DNA copies for a 10 µl analyte). The rapid, sensitive, label-free detection method will benefit the applications in point-of-care molecular diagnostic technologies.

Rapid detection of the Klebsiella pneumoniae carbapenemase (KPC) gene by loop-mediated isothermal amplification (LAMP).

PubMed

Nakano, Ryuichi; Nakano, Akiyo; Ishii, Yoshikazu; Ubagai, Tsuneyuki; Kikuchi-Ueda, Takane; Kikuchi, Hirotoshi; Tansho-Nagakawa, Shigeru; Kamoshida, Go; Mu, Xiaoqin; Ono, Yasuo

2015-03-01

Klebsiella pneumoniae carbapenemases (KPC), which are associated with resistance to carbapenem, have recently spread worldwide and have become a global concern. It is necessary to detect KPC-producing organisms in clinical settings to be able to control the spread of this resistance. We have developed a loop-mediated isothermal amplification (LAMP) method for rapid detection of KPC producers. LAMP primer sets were designed to recognize the homologous regions of blaKPC-2 to blaKPC-17 and could amplify blaKPC rapidly. The specificity and sensitivity of the primers in the LAMP reactions for blaKPC detection were determined. This LAMP assay was able to specifically detect KPC producers at 68 °C, and no cross-reactivity was observed for other types of β-lactamase (class A, B, C, or D) producers. The detection limit for this assay was found to be 10(0) CFU per tube, in 25 min, which was 10-fold more sensitive than a PCR assay for blaKPC detection. Then, the sensitivity of the LAMP reactions for blaKPC detection in human specimens (sputum samples, urine samples, fecal samples and blood samples) was analyzed; it was observed that the LAMP assay had almost the same sensitivity in these samples as when using purified DNA. The LAMP assay is easy to perform and rapid. It may therefore be routinely applied for detection of KPC producers in the clinical laboratory. Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

A multiplex PCR assay for the rapid and sensitive detection of methicillin-resistant Staphylococcus aureus and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci.

PubMed

Xu, Benjin; Liu, Ling; Liu, Li; Li, Xinping; Li, Xiaofang; Wang, Xin

2012-11-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a global health concern, which had been detected in food and food production animals. Conventional testing for detection of MRSA takes 3 to 5 d to yield complete information of the organism and its antibiotic sensitivity pattern. So, a rapid method is needed to diagnose and treat the MRSA infections. The present study focused on the development of a multiplex PCR assay for the rapid and sensitive detection of MRSA. The assay simultaneously detected 4 genes, namely, 16S rRNA of the Staphylococcus genus, femA of S. aureus, mecA that encodes methicillin resistance, and one internal control. It was rapid and yielded results within 4 h. The analytical sensitivity and specificity of the multiplex PCR assay was evaluated by comparing it with the conventional method. The analytical sensitivity of the multiplex PCR assay at the DNA level was 10 ng DNA. The analytical specificity was evaluated with 10 reference staphylococci strains and was 100%. The diagnostic evaluation of MRSA was carried out using 360 foodborne staphylococci isolates, and showed 99.1% of specificity, 96.4% of sensitivity, 97.5% of positive predictive value, and 97.3% of negative predictive value compared to the conventional method. The inclusion of an internal control in the multiplex PCR assay is important to exclude false-negative cases. This test can be used as an effective diagnostic and surveillance tool to investigate the spread and emergence of MRSA. © 2012 Institute of Food Technologists®

Aptamer-Nanoparticle Strip Biosensor for Rapid and Sensitive Detection of Cancer Cells

PubMed Central

Mao, Xun; Phillips, Joseph A.; Xu, Hui; Tan, Weihong; Zeng, Lingwen; Liu, Guodong

2009-01-01

We report an aptamer-nanoparticle strip biosensor (ANSB) for the rapid, specific, sensitive and low-cost detection of circulating cancer cells. Known for their high specificity and affinity, aptamers were first selected from live cells by the cell-SELEX (systematic evolution of ligands by exponential enrichment) process. When next combined with the unique optical properties of gold nanoparticles (Au-NPs), ANSBs were prepared on a lateral flow device. Ramos cells were used as a model target cell to demonstrate proof of principle. Under optimal conditions, the ANSB was capable of detecting a minimum of 4000 Ramos cells without instrumentation (visual judgment) and 800 Ramos cells with a portable strip reader within 15 minutes. Importantly, ANSB has successfully detected Ramos cells in human blood, thus providing a rapid, sensitive and low-cost quantitative tool for the detection of circulating cancer cells. ANSB therefore shows great promise for in-field and point-of-care cancer diagnosis and therapy. PMID:19904989

Comparing rapid methods for detecting Listeria in seafood and environmental samples using the most probably number (MPN) technique.

PubMed

Cruz, Cristina D; Win, Jessicah K; Chantarachoti, Jiraporn; Mutukumira, Anthony N; Fletcher, Graham C

2012-02-15

The standard Bacteriological Analytical Manual (BAM) protocol for detecting Listeria in food and on environmental surfaces takes about 96 h. Some studies indicate that rapid methods, which produce results within 48 h, may be as sensitive and accurate as the culture protocol. As they only give presence/absence results, it can be difficult to compare the accuracy of results generated. We used the Most Probable Number (MPN) technique to evaluate the performance and detection limits of six rapid kits for detecting Listeria in seafood and on an environmental surface compared with the standard protocol. Three seafood products and an environmental surface were inoculated with similar known cell concentrations of Listeria and analyzed according to the manufacturers' instructions. The MPN was estimated using the MPN-BAM spreadsheet. For the seafood products no differences were observed among the rapid kits and efficiency was similar to the BAM method. On the environmental surface the BAM protocol had a higher recovery rate (sensitivity) than any of the rapid kits tested. Clearview™, Reveal®, TECRA® and VIDAS® LDUO detected the cells but only at high concentrations (>10(2) CFU/10 cm(2)). Two kits (VIP™ and Petrifilm™) failed to detect 10(4) CFU/10 cm(2). The MPN method was a useful tool for comparing the results generated by these presence/absence test kits. There remains a need to develop a rapid and sensitive method for detecting Listeria in environmental samples that performs as well as the BAM protocol, since none of the rapid tests used in this study achieved a satisfactory result. Copyright © 2011 Elsevier B.V. All rights reserved.

Rapid screening of toxigenic vibrio cholerae O1 strains from south Iran by PCR-ELISA.

PubMed

Mousavi, Seyed Latif; Nazarian, Shahram; Amani, Jafar; Rahgerdi, Ahmad Karimi

2008-01-01

The ability to sensitively detect Vibrio cholera with PCR-ELISA method represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen. The aim of this research is to evaluate the suitability of a PCR-enzyme-linked immunosorbent assay for sensitive and rapid detection of V. cholera O1. The 398-bp sequence of a gene that codes for the cholera toxin B subunit was amplified by PCR. The digoxigenin-labeled amplified products were coated on microplates and detected by ELISA. The PCR product was also hybridized with biotin labelled probe and detected by ELISA using streptavidin. The specificity of the PCR was determined using 10 bacterial strains and 50 samples from south Iran. The detection limit was 0.5 pg of the genomic DNA and five bacterial cells. Adaptation of PCR into PCR-ELISA assay format facilitates specific and sensitive detection and diagnosis of human cholera disease. We conclude that this PCR-ELISA is a diagnostic method that specifically detects toxin genes in V. cholera O1 strains. It is more rapid and less cumbersome than other diagnostic methods for detection of toxicity in these strains.

Rapid, Efficient Detection and Drug Susceptibility Testing of Mycobacterium tuberculosis in Sputum by Microscopic Observation of Broth Cultures

PubMed Central

Caviedes, Luz; Lee, Tien-Shun; Gilman, Robert H.; Sheen, Patricia; Spellman, Emily; Lee, Ellen H.; Berg, Douglas E.; Montenegro-James, Sonia

2000-01-01

Inexpensive, rapid, and reliable methods of detecting infection by and drug susceptibility of Mycobacterium tuberculosis (MTB) are crucial to the control of tuberculosis. The novel microscopic observation broth-drug susceptibility assay (MODS) detects early growth of MTB in liquid medium, allowing more timely diagnosis and drug susceptibility testing. Sputum samples from hospitalized patients in Peru were analyzed by using stains, culture, and PCR. Sensitivity of MODS (92%) compared favorably with the most sensitive of the other culture methods (93%). Sputum samples positive for tuberculosis were tested for susceptibility to isoniazid and rifampin with the microwell alamar blue assay (MABA) and MODS. In 89% of cases, there was concordance between MODS and MABA. Of the diagnostic and susceptibility testing methods used, MODS yielded results most rapidly (median, 9.0 and 9.5 days, respectively). MODS is a rapid, inexpensive, sensitive, and specific method for MTB detection and susceptibility testing; it is particularly appropriate for use in developing countries burdened by significant infection rates and increasing numbers of multiple-drug-resistant cases. PMID:10699023

Rapid, efficient detection and drug susceptibility testing of Mycobacterium tuberculosis in sputum by microscopic observation of broth cultures. The Tuberculosis Working Group in Peru.

PubMed

Caviedes, L; Lee, T S; Gilman, R H; Sheen, P; Spellman, E; Lee, E H; Berg, D E; Montenegro-James, S

2000-03-01

Inexpensive, rapid, and reliable methods of detecting infection by and drug susceptibility of Mycobacterium tuberculosis (MTB) are crucial to the control of tuberculosis. The novel microscopic observation broth-drug susceptibility assay (MODS) detects early growth of MTB in liquid medium, allowing more timely diagnosis and drug susceptibility testing. Sputum samples from hospitalized patients in Peru were analyzed by using stains, culture, and PCR. Sensitivity of MODS (92%) compared favorably with the most sensitive of the other culture methods (93%). Sputum samples positive for tuberculosis were tested for susceptibility to isoniazid and rifampin with the microwell alamar blue assay (MABA) and MODS. In 89% of cases, there was concordance between MODS and MABA. Of the diagnostic and susceptibility testing methods used, MODS yielded results most rapidly (median, 9.0 and 9.5 days, respectively). MODS is a rapid, inexpensive, sensitive, and specific method for MTB detection and susceptibility testing; it is particularly appropriate for use in developing countries burdened by significant infection rates and increasing numbers of multiple-drug-resistant cases.

Development of a highly sensitive lateral immunochromatographic assay for rapid detection of Vibrio parahaemolyticus.

PubMed

Liu, Xinfeng; Guan, Yuyao; Cheng, Shiliang; Huang, Yidan; Yan, Qin; Zhang, Jun; Huang, Guanjun; Zheng, Jian; Liu, Tianqiang

2016-12-01

Vibrio parahaemolyticus is widely present in brackish water all over the world, causing infections in certain aquatic animals. It is also a foodborne pathogen that causes diarrhea in humans. The aim of this study is to develop an immunochromatographic lateral flow assay (LFA) for rapid detection of V. parahaemolyticus in both aquatic products and human feces of diarrheal patients. Two monoclonal antibody (MAb) pairs, GA1a-IC9 and IC9-KB4c, were developed and proven to be highly specific and sensitive to V. parahaemolyticus. Based on the two MAb pairs, two types of LFA strips were prepared. Their testing limits for V. parahaemolyticus culture were both 1.2×10 3 CFU/ml. The diagnostic sensitivities and specificities were both 100% for the 32 tested microbial species, including 6 Vibrio species. Subsequently, the LFA strips were used to test Whiteleg shrimps and human feces. The type II strip showed a higher diagnostic sensitivity. Its sensitivity and specificity for hepatopancreas and fecal samples from 13 Whiteleg shrimps and fecal samples from 146 human diarrheal patients were all 100%. In conclusion, our homemade type II LFA is a very promising testing device for rapid and convenient detection of V. parahaemolyticus infection not only in aquatic animals, but also in human diarrheal patients. This sensitive immunochromtographic LFA allows rapid detection of V. parahaemolyticus without requirement of culture enrichment. Copyright © 2016. Published by Elsevier B.V.

Rapid and accurate detection of KRAS mutations in colorectal cancers using the isothermal-based optical sensor for companion diagnostics

PubMed Central

Koo, Bonhan; Lee, Tae Yoon; Lee, Jeong Hoon; Shin, Yong; Lim, Seok-Byung

2017-01-01

Although KRAS mutational status testing is becoming a companion diagnostic tool for managing patients with colorectal cancer (CRC), there are still several difficulties when analyzing KRAS mutations using the existing assays, particularly with regard to low sensitivity, its time-consuming, and the need for large instruments. We developed a rapid, sensitive, and specific mutation detection assay based on the bio-photonic sensor termed ISAD (isothermal solid-phase amplification/detection), and used it to analyze KRAS gene mutations in human clinical samples. To validate the ISAD-KRAS assay for use in clinical diagnostics, we examined for hotspot KRAS mutations (codon 12 and codon 13) in 70 CRC specimens using PCR and direct sequencing methods. In a serial dilution study, ISAD-KRAS could detect mutations in a sample containing only 1% of the mutant allele in a mixture of wild-type DNA, whereas both PCR and direct sequencing methods could detect mutations in a sample containing approximately 30% of mutant cells. The results of the ISAD-KRAS assay from 70 clinical samples matched those from PCR and direct sequencing, except in 5 cases, wherein ISAD-KRAS could detect mutations that were not detected by PCR and direct sequencing. We also found that the sensitivity and specificity of ISAD-KRAS were 100% within 30 min. The ISAD-KRAS assay provides a rapid, highly sensitive, and label-free method for KRAS mutation testing, and can serve as a robust and near patient testing approach for the rapid detection of patients most likely to respond to anti-EGFR drugs. PMID:29137388

Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR.

PubMed

Sergueev, Kirill V; He, Yunxiu; Borschel, Richard H; Nikolich, Mikeljon P; Filippov, Andrey A

2010-06-28

Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics. The objective of this work was to develop an alternative to conventional phage lysis tests--a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages phiA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. phiA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10(3) CFU/ml (equivalent to as few as one Y. pestis cell per 1-microl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, phiA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR. Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria.

A rapid molecular diagnosis of cutaneous leishmaniasis by colorimetric malachite green-loop-mediated isothermal amplification (LAMP) combined with an FTA card as a direct sampling tool.

PubMed

Nzelu, Chukwunonso O; Cáceres, Abraham G; Guerrero-Quincho, Silvia; Tineo-Villafuerte, Edwin; Rodriquez-Delfin, Luis; Mimori, Tatsuyuki; Uezato, Hiroshi; Katakura, Ken; Gomez, Eduardo A; Guevara, Angel G; Hashiguchi, Yoshihisa; Kato, Hirotomo

2016-01-01

Leishmaniasis remains one of the world's most neglected diseases, and early detection of the infectious agent, especially in developing countries, will require a simple and rapid test. In this study, we established a quick, one-step, single-tube, highly sensitive loop-mediated isothermal amplification (LAMP) assay for rapid detection of Leishmania DNA from tissue materials spotted on an FTA card. An FTA-LAMP with pre-added malachite green was performed at 64°C for 60min using a heating block and/or water bath and DNA amplification was detected immediately after incubation. The LAMP assay had high detection sensitivity down to a level of 0.01 parasites per μl. The field- and clinic-applicability of the colorimetric FTA-LAMP assay was demonstrated with 122 clinical samples collected from patients suspected of having cutaneous leishmaniasis in Peru, from which 71 positives were detected. The LAMP assay in combination with an FTA card described here is rapid and sensitive, as well as simple to perform, and has great potential usefulness for diagnosis and surveillance of leishmaniasis in endemic areas. Copyright © 2015 Elsevier B.V. All rights reserved.

ELISA-PLA: A novel hybrid platform for the rapid, highly sensitive and specific quantification of proteins and post-translational modifications.

PubMed

Tong, Qing-He; Tao, Tao; Xie, Li-Qi; Lu, Hao-Jie

2016-06-15

Detection of low-abundance proteins and their post-translational modifications (PTMs) remains a great challenge. A conventional enzyme-linked immunosorbent assay (ELISA) is not sensitive enough to detect low-abundance PTMs and suffers from nonspecific detection. Herein, a rapid, highly sensitive and specific platform integrating ELISA with a proximity ligation assay (PLA), termed ELISA-PLA, was developed. Using ELISA-PLA, the specificity was improved by the simultaneous and proximate recognition of targets through multiple probes, and the sensitivity was significantly improved by rolling circle amplification (RCA). For GFP, the limit of detection (LOD) was decreased by two orders of magnitude compared to that of ELISA. Using site-specific phospho-antibody and pan-specific phospho-antibody, ELISA-PLA was successfully applied to quantify the phosphorylation dynamics of ERK1/2 and the overall tyrosine phosphorylation level of ERK1/2, respectively. ELISA-PLA was also used to quantify the O-GlcNAcylation of AKT, c-Fos, CREB and STAT3, which is faster and more sensitive than the conventional immunoprecipitation and western blotting (IP-WB) method. As a result, the sample consumption of ELISA-PLA was reduced 40-fold compared to IP-WB. Therefore, ELISA-PLA could be a promising platform for the rapid, sensitive and specific detection of proteins and PTMs. Copyright © 2016 Elsevier B.V. All rights reserved.

Rapid and Sensitive Detection of Protein Biomarker Using a Portable Fluorescence Biosensor based on Quantum Dots and a Lateral Flow Test Strip

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Zhaohui; Wang, Ying; Wang, Jun

2010-08-15

A portable fluorescence biosensor with rapid and ultrasensitive response for trace protein has been built up with quantum dots and lateral flow test strip. The superior signal brightness and high photostability of quantum dots are combined with the promising advantages of lateral flow test strip and resulted in high sensitivity, selectivity and speedy for protein detection. Nitrated ceruloplasmin, a significant biomarker for cardiovascular disease, lung cancer and stress response to smoking, was used as model protein to demonstrate the good performances of this proposed Qdot-based lateral flow test strip. Quantitative detection of nitrated ceruloplasmin was realized by recording the fluorescencemore » intensity of quantum dots captured on the test line. Under optimal conditions, this portable fluorescence biosensor displays rapid responses for nitrated ceruloplasmin in wide dynamic range with a detection limit of 0.1ng/mL (S/N=3). Furthermore, the biosensor was successfully utilized for spiked human plasma sample detection with the concentration as low as 1ng/mL. The results demonstrate that the quantum dot-based lateral flow test strip is capable for rapid, sensitive, and quantitative detection of nitrated ceruloplasmin and hold a great promise for point-of-care and in field analysis of other protein biomarkers.« less

Combining magnetic nanoparticle with biotinylated nanobodies for rapid and sensitive detection of influenza H3N2

PubMed Central

2014-01-01

Our objective is to develop a rapid and sensitive assay based on magnetic beads to detect the concentration of influenza H3N2. The possibility of using variable domain heavy-chain antibodies (nanobody) as diagnostic tools for influenza H3N2 was investigated. A healthy camel was immunized with inactivated influenza H3N2. A nanobody library of 8 × 108 clones was constructed and phage displayed. After three successive biopanning steps, H3N2-specific nanobodies were successfully isolated, expressed in Escherichia coli, and purified. Sequence analysis of the nanobodies revealed that we possessed four classes of nanobodies against H3N2. Two nanobodies were further used to prepare our rapid diagnostic kit. Biotinylated nanobody was effectively immobilized onto the surface of streptavidin magnetic beads. The modified magnetic beads with nanobody capture specifically influenza H3N2 and can still be recognized by nanobodies conjugated to horseradish peroxidase (HRP) conjugates. Under optimized conditions, the present immunoassay exhibited a relatively high sensitive detection with a limit of 50 ng/mL. In conclusion, by combining magnetic beads with specific nanobodies, this assay provides a promising influenza detection assay to develop a potential rapid, sensitive, and low-cost diagnostic tool to screen for influenza infections. PMID:25328501

Combining magnetic nanoparticle with biotinylated nanobodies for rapid and sensitive detection of influenza H3N2

NASA Astrophysics Data System (ADS)

Zhu, Min; Hu, Yonghong; Li, Guirong; Ou, Weijun; Mao, Panyong; Xin, Shaojie; Wan, Yakun

2014-09-01

Our objective is to develop a rapid and sensitive assay based on magnetic beads to detect the concentration of influenza H3N2. The possibility of using variable domain heavy-chain antibodies (nanobody) as diagnostic tools for influenza H3N2 was investigated. A healthy camel was immunized with inactivated influenza H3N2. A nanobody library of 8 × 108 clones was constructed and phage displayed. After three successive biopanning steps, H3N2-specific nanobodies were successfully isolated, expressed in Escherichia coli, and purified. Sequence analysis of the nanobodies revealed that we possessed four classes of nanobodies against H3N2. Two nanobodies were further used to prepare our rapid diagnostic kit. Biotinylated nanobody was effectively immobilized onto the surface of streptavidin magnetic beads. The modified magnetic beads with nanobody capture specifically influenza H3N2 and can still be recognized by nanobodies conjugated to horseradish peroxidase (HRP) conjugates. Under optimized conditions, the present immunoassay exhibited a relatively high sensitive detection with a limit of 50 ng/mL. In conclusion, by combining magnetic beads with specific nanobodies, this assay provides a promising influenza detection assay to develop a potential rapid, sensitive, and low-cost diagnostic tool to screen for influenza infections.

Spray Inlet Proton Transfer Reaction Mass Spectrometry (SI-PTR-MS) for Rapid and Sensitive Online Monitoring of Benzene in Water.

PubMed

Zou, Xue; Kang, Meng; Li, Aiyue; Shen, Chengyin; Chu, Yannan

2016-03-15

Rapid and sensitive monitoring of benzene in water is very important to the health of people and for environmental protection. A novel and online detection method of spray inlet proton transfer reaction mass spectrometry (SI-PTR-MS) was introduced for rapid and sensitive monitoring of trace benzene in water. A spraying extraction system was coupled with the self-developed PTR-MS. The benzene was extracted from the water sample in the spraying extraction system and continuously detected with PTR-MS. The flow of carrier gas and salt concentration in water were optimized to be 50 sccm and 20% (w/v), respectively. The response time and the limit of detection of the SI-PTR-MS for detection of benzene in water were 55 s and 0.14 μg/L at 10 s integration time, respectively. The repeatability of the SI-PTR-MS was evaluated, and the relative standard deviation of five replicate determinations was 4.3%. The SI-PTR-MS system was employed for monitoring benzene in different water matrices, such as tap water, lake water, and wastewater. The results indicated that the online SI-PTR-MS can be used for rapid and sensitive monitoring of trace benzene in water.

Highly sensitive chemiluminescent aptasensor for detecting HBV infection based on rapid magnetic separation and double-functionalized gold nanoparticles.

PubMed

Xi, Zhijiang; Gong, Quan; Wang, Chao; Zheng, Bing

2018-06-21

Hepatitis B virus (HBV) infection is a major global public health problem and one of the leading causes of chronic liver disease. HBsAg is the first serological marker to appear in the blood and is the most important marker of HBV infection. Detection of HBsAg in serum samples is commonly carried out using an immunoassay such as an enzyme-linked immunosorbent assay (ELISA), which is complex to perform, time-consuming, and unsatisfactory for testing sensitivity. Therefore, new methods for highly sensitive detection of HBV infection are urgently needed. Aptamers are specific recognition molecules with high affinity and specificity toward their targets. Biosensors that employ aptamers as biorecognition elements are known as aptasensors. In this study, we select an HBsAg-specific aptamer and use it to develop a new chemiluminescent aptasensor based on rapid magnetic separation and double-functionalized gold nanoparticles. This sensor enables rapid magnetic separation and highly sensitive detection of HBsAg in HBV-positive serum. The detection limit of this HBsAg-detecting chemiluminescent aptasensor is as low as 0.05 ng/mL, which is much lower than the 0.5 ng/mL limit of a typical ELISA used in hospitals. Furthermore, this aptasensor works well and is highly specific to HBV infection.

Engineered nanoconstructs for the multiplexed and sensitive detection of high-risk pathogens

NASA Astrophysics Data System (ADS)

Seo, Youngmin; Kim, Ji-Eun; Jeong, Yoon; Lee, Kwan Hong; Hwang, Jangsun; Hong, Jongwook; Park, Hansoo; Choi, Jonghoon

2016-01-01

Many countries categorize the causative agents of severe infectious diseases as high-risk pathogens. Given their extreme infectivity and potential to be used as biological weapons, a rapid and sensitive method for detection of high-risk pathogens (e.g., Bacillus anthracis, Francisella tularensis, Yersinia pestis, and Vaccinia virus) is highly desirable. Here, we report the construction of a novel detection platform comprising two units: (1) magnetic beads separately conjugated with multiple capturing antibodies against four different high-risk pathogens for simple and rapid isolation, and (2) genetically engineered apoferritin nanoparticles conjugated with multiple quantum dots and detection antibodies against four different high-risk pathogens for signal amplification. For each high-risk pathogen, we demonstrated at least 10-fold increase in sensitivity compared to traditional lateral flow devices that utilize enzyme-based detection methods. Multiplexed detection of high-risk pathogens in a sample was also successful by using the nanoconstructs harboring the dye molecules with fluorescence at different wavelengths. We ultimately envision the use of this novel nanoprobe detection platform in future applications that require highly sensitive on-site detection of high-risk pathogens.

The failure of routine rapid HIV testing: a case study of improving low sensitivity in the field.

PubMed

Wolpaw, Benjamin J; Mathews, Catherine; Chopra, Mickey; Hardie, Diana; de Azevedo, Virginia; Jennings, Karen; Lurie, Mark N

2010-03-22

The rapid HIV antibody test is the diagnostic tool of choice in low and middle-income countries. Previous evidence suggests that rapid HIV diagnostic tests may underperform in the field, failing to detect a substantial number of infections. A research study inadvertently discovered that a clinic rapid HIV testing process was failing to detect cases of established (high antibody titer) infection, exhibiting an estimated 68.7% sensitivity (95% CI [41.3%-89.0%]) over the course of the first three weeks of observation. The setting is a public service clinic that provides STI diagnosis and treatment in an impoverished, peri-urban community outside of Cape Town, South Africa. The researchers and local health administrators collaborated to investigate the cause of the poor test performance and make necessary corrections. The clinic changed the brand of rapid test being used and later introduced quality improvement measures. Observations were made of the clinic staff as they administered rapid HIV tests to real patients. Estimated testing sensitivity was calculated as the number of rapid HIV test positive individuals detected by the clinic divided by this number plus the number of PCR positive, highly reactive 3rd generation ELISA patients identified among those who were rapid test negative at the clinic. In the period of five months after the clinic made the switch of rapid HIV tests, estimated sensitivity improved to 93.5% (95% CI [86.5%-97.6%]), during which time observations of counselors administering tests at the clinic found poor adherence to the recommended testing protocol. Quality improvement measures were implemented and estimated sensitivity rose to 95.1% (95% CI [83.5%-99.4%]) during the final two months of full observation. Poor testing procedure in the field can lead to exceedingly low levels of rapid HIV test sensitivity, making it imperative that stringent quality control measures are implemented where they do not already exist. Certain brands of rapid-testing kits may perform better than others when faced with sub-optimal use.

Rapid detection of Mycobacterium tuberculosis and rifampicin resistance in extrapulmonary tuberculosis and sputum smear-negative pulmonary suspects using Xpert MTB/RIF.

PubMed

Ullah, Irfan; Javaid, Arshad; Masud, Haleema; Ali, Mazhar; Basit, Anila; Ahmad, Waqas; Younis, Faisal; Yasmin, Rehana; Khan, Afsar; Jabbar, Abdul; Husain, Masroor; Butt, Zahid Ahmad

2017-04-01

Tuberculosis (TB) is a serious public health problem in developing countries such as Pakistan. Rapid diagnosis of TB and detection of drug resistance are very important for timely and appropriate management of multidrug-resistant TB (MDR-TB). The purpose of this study was to determine the diagnostic efficacy of the Xpert MTB/RIF assay for rapid diagnosis of TB and detection of rifampicin (RIF) resistance in extrapulmonary and smear-negative pulmonary TB suspects. A total of 98 bronchoalveolar lavage fluid (BALF) and 168 extrapulmonary specimens were processed by Xpert MTB/RIF. Culture results are considered as the gold standard for diagnosis of TB, and drug susceptibility testing for detection of RIF resistance. Diagnostic efficacy was measured in terms of sensitivity, specificity and positive and negative predictive values. The Xpert MTB/RIF assay detected 40 (40.8 %) of 98 BALF of presumptive pulmonary TB and 60 (35.7 %) of 168 extrapulmonary specimens. Sensitivity and specificity of the Xpert MTB/RIF assay for detection of TB was 86 and 88.4 %, respectively. The positive predictive value was 71.5 % while negative predictive value was 95.1 %. The Xpert MTB/RIF assay is a rapid and simple technique with high sensitivity and specificity for diagnosing TB and detecting drug resistance in extrapulmonary and smear-negative TB cases.

Biotunable Nanoplasmonic Filter on Few-Layer MoS2 for Rapid and Highly Sensitive Cytokine Optoelectronic Immunosensing.

PubMed

Park, Younggeun; Ryu, Byunghoon; Oh, Bo-Ram; Song, Yujing; Liang, Xiaogan; Kurabayashi, Katsuo

2017-06-27

Monitoring of the time-varying immune status of a diseased host often requires rapid and sensitive detection of cytokines. Metallic nanoparticle-based localized surface plasmon resonance (LSPR) biosensors hold promise to meet this clinical need by permitting label-free detection of target biomolecules. These biosensors, however, continue to suffer from relatively low sensitivity as compared to conventional immunoassay methods that involve labeling processes. Their response speeds also need to be further improved to enable rapid cytokine quantification for critical care in a timely manner. In this paper, we report an immunobiosensing device integrating a biotunable nanoplasmonic optical filter and a highly sensitive few-layer molybdenum disulfide (MoS 2 ) photoconductive component, which can serve as a generic device platform to meet the need of rapid cytokine detection with high sensitivity. The nanoplasmonic filter consists of anticytokine antibody-conjugated gold nanoparticles on a SiO 2 thin layer that is placed 170 μm above a few-layer MoS 2 photoconductive flake device. The principle of the biosensor operation is based on tuning the delivery of incident light to the few-layer MoS 2 photoconductive flake thorough the nanoplasmonic filter by means of biomolecular surface binding-induced LSPR shifts. The tuning is dependent on cytokine concentration on the nanoplasmonic filter and optoelectronically detected by the few-layer MoS 2 device. Using the developed optoelectronic biosensor, we have demonstrated label-free detection of IL-1β, a pro-inflammatory cytokine, with a detection limit as low as 250 fg/mL (14 fM), a large dynamic range of 10 6 , and a short assay time of 10 min. The presented biosensing approach could be further developed and generalized for point-of-care diagnosis, wearable bio/chemical sensing, and environmental monitoring.

Rapid, Sensitive Detection of Botulinum Toxin on a Flexible Microfluidics Platform

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warner, Marvin G.; Dockendorff, Brian P.; Feldhaus, Michael J.

2004-10-30

In this paper we will describe how high affinity reagents and a sensor configuration enabling rapid mass transport can be combined for rapid, sensitive biodetection. The system that we have developed includes a renewable surface immunoassay, which involves on-column detection of a fluorescently labeled secondary antibody in a sandwich immunoassay. Yeast display and directed molecular evolution were used to create high affinity antibodies to the botulinum toxin heavy chain receptor binding domain, AR1 and 3D12. A rotating rod renewable surface microcolumn was used to form a microliter-sized column containing beads functionalized with the capture antibody (AR1). The column was perfusedmore » with sample, wash solutions, and a fluorescently labeled secondary antibody (3D12) while the on-column fluorescence was monitored. Detection was accomplished in less than 5 minutes, with a total processing time of about 10 minutes. On-column detection of botulinum toxin was more sensitive and much faster than flow cytometry analysis on microbeads using the same reagents.« less

[Multiplex real-time PCR method for rapid detection of Marburg virus and Ebola virus].

PubMed

Yang, Yu; Bai, Lin; Hu, Kong-Xin; Yang, Zhi-Hong; Hu, Jian-Ping; Wang, Jing

2012-08-01

Marburg virus and Ebola virus are acute infections with high case fatality rates. A rapid, sensitive detection method was established to detect Marburg virus and Ebola virus by multiplex real-time fluorescence quantitative PCR. Designing primers and Taqman probes from highly conserved sequences of Marburg virus and Ebola virus through whole genome sequences alignment, Taqman probes labeled by FAM and Texas Red, the sensitivity of the multiplex real-time quantitative PCR assay was optimized by evaluating the different concentrations of primers and Probes. We have developed a real-time PCR method with the sensitivity of 30.5 copies/microl for Marburg virus positive plasmid and 28.6 copies/microl for Ebola virus positive plasmids, Japanese encephalitis virus, Yellow fever virus, Dengue virus were using to examine the specificity. The Multiplex real-time PCR assays provide a sensitive, reliable and efficient method to detect Marburg virus and Ebola virus simultaneously.

Rapid and sensitive detection of synthetic cannabinoids AMB-FUBINACA and α-PVP using surface enhanced Raman scattering (SERS)

NASA Astrophysics Data System (ADS)

Islam, Syed K.; Cheng, Yin Pak; Birke, Ronald L.; Green, Omar; Kubic, Thomas; Lombardi, John R.

2018-04-01

The application of surface enhanced Raman scattering (SERS) has been reported as a fast and sensitive analytical method in the trace detection of the two most commonly known synthetic cannabinoids AMB-FUBINACA and alpha-pyrrolidinovalerophenone (α-PVP). FUBINACA and α-PVP are two of the most dangerous synthetic cannabinoids which have been reported to cause numerous deaths in the United States. While instruments such as GC-MS, LC-MS have been traditionally recognized as analytical tools for the detection of these synthetic drugs, SERS has been recently gaining ground in the analysis of these synthetic drugs due to its sensitivity in trace analysis and its effectiveness as a rapid method of detection. This present study shows the limit of detection of a concentration as low as picomolar for AMB-FUBINACA while for α-PVP, the limit of detection is in nanomolar concentration using SERS.

Comparison of Conventional PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Arcobacter Species

PubMed Central

Wang, Xiaoyu; Seo, Dong Joo; Lee, Min Hwa

2014-01-01

This study aimed to develop a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Arcobacter species. Specific primers targeting the 23S ribosomal RNA gene were used to detect Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. The specificity of the LAMP primer set was assessed using DNA samples from a panel of Arcobacter and Campylobacter species, and the sensitivity was determined using serial dilutions of Arcobacter species cultures. LAMP showed a 10- to 1,000-fold-higher sensitivity than multiplex PCR, with a detection limit of 2 to 20 CFU per reaction in vitro. Whereas multiplex PCR showed cross-reactivity with Campylobacter species, the LAMP method developed in this study was more sensitive and reliable than conventional PCR or multiplex PCR for the detection of Arcobacter species. PMID:24478488

A loop-mediated isothermal amplification assay for rapid and sensitive detection of bovine papular stomatitis virus.

PubMed

Kurosaki, Yohei; Okada, Sayaka; Nakamae, Sayuri; Yasuda, Jiro

2016-12-01

Bovine papular stomatitis virus (BPSV) causes pustular cutaneous disease in cattle worldwide. This paper describes the development of a specific loop-mediated isothermal amplification (LAMP) assay to detect BPSV which did not cross-react with other parapoxviruses. To assess analytical sensitivity of this LAMP assay, DNA was extracted from serially diluted BPSV from which the infectious titer was determined by a novel assay based on calf kidney epithelial cells. The LAMP assay had equivalent analytical sensitivity to quantitative PCR, and could detect as few as 86 copies of viral DNA per reaction. These results suggest that the assay is a specific and sensitive technique to rapidly diagnose bovine papular stomatitis in domestic animals. Copyright © 2016 Elsevier B.V. All rights reserved.

Impact of the rapid antigen detection test in diagnosis and treatment of acute pharyngotonsillitis in a pediatric emergency room.

PubMed

Cardoso, Débora Morais; Gilio, Alfredo Elias; Hsin, Shieh Huei; Machado, Beatriz Marcondes; de Paulis, Milena; Lotufo, João Paulo B; Martinez, Marina Baquerizo; Grisi, Sandra Josefina E

2013-01-01

To evaluate the impact of the routine use of rapid antigen detection test in the diagnosis and treatment of acute pharyngotonsillitis in children. This is a prospective and observational study, with a protocol compliance design established at the Emergency Unit of the University Hospital of Universidade de São Paulo for the care of children and adolescents diagnosed with acute pharyngitis. 650 children and adolescents were enrolled. Based on clinical findings, antibiotics would be prescribed for 389 patients (59.8%); using the rapid antigen detection test, they were prescribed for 286 patients (44.0%). Among the 261 children who would not have received antibiotics based on the clinical evaluation, 111 (42.5%) had positive rapid antigen detection test. The diagnosis based only on clinical evaluation showed 61.1% sensitivity, 47.7% specificity, 44.9% positive predictive value, and 57.5% negative predictive value. The clinical diagnosis of streptococcal pharyngotonsillitis had low sensitivity and specificity. The routine use of rapid antigen detection test led to the reduction of antibiotic use and the identification of a risk group for complications of streptococcal infection, since 42.5% positive rapid antigen detection test patients would not have received antibiotics based only on clinical diagnosis.

A biolayer interferometry-based assay for rapid and highly sensitive detection of biowarfare agents.

PubMed

Mechaly, Adva; Cohen, Hila; Cohen, Ofer; Mazor, Ohad

2016-08-01

Biolayer interferometry (BLI) is an optical technique that uses fiber-optic biosensors for label-free real-time monitoring of protein-protein interactions. In this study, we coupled the advantages of the Octet Red BLI system (automation, fluidics-free, and on-line monitoring) with a signal enhancement step and developed a rapid and sensitive immunological-based method for detection of biowarfare agents. As a proof of concept, we chose to demonstrate the efficacy of this novel assay for the detection of agents representing two classes of biothreats, proteinaceous toxins, and bacterial pathogens: ricin, a lethal plant toxin, and the gram-negative bacterium Francisella tularensis, the causative agent of tularemia. The assay setup consisted of biotinylated antibodies immobilized to the biosensor coupled with alkaline phosphatase-labeled antibodies as the detection moiety to create nonsoluble substrate crystals that precipitate on the sensor surface, thereby inducing a significant wavelength interference. It was found that this BLI-based assay enables sensitive detection of these pathogens (detection limits of 10 pg/ml and 1 × 10(4) pfu/ml ricin and F. tularensis, respectively) within a very short time frame (17 min). Owing to its simplicity, this assay can be easily adapted to detect other analytes in general, and biowarfare agents in particular, in a rapid and sensitive manner. Copyright © 2016 Elsevier Inc. All rights reserved.

Electrochemical Impedance Immunosensor Based on Self-Assembled Monolayers for Rapid Detection of Escherichia coli O157:H7 with Signal Amplification Using Lectin

PubMed Central

Li, Zhanming; Fu, Yingchun; Fang, Weihuan; Li, Yanbin

2015-01-01

Escherichia coli O157:H7 is a predominant foodborne pathogen with severe pathogenicity, leading to increasing attention given to rapid and sensitive detection. Herein, we propose an impedance biosensor using new kinds of screen-printed interdigitated microelectrodes (SPIMs) and wheat germ agglutinin (WGA) for signal amplification to detect E. coli O157:H7 with high sensitivity and time-efficiency. The SPIMs integrate the high sensitivity and short response time of the interdigitated electrodes and the low cost of the screen-printed electrodes. Self-assembling of bi-functional 3-dithiobis-(sulfosuccinimidyl-propionate) (DTSP) on the SPIMs was investigated and was proved to be able to improve adsorption quantity and stability of biomaterials. WGA was further adopted to enhance the signal taking advantage of the abundant lectin-binding sites on the bacteria surface. The immunosensor exhibited a detection limit of 102 cfu·mL−1, with a linear detection range from 102 to 107 cfu·mL−1 (r2 = 0.98). The total detection time was less than 1 h, showing its comparable sensitivity and rapid response. Furthermore, the low cost of one SPIM significantly reduced the detection cost of the biosensor. The biosensor may have great promise in food safety analysis and lead to a portable biosensing system for routine monitoring of foodborne pathogens. PMID:26251911

Loop-mediated isothermal amplification for detection of Staphylococcus aureus in dairy cow suffering from mastitis.

PubMed

Tie, Zhang; Chunguang, Wang; Xiaoyuan, Wei; Xinghua, Zhao; Xiuhui, Zhong

2012-01-01

To develop a rapid detection method of Staphylococcus aureus using loop-mediated isothermal amplification (LAMP), four specific primers were designed according to six distinct sequences of the nuc gene. In addition, the specificity and sensitivity of LAMP were verified and compared with those of PCR. Results showed that the LAMP reaction was completed within 45 min at 62.5°C, and ladder bands were appeared in LAMP products analyzed by gel electrophoresis. After adding 1x SYBR Green l, the positive reaction tube showed green color and the negative reaction tube remained orange, indicating that the LAMP has high specificity. The minimal detectable concentration of LAMP was 1 × 10² CFU/mL and that of PCR was 1 × 10⁴ CFU/mL, indicating that the LAMP was 100 times more sensitive than the PCR. The LAMP method for detection of Staphylococcus aureus has many advantages, such as simple operation, high sensitivity, high specificity, and rapid analysis. Therefore, this method is more suitable for the rapid on-site detection of Staphylococcus aureus.

Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood

PubMed Central

Bacconi, Andrea; Richmond, Gregory S.; Baroldi, Michelle A.; Laffler, Thomas G.; Blyn, Lawrence B.; Carolan, Heather E.; Frinder, Mark R.; Toleno, Donna M.; Metzgar, David; Gutierrez, Jose R.; Massire, Christian; Rounds, Megan; Kennel, Natalie J.; Rothman, Richard E.; Peterson, Stephen; Carroll, Karen C.; Wakefield, Teresa; Ecker, David J.

2014-01-01

The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml whole-blood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections. PMID:24951806

Nucleic acid detection system and method for detecting influenza

DOEpatents

Cai, Hong; Song, Jian

2015-03-17

The invention provides a rapid, sensitive and specific nucleic acid detection system which utilizes isothermal nucleic acid amplification in combination with a lateral flow chromatographic device, or DNA dipstick, for DNA-hybridization detection. The system of the invention requires no complex instrumentation or electronic hardware, and provides a low cost nucleic acid detection system suitable for highly sensitive pathogen detection. Hybridization to single-stranded DNA amplification products using the system of the invention provides a sensitive and specific means by which assays can be multiplexed for the detection of multiple target sequences.

Novel approach based on one-tube nested PCR and a lateral flow strip for highly sensitive diagnosis of tuberculous meningitis.

PubMed

Sun, Yajuan; Chen, Jiajun; Li, Jia; Xu, Yawei; Jin, Hui; Xu, Na; Yin, Rui; Hu, Guohua

2017-01-01

Rapid and sensitive detection of Mycobacterium tuberculosis (M. Tb) in cerebrospinal fluid is crucial in the diagnosis of tuberculous meningitis (TBM), but conventional diagnostic technologies have limited sensitivity and specificity or are time-consuming. In this work, a novel, highly sensitive molecular diagnostic method, one-tube nested PCR-lateral flow strip test (OTNPCR-LFST), was developed for detecting M. tuberculosis. This one-tube nested PCR maintains the sensitivity of conventional two-step nested PCR and reduces both the chance of cross-contamination and the time required for analysis. The PCR product was detected by a lateral flow strip assay, which provided a basis for migration of the test to a point-of-care (POC) microfluidic format. The developed assay had an improved sensitivity compared with traditional PCR, and the limit of detection was up to 1 fg DNA isolated from M. tuberculosis. The assay was also specific for M. tuberculosis, and no cross-reactions were found in other non-target bacteria. The application of this technique to clinical samples was successfully evaluated, and OTNPCR-LFST showed 89% overall sensitivity and 100% specificity for TBM patients. This one-tube nested PCR-lateral flow strip assay is useful for detecting M. tuberculosis in TBM due to its rapidity, high sensitivity and simple manipulation.

A novel strategy for rapid detection of NT-proBNP

NASA Astrophysics Data System (ADS)

Cui, Qiyao; Sun, Honghao; Zhu, Hui

2017-09-01

In order to establish a simple, rapid, sensitive, and specific quantitative assay to detect the biomarkers of heart failure, in this study, biotin-streptavidin technology was employed with fluorescence immunochromatographic assay to detect the concentration of the biomarkers in serum, and this method was applied to detect NT-proBNP, which is valuable for diagnostic evaluation of heart failure.

Development of an immunochromatographic strip test for rapid detection of melamine in raw milk, milk products, and animal feed

USDA-ARS?s Scientific Manuscript database

A simple, rapid and sensitive immunogold chromatographic strip test based on a monoclonal antibody was developed for the detection of melamine (MEL) residues in raw milk, milk products and animal feed. The limit of detection was estimated to be 0.05 µg/mL in raw milk, since the detection test line ...

Rapid and sensitive detection of Lily symptomless virus by reverse transcription loop-mediated isothermal amplification.

PubMed

He, Xiangfeng; Xue, Fei; Xu, Shufa; Wang, Wenhe

2016-12-01

Lily symptomless virus (LSV) is one of the most prevalent viruses that infect lily plants worldwide. A rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of LSV, using two primer pairs that specifically amplified the conserved sequence of LSV coat protein. The optimum reaction conditions were as follows: 4mM MgCl 2 and 0.8M betaine with incubation at 64°C for 30min. The limit of detection of LSV from infected lily leaves was 10-fold higher for RT-LAMP than for conventional RT-PCR. Moreover, RT-LAMP detected LSV in not only symptomatic, but also in symptomless tissues of infected plants. These findings indicate that our RT-LAMP method for LSV can serve as a low-cost, simple, and rapid alternative to conventional detection assays. Copyright © 2016 Elsevier B.V. All rights reserved.

Rapid and specific detection of Asian- and African-lineage Zika viruses

PubMed Central

Chotiwan, Nunya; Brewster, Connie D.; Magalhaes, Tereza; Weger-Lucarelli, James; Duggal, Nisha K.; Rückert, Claudia; Nguyen, Chilinh; Garcia Luna, Selene M.; Fauver, Joseph R.; Andre, Barb; Gray, Meg; Black, William C.; Kading, Rebekah C.; Ebel, Gregory D.; Kuan, Guillermina; Balmaseda, Angel; Jaenisch, Thomas; Marques, Ernesto T. A.; Brault, Aaron C.; Harris, Eva; Foy, Brian D.; Quackenbush, Sandra L.; Perera, Rushika; Rovnak, Joel

2017-01-01

Understanding the dynamics of Zika virus transmission and formulating rational strategies for its control require precise diagnostic tools that are also appropriate for resource-poor environments. We have developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay that distinguishes Zika viruses of Asian and African lineages. The assay does not detect chikungunya virus or flaviviruses such as dengue, yellow fever, or West Nile viruses. The assay conditions allowed direct detection of Zika virus RNA in cultured infected cells; in mosquitoes; in virus-spiked samples of human blood, plasma, saliva, urine, and semen; and in infected patient serum, plasma, and semen samples without the need for RNA isolation or reverse transcription. The assay offers rapid, specific, sensitive, and inexpensive detection of the Asian-lineage Zika virus strain that is currently circulating in the Western hemisphere, and can also detect the African-lineage Zika virus strain using separate, specific primers. PMID:28469032

Molecularly imprinted fluorescent hollow nanoparticles as sensors for rapid and efficient detection λ-cyhalothrin in environmental water.

PubMed

Wang, Jixiang; Qiu, Hao; Shen, Hongqiang; Pan, Jianming; Dai, Xiaohui; Yan, Yongsheng; Pan, Guoqing; Sellergren, Börje

2016-11-15

Molecularly imprinted fluorescent polymers have shown great promise in biological or chemical separations and detections, due to their high stability, selectivity and sensitivity. In this work, molecularly imprinted fluorescent hollow nanoparticles, which could rapidly and efficiently detect λ-cyhalothrin (a toxic insecticide) in water samples, was reported. The molecularly imprinted fluorescent sensor showed excellent sensitivity (the limit of detection low to 10.26nM), rapid detection rate (quantitative detection of λ-cyhalothrin within 8min), regeneration ability (maintaining good fluorescence properties after 8 cycling operation) and appreciable selectivity over several structural analogs. Moreover, the fluorescent sensor was further used to detect λ-cyhalothrin in real samples form the Beijing-Hangzhou Grand Canal Water. Despite the relatively complex components of the environmental water, the molecularly imprinted fluorescent hollow nanosensor still showed good recovery, clearly demonstrating the potential value of this smart sensor nanomaterial in environmental monitoring. Copyright © 2016 Elsevier B.V. All rights reserved.

Rationalizing and advancing the 3-MPBA SERS sandwich assay for rapid detection of bacteria in environmental and food matrices.

PubMed

Pearson, Brooke; Mills, Alexander; Tucker, Madeline; Gao, Siyue; McLandsborough, Lynne; He, Lili

2018-06-01

Bacterial foodborne illness continues to be a pressing issue in our food supply. Rapid detection methods are needed for perishable foods due to their short shelf lives and significant contribution to foodborne illness. Previously, a sensitive and reliable surface-enhanced Raman spectroscopy (SERS) sandwich assay based on 3-mercaptophenylboronic acid (3-MBPA) as a capturer and indicator molecule was developed for rapid bacteria detection. In this study, we explored the advantages and constraints of this assay over the conventional aerobic plate count (APC) method and further developed methods for detection in real environmental and food matrices. The SERS sandwich assay was able to detect environmental bacteria in pond water and on spinach leaves at higher levels than the APC method. In addition, the SERS assay appeared to have higher sensitivity to quantify bacteria in the stationary phase. On the other hand, the APC method was more sensitive to cell viability. Finally, a method to detect bacteria in a challenging high-sugar juice matrix was developed to enhance bacteria capture. This study advanced the SERS technique for real applications in environment and food matrices. Copyright © 2017 Elsevier Ltd. All rights reserved.

Correlation between the immunological condition and the results of immunoenzymatic tests in diagnosing infectious mononucleosis.

PubMed

Tamaro, Giorgio; Donato, Michela; Princi, Tanja; Parco, Sergio

2009-04-01

A symptom-based diagnosis of infectious mononucleosis is not sufficiently accurate, since some clinical symptoms of infectious mononucleosis are also detected in other virally induced diseases. Moreover, not all patients suffering from infectious mononucleosis show circulating atypical lymphocytes, which are considered characteristic of this disease. Therefore, when this disorder is suspected, serum analyses are carried out to detect the presence of certain immunoglobulins associated with infectious mononucleosis in the patient's blood. The aim of this study was to evaluate the sensitivity and the specificity of a rapid test detecting heterophil antibodies in diagnosing infectious mononucleosis in a paediatric population. We considered 163 paediatric patients with suspected infectious mononucleosis and we tested their serums to detect heterophil antibodies (using an inexpensive and rapid test) and specific immunoglobulins directed against Epstein-Barr virus (EBV) (these assays are known to be characterized by high sensitivity and specificity, but are more expensive and time-consuming). By comparing the results of the rapid test with those of the other assays, we found that the sensitivity of the first test was 61.8%, whereas its specificity was sufficiently high (about 90%). We show that, in paediatric patients, the detection of heterophil antibodies is not a very sensitive test, therefore the determination of immunoglobulins against specific antigens of EBV is recommended.

Validity of rapid antigen detection testing in group A beta-hemolytic streptococcal tonsillopharyngitis.

PubMed

Küçük, Oznur; Biçer, Suat; Giray, Tuba; Cöl, Defne; Erdağ, Gülay Ciler; Gürol, Yeşim; Kaspar, Ciğdem E; Vitrinel, Ayça

2014-02-01

To evaluate the utility of rapid antigen detection testing (RADT) for the diagnosis of group A beta-hemolytic streptococcal tonsillopharyngitis in children, and to detect the sensitivity and specificity of rapid antigen detection of group A beta-hemolytic streptococci from throat specimen compared with throat culture. Rapid antigen detection and throat culture results for group A beta-hemolytic streptococci from outpatients attending university hospital between 1st January 2011 and 31st of December 2011 were evaluated retrospectively. The antigen test negative-throat culture positive patients were investigated for streptococcal carriage. For this purpose, the throat culture results taken from these patients were reviewed after treatment. Eight hundred and ninetytwo children were included in the studywith a mean age of 5.34 y. There were 639 and 253 children in two groups with age of 0-6 and 7-17 y, RADT sensitivity and specificity were found to be 59.5 % and 97.2 %, respectively. The positive predictive value was 87.1 %, whereas negative predictive value was 88.4 %. After treatment of 74 patients with throat culture positive and antigen test negative. Group A beta-hemolytic streptococci were isolated in 12 of them (16.2 %) and accepted as a carrier. The low sensitivity of the RADT may be related to streptococcal carriage in some patients. The throat culture should be repeated after treatment to detect streptococcal carriage.

Simple, rapid, and cost-effective modified Carba NP test for carbapenemase detection among Gram-negative bacteria

PubMed Central

Rudresh, Shoorashetty Manohara; Ravi, Giriyapur Siddappa; Sunitha, Lakshminarayanappa; Hajira, Sadiya Noor; Kalaiarasan, Ellappan; Harish, Belgode Narasimha

2017-01-01

PURPOSE: Detection of carbapenemases among Gram-negative bacteria (GNB) is important for both clinicians and infection control practitioners. The Clinical and Laboratory Standards Institute recommends Carba NP (CNP) as confirmatory test for carbapenemase production. The reagents required for CNP test are costly and hence the test cannot be performed on a routine basis. The present study evaluates modifications of CNP test for rapid detection of carbapenemases among GNB. MATERIALS AND METHODS: The GNB were screened for carbapenemase production using CNP, CarbAcineto NP (CANP), and modified CNP (mCNP) test. A multiplex polymerase chain reaction (PCR) was performed on all the carbapenem-resistant bacteria for carbapenemase genes. The results of three phenotypic tests were compared with PCR. RESULTS: A total of 765 gram negative bacteria were screened for carbapenem resistance. Carbapenem resistance was found in 144 GNB. The metallo-β-lactamases were most common carbapenemases followed by OXA-48-like enzymes. The CANP test was most sensitive (80.6%) for carbapenemases detection. The mCNP test was 62.1% sensitive for detection of carbapenemases. The mCNP, CNP, and CANP tests were equally sensitive (95%) for detection of NDM enzymes among Enterobacteriaceae. The mCNP test had poor sensitivity for detection of OXA-48-like enzymes. CONCLUSION: The mCNP test was rapid, cost-effective, and easily adoptable on routine basis. The early detection of carbapenemases using mCNP test will help in preventing the spread of multidrug-resistant organisms in the hospital settings. PMID:28966495

Simple, rapid, and cost-effective modified Carba NP test for carbapenemase detection among Gram-negative bacteria.

PubMed

Rudresh, Shoorashetty Manohara; Ravi, Giriyapur Siddappa; Sunitha, Lakshminarayanappa; Hajira, Sadiya Noor; Kalaiarasan, Ellappan; Harish, Belgode Narasimha

2017-01-01

Detection of carbapenemases among Gram-negative bacteria (GNB) is important for both clinicians and infection control practitioners. The Clinical and Laboratory Standards Institute recommends Carba NP (CNP) as confirmatory test for carbapenemase production. The reagents required for CNP test are costly and hence the test cannot be performed on a routine basis. The present study evaluates modifications of CNP test for rapid detection of carbapenemases among GNB. The GNB were screened for carbapenemase production using CNP, CarbAcineto NP (CANP), and modified CNP (mCNP) test. A multiplex polymerase chain reaction (PCR) was performed on all the carbapenem-resistant bacteria for carbapenemase genes. The results of three phenotypic tests were compared with PCR. A total of 765 gram negative bacteria were screened for carbapenem resistance. Carbapenem resistance was found in 144 GNB. The metallo-β-lactamases were most common carbapenemases followed by OXA-48-like enzymes. The CANP test was most sensitive (80.6%) for carbapenemases detection. The mCNP test was 62.1% sensitive for detection of carbapenemases. The mCNP, CNP, and CANP tests were equally sensitive (95%) for detection of NDM enzymes among Enterobacteriaceae. The mCNP test had poor sensitivity for detection of OXA-48-like enzymes. The mCNP test was rapid, cost-effective, and easily adoptable on routine basis. The early detection of carbapenemases using mCNP test will help in preventing the spread of multidrug-resistant organisms in the hospital settings.

Gold Nanoparticles as a Direct and Rapid Sensor for Sensitive Analytical Detection of Biogenic Amines

NASA Astrophysics Data System (ADS)

El-Nour, K. M. A.; Salam, E. T. A.; Soliman, H. M.; Orabi, A. S.

2017-03-01

A new optical sensor was developed for rapid screening with high sensitivity for the existence of biogenic amines (BAs) in poultry meat samples. Gold nanoparticles (GNPs) with particle size 11-19 nm function as a fast and sensitive biosensor for detection of histamine resulting from bacterial decarboxylation of histidine as a spoilage marker for stored poultry meat. Upon reaction with histamine, the red color of the GNPs converted into deep blue. The appearance of blue color favorably coincides with the concentration of BAs that can induce symptoms of poisoning. This biosensor enables a semi-quantitative detection of analyte in real samples by eye-vision. Quality evaluation is carried out by measuring histamine and histidine using different analytical techniques such as UV-vis, FTIR, and fluorescence spectroscopy as well as TEM. A rapid quantitative readout of samples by UV-vis and fluorescence methods with standard instrumentation were proposed in a short time unlike chromatographic and electrophoretic methods. Sensitivity and limit of detection (LOD) of 6.59 × 10-4 and 0.6 μM, respectively, are determined for histamine as a spoilage marker with a correlation coefficient ( R 2) of 0.993.

Synthesis of molecularly imprinted dye-silica nanocomposites with high selectivity and sensitivity: Fluorescent imprinted sensor for rapid and efficient detection of τ-fluvalinate in vodka.

PubMed

Wang, Yunyun; Wang, Jixiang; Cheng, Rujia; Sun, Lin; Dai, Xiaohui; Yan, Yongsheng

2018-04-01

An imprinted fluorescent sensor was fabricated based on SiO 2 nanoparticles encapsulated with a molecularly imprinted polymer containing allyl fluorescein. High fluorine cypermethirin as template molecules, methyl methacrylate as functional monomer, and allyl fluorescein as optical materials synthesized a core-shell fluorescent molecular imprinted sensor, which showed a high and rapid sensitivity and selectivity for the detection of τ-fluvalinate. The sensor presented appreciable sensitivity with a limit of 13.251 nM, rapid detection that reached to equilibrium within 3 min, great linear relationship in the relevant concentration range from 0 to 150 nM, and excellent selectivity over structural analogues. In addition, the fluorescent sensor demonstrated desirable regeneration ability (eight cycling operations). The molecularly imprinted polymers ensured specificity, while the fluorescent dyes provided the stabile sensitivity. Finally, an effective application of the sensor was implemented by the detection of τ-fluvalinate in real samples from vodka. The molecularly imprinted fluorescent sensor showed a promising potential in environmental monitoring and food safety. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lab on a chip sensor for rapid detection and antibiotic resistance determination of Staphylococcus aureus.

PubMed

Abeyrathne, Chathurika D; Huynh, Duc H; Mcintire, Thomas W; Nguyen, Thanh C; Nasr, Babak; Zantomio, Daniela; Chana, Gursharan; Abbott, Iain; Choong, Peter; Catton, Mike; Skafidas, Efstratios

2016-03-21

The Gram-positive bacterium, Staphylococcus aureus (S. aureus), is a major pathogen responsible for a variety of infectious diseases ranging from cellulitis to more serious conditions such as septic arthritis and septicaemia. Timely treatment with appropriate antibiotic therapy is essential to ensure clinical defervescence and to prevent further complications such as infective endocarditis or organ impairment due to septic shock. To date, initial antibiotic choice is empirical, using a "best guess" of likely organism and sensitivity- an approach adopted due to the lack of rapid identification methods for bacteria. Current culture based methods take up to 5 days to identify the causative bacterial pathogen and its antibiotic sensitivity. This paper provides proof of concept for a biosensor, based on interdigitated electrodes, to detect the presence of S. aureus and ascertain its sensitivity to flucloxacillin rapidly (within 2 hours) in a cost effective manner. The proposed method is label-free and uses non-faradic measurements. This is the first study to successfully employ interdigitated electrodes for the rapid detection of antibiotic resistance. The method described has important potential outcomes of faster definitive antibiotic treatment and more rapid clinical response to treatment.

Rapid diagnosis of equine influenza by highly sensitive silver amplification immunochromatography system.

PubMed

Yamanaka, Takashi; Nemoto, Manabu; Bannai, Hiroshi; Tsujimura, Koji; Kondo, Takashi; Matsumura, Tomio; Fu, Tao Qi Huang; Fernandez, Charlene Judith; Gildea, Sarah; Cullinane, Ann

2017-06-16

Equine influenza (EI) is a respiratory disease caused by equine influenza A virus (EIV, H3N8) infection. Rapid diagnosis is essential to limit the disease spread. We previously reported that some rapid antigen detection (RAD) tests are fit for diagnosing EI although their sensitivity is not optimal. Here, we evaluated the performance of the newly developed RAD test using silver amplification immunochromatography (Quick Chaser Auto Flu A, B: QCA) to diagnose EI. The detection limits of QCA for EIVs were five-fold lower than the conventional RAD tests. The duration of virus antigen detection in the infected horses was longer than the conventional RAD tests. We conclude that QCA could be a valuable diagnostic method for EI.

Development of one-step quantitative reverse transcription PCR for the rapid detection of flaviviruses.

PubMed

Patel, Pranav; Landt, Olfert; Kaiser, Marco; Faye, Oumar; Koppe, Tanja; Lass, Ulrich; Sall, Amadou A; Niedrig, Matthias

2013-02-14

The genus Flavivirus includes several pathogenic agents that cause severe illness in humans. Re-emergence of West Nile virus in Europe and continuous spread of certain flaviviruses such as dengue, yellow fever and Japanese encephalitis viruses represent a global danger to public health. Therefore, a rapid and accurate molecular method is required for diagnostics and epidemiological surveillance of flaviviruses. A Pan-Flavi quantitative RT-PCR assay using a Locked-Nucleic Acid probe targeting the flavivirus NS5 gene was developed and optimized to detect a wide range of flaviviruses simultaneously. The specificity and sensitivity of the Pan-Flavi assay were tested using RNA of different flaviviruses and non-flaviviruses. Furthermore, the assay was compared directly to flavivirus species-specific assays for the ability to detect flaviviruses sensitively. Two degenerate primers and one Locked-Nucleic Acids probe were designed to amplify most of the flaviviruses. To increase the specificity and fluorescence signal of the Pan-Flavi assay for detection of yellow fever virus and dengue virus 4, additional primers and probes were included. Viral RNA of thirty different flaviviruses was detected, verifying the broad range specificity. The testing of this assay was successful, using standard plasmid and RNA dilutions of yellow fever virus vaccine strain, dengue virus 1 and tick-borne encephalitis virus, with a sensitivity limit of 10-100 genome copies/reaction. Also comparatively good results were achieved for detecting different flaviviruses by the Pan-Flavi assay when compared to the flavivirus species-specific assays. The assay is rapid, broad-range flavivirus-specific and highly sensitive making it a valuable tool for rapid detection of flaviviruses in livestock samples, epidemiological studies or as useful complement to single flavivirus-specific assays for clinical diagnosis.

A Novel Sensitive Luminescence Probe Microspheres for Rapid and Efficient Detection of τ-Fluvalinate in Taihu Lake

PubMed Central

Wang, Jixiang; Wang, Yunyun; Qiu, Hao; Sun, Lin; Dai, Xiaohui; Pan, Jianming; Yan, Yongsheng

2017-01-01

Fluorescent molecularly imprinted polymers have shown great promise in biological or chemical separations and detection, due to their high stability, selectivity and sensitivity. In this work, fluorescent molecularly imprinted microsphere was synthesized via precipitation polymerization, which could separate efficiently and rapidly detect τ-fluvalinate (a toxic insecticide) in water samples, was reported. The fluorescent imprinted sensor showed excellent stability, outstanding selectivity and the limit of detection low to 12.14 nM, good regeneration ability which still kept good sensitivity after 8 cycling experiments and fluorescence quenching mechanism was illustrated in details. In addition, the fluorescent sensor was further used to detect τ-fluvalinate in real samples from Taihu Lake. Despite the relatively complex components of the environment water, the fluorescent imprinted microspheres sitll showed good recovery, clearly demonstrating the potental value of this smart sensor nanomaterial in environment monitoring. PMID:28485402

Rapid and Sensitive Detection of Lymphocystis Disease Virus Genotype VII by Loop-Mediated Isothermal Amplification.

PubMed

Valverde, Estefanía J; Cano, Irene; Castro, Dolores; Paley, Richard K; Borrego, Juan J

2017-03-01

Lymphocystis disease virus (LCDV) infections have been described in gilthead seabream (Sparus aurata L.) and Senegalese sole (Solea senegalensis, Kaup), two of the most important marine fish species in the Mediterranean aquaculture. In this study, a rapid, specific, and sensitive detection method for LCDV genotype VII based on loop-mediated isothermal amplification (LAMP) was developed. The LAMP assay, performed using an apparatus with real-time amplification monitoring, was able to specifically detect LCDV genotype VII from clinically positive samples in less than 12 min. In addition, the assay allowed the detection of LCDV in all asymptomatic carrier fish analysed, identified by qPCR, showing an analytical sensitivity of ten copies of viral DNA per reaction. The LCDV LAMP assay has proven to be a promising diagnostic method that can be used easily in fish farms to detect the presence and spread of this iridovirus.

A rapid method for the detection of foodborne pathogens by extraction of a trace amount of DNA from raw milk based on amino-modified silica-coated magnetic nanoparticles and polymerase chain reaction.

PubMed

Bai, Yalong; Song, Minghui; Cui, Yan; Shi, Chunlei; Wang, Dapeng; Paoli, George C; Shi, Xianming

2013-07-17

A method based on amino-modified silica-coated magnetic nanoparticles (ASMNPs) and polymerase chain reaction (PCR) was developed to rapidly and sensitively detect foodborne pathogens in raw milk. After optimizing parameters such as pH, temperature, and time, a trace amount of genomic DNA of pathogens could be extracted directly from complex matrices such as raw milk using ASMNPs. The magnetically separated complexes of genomic DNA and ASMNPs were directly subjected to single PCR (S-PCR) or multiplex PCR (M-PCR) to detect single or multiple pathogens from raw milk samples. Salmonella Enteritidis (Gram-negative) and Listeria monocytogenes (Gram-positive) were used as model organisms to artificially contaminate raw milk samples. After magnetic separation and S-PCR, the detection sensitivities were 8 CFU mL(-1) and 13 CFU mL(-1) respectively for these two types of pathogens. Furthermore, this method was successfully used to detect multiple pathogens (S. Enteritidis and L. monocytogenes) from artificially contaminated raw milk using M-PCR at sensitivities of 15 CFU mL(-1) and 25 CFU mL(-1), respectively. This method has great potential to rapidly and sensitively detect pathogens in raw milk or other complex food matrices. Copyright © 2013 Elsevier B.V. All rights reserved.

Rapid and Highly Sensitive Detection of Lead Ions in Drinking Water Based on a Strip Immunosensor

PubMed Central

Kuang, Hua; Xing, Changrui; Hao, Changlong; Liu, Liqiang; Wang, Libing; Xu, Chuanlai

2013-01-01

In this study, we have first developed a rapid and sensitive strip immunosensor based on two heterogeneously-sized gold nanoparticles (Au NPs) probes for the detection of trace lead ions in drinking water. The sensitivity was 4-fold higher than that of the conventional LFA under the optimized conditions. The visual limit of detection (LOD) of the amplified method for qualitative detection lead ions was 2 ng/mL and the LOD for semi-quantitative detection could go down to 0.19 ng/mL using a scanning reader. The method suffered from no interference from other metal ions and could be used to detect trace lead ions in drinking water without sample enrichment. The recovery of the test samples ranged from 96% to 103%. As the detection method could be accomplished within 15 min, this method could be used as a potential tool for preliminary monitoring of lead contamination in drinking water. PMID:23539028

Rapid and sensitive detection of Zika virus by reverse transcription loop-mediated isothermal amplification.

PubMed

Wang, Xuan; Yin, Fenggui; Bi, Yuhai; Cheng, Gong; Li, Jing; Hou, Lidan; Li, Yunlong; Yang, Baozhi; Liu, Wenjun; Yang, Limin

2016-12-01

Zika virus (ZIKV) is an arbovirus that recently emerged and has expanded worldwide, causing a global threat and raising international concerns. Current molecular diagnostics, e.g., real-time PCR and reverse transcription PCR (RT-PCR), are time consuming, expensive, and can only be deployed in a laboratory instead of for field diagnostics. This study aimed to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform showing sensitivity, specificity, and more convenience than previous methods, being easily distributed and implemented. Specific primers were designed and screened to target the entire ZIKV genome. The analytical sensitivity and specificity of the assay were evaluated and compared with traditional PCR and quantitative real-time PCR. Three different simulated clinical sample quick preparation protocols were evaluated to establish a rapid and straightforward treatment procedure for clinical specimens in open field detection. The RT-LAMP assay for detection of ZIKV demonstrated superior specificity and sensitivity compared to traditional PCR at the optimum reaction temperature. For the ZIKV RNA standard, the limit of detection was 20 copies/test. For the simulated ZIKV clinical samples, the limit of detection was 0.02 pfu/test, which was one order of magnitude higher than RT-PCR and similar to real-time PCR. The detection limit of simulated ZIKV specimens prepared using a protease quick processing method was consistent with that of samples prepared using commercial nucleic acid extraction kits, indicating that our ZIKV detection method could be used in point-of-care testing. The RT-LAMP assay had excellent sensitivity and specificity for detecting ZIKV and can be deployed together with a rapid specimen processing method, offering the possibility for ZIKV diagnosis outside of the laboratory. Copyright © 2016 Elsevier B.V. All rights reserved.

Highly sensitive and rapid bacteria detection using molecular beacon-Au nanoparticles hybrid nanoprobes.

PubMed

Cao, Jing; Feng, Chao; Liu, Yan; Wang, Shouyu; Liu, Fei

2014-07-15

Since many diseases are caused by pathogenic bacterial infections, accurate and rapid detection of pathogenic bacteria is in urgent need to timely apply appropriate treatments and to reduce economic costs. To end this, we designed molecular beacon-Au nanoparticle hybrid nanoprobes to improve the bacterial detection efficiency and sensitivity. Here, we show that the designed molecular beacon modified Au nanoparticles could specifically recognize synthetic DNAs targets and can readily detect targets in clinical samples. Moreover, the hybrid nanoprobes can recognize Escherichia coli within an hour at a concentration of 10(2) cfu/ml, which is 1000-folds sensitive than using molecular beacon directly. Our results show that the molecular beacon-Au nanoparticle hybrid nanoprobes have great potential in medical and biological applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Sensitive detection of viable Escherichia coli O157:H7 from foods using a luciferase-reporter phage phiV10lux.

PubMed

Kim, Jinwoo; Kim, Minsik; Kim, Seongmi; Ryu, Sangryeol

2017-08-02

Escherichia coli O157:H7, a major foodborne pathogen, is a major public health concern associated with life-threatening diseases such as hemolytic uremic syndrome. To alleviate this burden, a sensitive and rapid system is required to detect this pathogen in various kinds of foods. Herein, we propose a phage-based pathogen detection method to replace laborious and time-consuming conventional methods. We engineered an E. coli O157:H7-specific phage phiV10 to rapidly and sensitively detect this notorious pathogen. The luxCDABE operon was introduced into the phiV10 genome and allowed the engineered phage phiV10lux to generate bioluminescence proportional to the number of viable E. coli O157:H7 cells without any substrate addition. The phage phiV10lux was able to detect at least 1CFU/ml of E. coli O157:H7 in a pure culture within 40min after 5h of pre-incubation. In artificially contaminated romaine lettuce, apple juice (pH3.51), and ground beef, the reporter phage could detect approximately 10CFU/cm 2 , 13CFU/ml, and 17CFU/g of E. coli O157:H7, respectively. Taken together, the constructed reporter phage phiV10lux could be applied as a powerful tool for rapid and sensitive detection of live E. coli O157:H7 in foods. Copyright © 2017 Elsevier B.V. All rights reserved.

Reverse transcription strand invasion based amplification (RT-SIBA): a method for rapid detection of influenza A and B.

PubMed

Eboigbodin, Kevin; Filén, Sanna; Ojalehto, Tuomas; Brummer, Mirko; Elf, Sonja; Pousi, Kirsi; Hoser, Mark

2016-06-01

Rapid and accurate diagnosis of influenza viruses plays an important role in infection control, as well as in preventing the misuse of antibiotics. Isothermal nucleic acid amplification methods offer significant advantages over the polymerase chain reaction (PCR), since they are more rapid and do not require the sophisticated instruments needed for thermal cycling. We previously described a novel isothermal nucleic acid amplification method, 'Strand Invasion Based Amplification' (SIBA®), with high analytical sensitivity and specificity, for the detection of DNA. In this study, we describe the development of a variant of the SIBA method, namely, reverse transcription SIBA (RT-SIBA), for the rapid detection of viral RNA targets. The RT-SIBA method includes a reverse transcriptase enzyme that allows one-step reverse transcription of RNA to complementary DNA (cDNA) and simultaneous amplification and detection of the cDNA by SIBA under isothermal reaction conditions. The RT-SIBA method was found to be more sensitive than PCR for the detection of influenza A and B and could detect 100 copies of influenza RNA within 15 min. The development of RT-SIBA will enable rapid and accurate diagnosis of viral RNA targets within point-of-care or central laboratory settings.

Rapid analysis of N-linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) by reversed-phase ultra-performance liquid chromatography with fluorescence detection and electrospray ionization time-of-flight mass spectrometry.

PubMed

Kurihara, Takamasa; Min, Jun Zhe; Hirata, Asuka; Toyo'oka, Toshimasa; Inagaki, Shinsuke

2009-05-01

Rapid, selective and sensitive determination of N-linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) was performed by ultra-performance liquid chromatography (UPLC) with fluorescence (FL) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). The asparaginyl-oligosaccharide moiety was first liberated from each glycoprotein by pronase E (a proteolitic enzyme). The oligosaccharide fractions separated by gel-permeation chromatography were labeled with 1-pyrenesulfonyl chloride (PSC, a fluorescence reagent), separated by UPLC in a short run time, and then detected by FL and TOF-MS. The PSC-labeled oligosaccharides were selectively identified from the FL detection and then sensitively determined by ESI-TOF-MS. As the results, 15, eight and four kinds of N-linked oligosaccharides were detected from ovalbumin, ribonuclease B and fetuin, respectively. Because the present method is rapid (within 9 min), selective and sensitive (approximate 60 fmol, S/N = 5), the determination of N-linked oligosaccharides in various glycoproteins seems to be possible.

A novel immunochromatographic electrochemical biosensor for highly sensitive and selective detection of trichloropyridinol, a biomarker of exposure to chlorpyrifos.

PubMed

Wang, Limin; Lu, Donglai; Wang, Jun; Du, Dan; Zou, Zhexiang; Wang, Hua; Smith, Jordan N; Timchalk, Charles; Liu, Fengquan; Lin, Yuehe

2011-02-15

We present a novel portable immunochromatographic electrochemical biosensor (IEB) for simple, rapid, and sensitive biomonitoring of trichloropyridinol (TCP), a metabolite biomarker of exposure to organophosphorus insecticides. Our new approach takes the advantage of immunochromatographic test strip for a rapid competitive immunoreaction and a disposable screen-printed carbon electrode for a rapid and sensitive electrochemical analysis of captured HRP labeling. Several key experimental parameters (e.g. immunoreaction time, the amount of HRP labeled TCP, concentration of the substrate for electrochemical measurements, and the blocking agents for the nitrocellulose membrane) were optimized to achieve a high sensitivity, selectivity and stability. Under optimal conditions, the IEB has demonstrated a wide linear range (0.1-100 ng/ml) with a detection limit as low as 0.1 ng/ml TCP. Furthermore, the IEB has been successfully applied for biomonitoring of TCP in the rat plasma samples with in vivo exposure to organophosphorus insecticides like Chlorpyrifos-oxon (CPF-oxon). The IEB thus opens up new pathways for designing a simple, rapid, clinically accurate, and quantitative tool for TCP detection, as well as holds a great promise for in-field screening of metabolite biomarkers, e.g., TCP, for humans exposed to organophosphorus insecticides. Copyright © 2010 Elsevier B.V. All rights reserved.

Use of rapid-scan EPR to improve detection sensitivity for spin-trapped radicals.

PubMed

Mitchell, Deborah G; Rosen, Gerald M; Tseitlin, Mark; Symmes, Breanna; Eaton, Sandra S; Eaton, Gareth R

2013-07-16

The short lifetime of superoxide and the low rates of formation expected in vivo make detection by standard continuous wave (CW) electron paramagnetic resonance (EPR) challenging. The new rapid-scan EPR method offers improved sensitivity for these types of samples. In rapid-scan EPR, the magnetic field is scanned through resonance in a time that is short relative to electron spin relaxation times, and data are processed to obtain the absorption spectrum. To validate the application of rapid-scan EPR to spin trapping, superoxide was generated by the reaction of xanthine oxidase and hypoxanthine with rates of 0.1-6.0 μM/min and trapped with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO). Spin trapping with BMPO to form the BMPO-OOH adduct converts the very short-lived superoxide radical into a more stable spin adduct. There is good agreement between the hyperfine splitting parameters obtained for BMPO-OOH by CW and rapid-scan EPR. For the same signal acquisition time, the signal/noise ratio is >40 times higher for rapid-scan than for CW EPR. Rapid-scan EPR can detect superoxide produced by Enterococcus faecalis at rates that are too low for detection by CW EPR. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Dual Electrophoresis Detection System for Rapid and Sensitive Immunoassays with Nanoparticle Signal Amplification

NASA Astrophysics Data System (ADS)

Zhang, Fangfang; Ma, Junjie; Watanabe, Junji; Tang, Jinlong; Liu, Huiyu; Shen, Heyun

2017-02-01

An electrophoretic technique was combined with an enzyme-linked immunosorbent assay (ELISA) system to achieve a rapid and sensitive immunoassay. A cellulose acetate filter modified with polyelectrolyte multilayer (PEM) was used as a solid substrate for three-dimensional antigen-antibody reactions. A dual electrophoresis process was used to induce directional migration and local condensation of antigens and antibodies at the solid substrate, avoiding the long diffusion times associated with antigen-antibody reactions in conventional ELISAs. The electrophoretic forces drove two steps in the ELISA process, namely the adsorption of antigen, and secondary antibody-labelled polystyrene nanoparticles (NP-Ab). The total time needed for dual electrophoresis-driven detection was just 4 min, nearly 2 h faster than a conventional ELISA system. Moreover, the rapid NP-Ab electrophoresis system simultaneously achieved amplification of the specific signal and a reduction in noise, leading to a more sensitive NP-Ab immunoassay with a limit of detection (LOD) of 130 fM, and wide range of detectable concentrations from 0.13 to 130 pM. These results suggest that the combination of dual electrophoresis detection and NP-Ab signal amplification has great potential for future immunoassay systems.

Single-layer MnO2 nanosheets for sensitive and selective detection of glutathione by a colorimetric method

NASA Astrophysics Data System (ADS)

Di, Weihua; Zhang, Xiang; Qin, Weiping

2017-04-01

The rapid, sensitive and selective detection of glutathione (GSH) is of great importance in the biological systems. In this work, a template-free and one-step method was used to synthesize the single-layer MnO2 nanosheets via a redox reaction. The resulting product was characterized by XRD, TEM, FTIR, XPS and UV-vis absorption. The addition of GSH results in the change of solution color depth owing to the occurrence of a redox reaction between MnO2 and GSH, enabling colorimetric detection of GSH. At a pH of 3.6, the proposed sensor gives a linear calibration over a GSH concentration range of 10-100 μM, with a rapid response of less than 2 min and a low detection limit of 0.5 μM. The relative standard deviation for seven repeated determinations of GSH is lower than 5.6%. Furthermore, the chemical response of the synthesized MnO2 nanosheets toward GSH is selective. Owing to the advantages with good water solubility, rapid response, high sensitivity, good biocompatibility and operation simplicity, this two-dimensional MnO2-based sensing material might be potential for detecting GSH in biological applications.

Development of an isothermal recombinase polymerase amplification assay for rapid detection of pseudorabies virus.

PubMed

Yang, Yang; Qin, Xiaodong; Zhang, Wei; Li, Zhiyong; Zhang, Shuaijun; Li, Yanmin; Zhang, Zhidong

2017-06-01

Recombinase polymerase amplification assays using real-time fluorescent detection (real-time RPA assay) and lateral flow dipstick (RPA LFD assay) were developed targeting the gD gene of pseudorabies virus (PRV). Both assays were performed at 39 °C within 20 min. The sensitivity of the real-time RPA assay and the RPA LFD assay was 100 copies per reaction and 160 copies per reaction, respectively. Both assays did not detect DNAs from other virus or PRV negative samples. Therefore, the developed RPA assays provide a rapid, simple, sensitive and specific alternative tool for detection of PRV. Copyright © 2017. Published by Elsevier Ltd.

The development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of abalone herpesvirus DNA.

PubMed

Chen, M H; Kuo, S T; Renault, T; Chang, P H

2014-02-01

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of abalone herpesvirus DNA. Two pairs of primers were designed, based on the sequence of the DNA polymerase gene of abalone herpesvirus. The reaction temperature and time were optimized to 63°C and 60min, respectively. LAMP amplicons were analyzed by 2% agarose gel electrophoresis or by visual inspection of a colour change emitted by fluorescent dye. The method developed was specific for the detection of abalone herpesvirus, without cross-reactions with other tested herpesviruses including ostreid herpesvirus 1 (OsHV-1), European eel herpesvirus, koi herpesvirus (KHV) and an avian herpesvirus. The LAMP assay was 100 folds more sensitive than a conventional PCR and 10 folds less sensitive than a SYBR Green PCR. These results indicate that the developed LAMP assay is a simple, rapid, sensitive, specific and reliable technique for the detection of abalone herpesvirus. Copyright © 2013 Elsevier B.V. All rights reserved.

Novel approach based on one-tube nested PCR and a lateral flow strip for highly sensitive diagnosis of tuberculous meningitis

PubMed Central

Sun, Yajuan; Chen, Jiajun; Li, Jia; Xu, Yawei; Jin, Hui; Xu, Na; Yin, Rui

2017-01-01

Rapid and sensitive detection of Mycobacterium tuberculosis (M. Tb) in cerebrospinal fluid is crucial in the diagnosis of tuberculous meningitis (TBM), but conventional diagnostic technologies have limited sensitivity and specificity or are time-consuming. In this work, a novel, highly sensitive molecular diagnostic method, one-tube nested PCR-lateral flow strip test (OTNPCR-LFST), was developed for detecting M. tuberculosis. This one-tube nested PCR maintains the sensitivity of conventional two-step nested PCR and reduces both the chance of cross-contamination and the time required for analysis. The PCR product was detected by a lateral flow strip assay, which provided a basis for migration of the test to a point-of-care (POC) microfluidic format. The developed assay had an improved sensitivity compared with traditional PCR, and the limit of detection was up to 1 fg DNA isolated from M. tuberculosis. The assay was also specific for M. tuberculosis, and no cross-reactions were found in other non-target bacteria. The application of this technique to clinical samples was successfully evaluated, and OTNPCR-LFST showed 89% overall sensitivity and 100% specificity for TBM patients. This one-tube nested PCR-lateral flow strip assay is useful for detecting M. tuberculosis in TBM due to its rapidity, high sensitivity and simple manipulation. PMID:29084241

Monitoring of rapid blood pH variations by CO detection in breath with tunable diode laser

NASA Astrophysics Data System (ADS)

Kouznetsov, Andrian I.; Stepanov, Eugene V.; Zyrianov, Pavel V.; Shulagin, Yurii A.; Diachenko, Alexander I.; Gurfinkel, Youri I.

1997-06-01

Detection of endogenous carbon monoxide content in breath with tunable diode lasers (TDL) was proposed for noninvasive monitoring of rapid blood pH variation. Applied approach is based on high sensitivity of the haemoglobin and myoglobin affinity for CO to blood pH value and an ability to detect rapidly small variations of CO content in expired air. Breath CO absorption in 4.7 micrometers spectral region was carefully measured using PbSSe tunable diode laser that can provide 1 ppb CO concentration sensitivity and 10 s time constant. Applied TDL gas analyzer was used to monitor expired air of studied persons in physiological tests including hyperventilation and physical load. Simultaneous blood tests were conducted to demonstrate correlation between blood and breath chemical parameters.

Rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) by real-time, isothermal recombinase polymerase amplification assay.

PubMed

Xia, Xiaoming; Yu, Yongxin; Hu, Linghao; Weidmann, Manfred; Pan, Yingjie; Yan, Shuling; Wang, Yongjie

2015-04-01

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) causes mortality or runt deformity syndrome in penaeid shrimps and is responsible for significant economic losses in the shrimp aquaculture industry. Here, we describe a novel real-time isothermal recombinase polymerase amplification (RPA) assay developed for IHHNV detection. Using IHHNV plasmid standards and DNA samples from a variety of organisms, we evaluated the ability of the IHHNV-RPA assay to detect IHHNV based on analysis of its sensitivity, specificity, rapidity, and reproducibility. Probit analysis of eight independent experimental replicates indicated satisfactory performance of the RPA assay, which is sufficiently sensitive to detect as few as 4 copies of the IHHNV genome within 7 min at 39 °C with 95 % reliability. Therefore, this rapid RPA method has great potential for applications, either in field use or as a point of care diagnostic technique.

Development of a rapid recombinase polymerase amplification assay for detection of Brucella in blood samples.

PubMed

Ren, Hang; Yang, Mingjuan; Zhang, Guoxia; Liu, Shiwei; Wang, Xinhui; Ke, Yuehua; Du, Xinying; Wang, Zhoujia; Huang, Liuyu; Liu, Chao; Chen, Zeliang

2016-04-01

A rapid and sensitive recombinase polymerase amplification (RPA) assay, Bruce-RPA, was developed for detection of Brucella. The assay could detect as few as 3 copies of Brucella per reaction within 20 min. Bruce-RPA represents a candidate point-of-care diagnosis assay for human brucellosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Advances in developing rapid, reliable and portable detection systems for alcohol.

PubMed

Thungon, Phurpa Dema; Kakoti, Ankana; Ngashangva, Lightson; Goswami, Pranab

2017-11-15

Development of portable, reliable, sensitive, simple, and inexpensive detection system for alcohol has been an instinctive demand not only in traditional brewing, pharmaceutical, food and clinical industries but also in rapidly growing alcohol based fuel industries. Highly sensitive, selective, and reliable alcohol detections are currently amenable typically through the sophisticated instrument based analyses confined mostly to the state-of-art analytical laboratory facilities. With the growing demand of rapid and reliable alcohol detection systems, an all-round attempt has been made over the past decade encompassing various disciplines from basic and engineering sciences. Of late, the research for developing small-scale portable alcohol detection system has been accelerated with the advent of emerging miniaturization techniques, advanced materials and sensing platforms such as lab-on-chip, lab-on-CD, lab-on-paper etc. With these new inter-disciplinary approaches along with the support from the parallel knowledge growth on rapid detection systems being pursued for various targets, the progress on translating the proof-of-concepts to commercially viable and environment friendly portable alcohol detection systems is gaining pace. Here, we summarize the progress made over the years on the alcohol detection systems, with a focus on recent advancement towards developing portable, simple and efficient alcohol sensors. Copyright © 2017 Elsevier B.V. All rights reserved.

Time-Resolved Fluorescent Immunochromatography of Aflatoxin B1 in Soybean Sauce: A Rapid and Sensitive Quantitative Analysis.

PubMed

Wang, Du; Zhang, Zhaowei; Li, Peiwu; Zhang, Qi; Zhang, Wen

2016-07-14

Rapid and quantitative sensing of aflatoxin B1 with high sensitivity and specificity has drawn increased attention of studies investigating soybean sauce. A sensitive and rapid quantitative immunochromatographic sensing method was developed for the detection of aflatoxin B1 based on time-resolved fluorescence. It combines the advantages of time-resolved fluorescent sensing and immunochromatography. The dynamic range of a competitive and portable immunoassay was 0.3-10.0 µg·kg(-1), with a limit of detection (LOD) of 0.1 µg·kg(-1) and recoveries of 87.2%-114.3%, within 10 min. The results showed good correlation (R² > 0.99) between time-resolved fluorescent immunochromatographic strip test and high performance liquid chromatography (HPLC). Soybean sauce samples analyzed using time-resolved fluorescent immunochromatographic strip test revealed that 64.2% of samples contained aflatoxin B1 at levels ranging from 0.31 to 12.5 µg·kg(-1). The strip test is a rapid, sensitive, quantitative, and cost-effective on-site screening technique in food safety analysis.

Rapid and sensitive microRNA detection with laminar flow-assisted dendritic amplification on power-free microfluidic chip.

PubMed

Arata, Hideyuki; Komatsu, Hiroshi; Hosokawa, Kazuo; Maeda, Mizuo

2012-01-01

Detection of microRNAs, small noncoding single-stranded RNAs, is one of the key topics in the new generation of cancer research because cancer in the human body can be detected or even classified by microRNA detection. This report shows rapid and sensitive microRNA detection using a power-free microfluidic device, which is driven by degassed poly(dimethylsiloxane), thus eliminating the need for an external power supply. MicroRNA is detected by sandwich hybridization, and the signal is amplified by laminar flow-assisted dendritic amplification. This method allows us to detect microRNA of specific sequences at a limit of detection of 0.5 pM from a 0.5 µL sample solution with a detection time of 20 min. Together with the advantages of self-reliance of this device, this method might contribute substantially to future point-of-care early-stage cancer diagnosis.

Establishment and Application of a Loop-Mediated Isothermal Amplification Method for Simple, Specific, Sensitive and Rapid Detection of Toxoplasma gondii

PubMed Central

CAO, Lili; CHENG, Ronghua; YAO, Lin; YUAN, Shuxian; YAO, Xinhua

2013-01-01

ABSTRACT The Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high simply, specificity, sensitivity and rapidity. In this study, A LAMP assay with 6 primers targeting a highly conserved region of the GRA1 gene was developed to diagnose Toxoplasma gondii. The reaction time of the LAMP assay was shortened to 30 min after optimizing the reaction system. The LAMP assay was found to be highly specific and stable. The detection limit of the LAMP assay was 10 copies, the same as that of the conventional PCR. We used the LAMP assay to develop a real-time fluorogenic protocol to quantitate T. gondii DNA and generated a log-linear regression plot by plotting the time-to-threshold values against genomic equivalent copies. Furthermore, the LAMP assay was applied to detect T. gondii DNA in 423 blood samples and 380 lymph node samples from 10 pig farms, and positive results were obtained for 7.8% and 8.2% of samples, respectively. The results showed that the LAMP method is slightly more sensitive than conventional PCR (6.1% and 7.6%). Positive samples obtained from 6 pig farms. The LAMP assay established in this study resulted in simple, specific, sensitive and rapid detection of T. gondii DNA and is expected to play an important role in clinical detection of T. gondii. PMID:23965849

Nanoswitch-linked immunosorbent assay (NLISA) for fast, sensitive, and specific protein detection.

PubMed

Hansen, Clinton H; Yang, Darren; Koussa, Mounir A; Wong, Wesley P

2017-09-26

Protein detection and quantification play critical roles in both basic research and clinical practice. Current detection platforms range from the widely used ELISA to more sophisticated, and more expensive, approaches such as digital ELISA. Despite advances, there remains a need for a method that combines the simplicity and cost-effectiveness of ELISA with the sensitivity and speed of modern approaches in a format suitable for both laboratory and rapid, point-of-care applications. Building on recent developments in DNA structural nanotechnology, we introduce the nanoswitch-linked immunosorbent assay (NLISA), a detection platform based on easily constructed DNA nanodevices that change conformation upon binding to a target protein with the results read out by gel electrophoresis. NLISA is surface-free and includes a kinetic-proofreading step for purification, enabling both enhanced sensitivity and reduced cross-reactivity. We demonstrate femtomolar-level detection of prostate-specific antigen in biological fluids, as well as reduced cross-reactivity between different serotypes of dengue and also between a single-mutation and wild-type protein. NLISA is less expensive, uses less sample volume, is more rapid, and, with no washes, includes fewer hands-on steps than ELISA, while also achieving superior sensitivity. Our approach also has the potential to enable rapid point-of-care assays, as we demonstrate by performing NLISA with an iPad/iPhone camera for imaging.

RAMA casein zymography: Time-saving and highly sensitive casein zymography for MMP7 and trypsin.

PubMed

Yasumitsu, Hidetaro; Ozeki, Yasuhiro; Kanaly, Robert A

2016-11-01

To detect metalloproteinase-7 (MMP7), zymography is conducted using a casein substrate and conventional CBB stain. It has disadvantages because it is time consuming and has low sensitivity. Previously, a sensitive method to detect MMP7 up to 30 pg was reported, however it required special substrates and complicated handlings. RAMA casein zymography described herein is rapid, sensitive, and reproducible. By applying high-sensitivity staining with low substrate conditions, the staining process is completed within 1 h and sensitivity was increased 100-fold. The method can detect 10 pg MMP7 by using commercially available casein without complicated handlings. Moreover, it increases detection sensitivity for trypsin. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of an immunochromatographic assay for the detection of anti-hepatitis A virus IgM

PubMed Central

2010-01-01

Background Hepatitis A virus (HAV) is a causative agent of acute hepatitis, which is transmitted by person-to-person contact and via the faecal-oral route. Acute HAV infection is usually confirmed by anti-HAV IgM detection. In order to detect anti-HAV IgM in the serum of patients infected with HAV, we developed a rapid assay based on immunochromatography (ICA) and evaluated the sensitivity of this assay by comparing it with a commercial microparticle enzyme immunoassay (MEIA) that is widely used for serological diagnosis. Results The newly developed ICA showed 100% sensitivity and specificity when used to test 150 anti-HAV IgM-positive sera collected from infected patients and 75 negative sera from healthy subjects. Also, the sensitivity of ICA is about 10 times higher than MEIA used in this study by determining end point to detect independent on infected genotype of HAV. In addition, the ICA was able to detect 1 positive sample from among 50 sera from acute hepatitis patients that had tested negative for anti-HAV IgM using the MEIA. Conclusion Conclusively, ICA for the detection of anti-HAV IgM will be very effective for rapid assay to apply clinical diagnosis and epidemiological investigation on epidemics due to the simplicity, rapidity and specificity. PMID:20637129

Bioengineering bacteriophages to enhance the sensitivity of phage amplification-based paper fluidic detection of bacteria.

PubMed

Alcaine, S D; Law, K; Ho, S; Kinchla, A J; Sela, D A; Nugen, S R

2016-08-15

Bacteriophage (phage) amplification is an attractive method for the detection of bacteria due to a narrow phage-host specificity, short amplification times, and the phages' ability to differentiate between viable and non-viable bacterial cells. The next step in phage-based bacteria detection is leveraging bioengineered phages to create low-cost, rapid, and easy-to-use detection platforms such as lateral flow assays. Our work establishes the proof-of-concept for the use of bioengineered T7 phage strains to increase the sensitivity of phage amplification-based lateral flow assays. We have demonstrated a greater than 10-fold increase in sensitivity using a phage-based protein reporter, maltose-binding protein, over the detection of replicated T7 phage viron itself, and a greater then 100-fold increase in sensitivity using a phage-based enzymatic reporter, alkaline phosphatase. This increase in sensitivity enabled us to detect 10(3)CFU/mL of Escherichia coli in broth after 7h, and by adding a filter concentration step, the ability to detect a regulatory relevant E. coli concentration of 100CFU/100mL in inoculated river water after 9h, where the current standard requires days for results. The combination of the paper fluidic format with phage-based detection provides a platform for the development of novel diagnostics that are sensitive, rapid, and easy to use. Copyright © 2016 Elsevier B.V. All rights reserved.

A rapid and sensitive dot-blot hybridization assay for the detection of citrus exocortis viroid in Citrus medica with digoxigenin-labelled RNA probes.

PubMed

Fonseca, M E; Marcellino, L H; Gander, E

1996-04-05

A rapid and sensitive dot-blot hybridization assay using in vitro-transcribed digoxigenin-labelled RNA probes (riboprobes) was developed aiming at detection of citrus exocortis viroid (CEVd) in crude sap of infected Citrus medica plants. The protocol includes a very quick and simple preparation of RNA extracts from samples using a denaturation step with formaldehyde. From our results, the employment of this step is highly recommended because the hybridization signals in formaldehyde-denatured samples were significantly stronger when compared with that of extracts without formaldehyde treatment. The assay was found to be sensitive enough to detect 0.1 ng of purified CEVd RNA and was able to detect viroid in 0.2 mg of symptomatic Citrus medica leaves. The use of riboprobes also allowed hybridization under high temperature conditions, avoiding non-specific background.

An insulated isothermal PCR method on a field-deployable device for rapid and sensitive detection of canine parvovirus type 2 at points of need.

PubMed

Wilkes, Rebecca P; Lee, Pei-Yu A; Tsai, Yun-Long; Tsai, Chuan-Fu; Chang, Hsiu-Hui; Chang, Hsiao-Fen G; Wang, Hwa-Tang T

2015-08-01

Canine parvovirus type 2 (CPV-2), including subtypes 2a, 2b and 2c, causes an acute enteric disease in both domestic and wild animals. Rapid and sensitive diagnosis aids effective disease management at points of need (PON). A commercially available, field-deployable and user-friendly system, designed with insulated isothermal PCR (iiPCR) technology, displays excellent sensitivity and specificity for nucleic acid detection. An iiPCR method was developed for on-site detection of all circulating CPV-2 strains. Limit of detection was determined using plasmid DNA. CPV-2a, 2b and 2c strains, a feline panleukopenia virus (FPV) strain, and nine canine pathogens were tested to evaluate assay specificity. Reaction sensitivity and performance were compared with an in-house real-time PCR using serial dilutions of a CPV-2b strain and 100 canine fecal clinical samples collected from 2010 to 2014, respectively. The 95% limit of detection of the iiPCR method was 13 copies of standard DNA and detection limits for CPV-2b DNA were equivalent for iiPCR and real-time PCR. The iiPCR reaction detected CPV-2a, 2b and 2c and FPV. Non-targeted pathogens were not detected. Test results of real-time PCR and iiPCR from 99 fecal samples agreed with each other, while one real-time PCR-positive sample tested negative by iiPCR. Therefore, excellent agreement (k = 0.98) with sensitivity of 98.41% and specificity of 100% in detecting CPV-2 in feces was found between the two methods. In conclusion, the iiPCR system has potential to serve as a useful tool for rapid and accurate PON, molecular detection of CPV-2. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Rapid and specific detection of Asian- and African-lineage Zika viruses.

PubMed

Chotiwan, Nunya; Brewster, Connie D; Magalhaes, Tereza; Weger-Lucarelli, James; Duggal, Nisha K; Rückert, Claudia; Nguyen, Chilinh; Garcia Luna, Selene M; Fauver, Joseph R; Andre, Barb; Gray, Meg; Black, William C; Kading, Rebekah C; Ebel, Gregory D; Kuan, Guillermina; Balmaseda, Angel; Jaenisch, Thomas; Marques, Ernesto T A; Brault, Aaron C; Harris, Eva; Foy, Brian D; Quackenbush, Sandra L; Perera, Rushika; Rovnak, Joel

2017-05-03

Understanding the dynamics of Zika virus transmission and formulating rational strategies for its control require precise diagnostic tools that are also appropriate for resource-poor environments. We have developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay that distinguishes Zika viruses of Asian and African lineages. The assay does not detect chikungunya virus or flaviviruses such as dengue, yellow fever, or West Nile viruses. The assay conditions allowed direct detection of Zika virus RNA in cultured infected cells; in mosquitoes; in virus-spiked samples of human blood, plasma, saliva, urine, and semen; and in infected patient serum, plasma, and semen samples without the need for RNA isolation or reverse transcription. The assay offers rapid, specific, sensitive, and inexpensive detection of the Asian-lineage Zika virus strain that is currently circulating in the Western hemisphere, and can also detect the African-lineage Zika virus strain using separate, specific primers. Copyright © 2017, American Association for the Advancement of Science.

Interdigitated microelectrode based impedance biosensor for detection of salmonella enteritidis in food samples

NASA Astrophysics Data System (ADS)

Kim, G.; Morgan, M.; Hahm, B. K.; Bhunia, A.; Mun, J. H.; Om, A. S.

2008-03-01

Salmonella enteritidis outbreaks continue to occur, and S. enteritidis-related outbreaks from various food sources have increased public awareness of this pathogen. Conventional methods for pathogens detection and identification are labor-intensive and take days to complete. Some immunological rapid assays are developed, but these assays still require prolonged enrichment steps. Recently developed biosensors have shown great potential for the rapid detection of foodborne pathogens. To develop the biosensor, an interdigitated microelectrode (IME) was fabricated by using semiconductor fabrication process. Anti-Salmonella antibodies were immobilized based on avidin-biotin binding on the surface of the IME to form an active sensing layer. To increase the sensitivity of the sensor, three types of sensors that have different electrode gap sizes (2 μm, 5 μm, 10 μm) were fabricated and tested. The impedimetric biosensor could detect 103 CFU/mL of Salmonella in pork meat extract with an incubation time of 5 minutes. This method may provide a simple, rapid and sensitive method to detect foodborne pathogens.

Electrochemical Biosensor for Rapid and Sensitive Detection of Magnetically Extracted Bacterial Pathogens

PubMed Central

Setterington, Emma B.; Alocilja, Evangelyn C.

2012-01-01

Biological defense and security applications demand rapid, sensitive detection of bacterial pathogens. This work presents a novel qualitative electrochemical detection technique which is applied to two representative bacterial pathogens, Bacillus cereus (as a surrogate for B. anthracis) and Escherichia coli O157:H7, resulting in detection limits of 40 CFU/mL and 6 CFU/mL, respectively, from pure culture. Cyclic voltammetry is combined with immunomagnetic separation in a rapid method requiring approximately 1 h for presumptive positive/negative results. An immunofunctionalized magnetic/polyaniline core/shell nano-particle (c/sNP) is employed to extract target cells from the sample solution and magnetically position them on a screen-printed carbon electrode (SPCE) sensor. The presence of target cells significantly inhibits current flow between the electrically active c/sNPs and SPCE. This method has the potential to be adapted for a wide variety of target organisms and sample matrices, and to become a fully portable system for routine monitoring or emergency detection of bacterial pathogens. PMID:25585629

Colorimetric and fluorescent detection of hydrazine with high sensitivity and excellent selectivity

NASA Astrophysics Data System (ADS)

Shi, Bingjie; Qi, Sujie; Yu, Mingming; Liu, Chunxia; Li, Zhanxian; Wei, Liuhe; Ni, Zhonghai

2018-01-01

It is critical to develop probes for rapid, selective, and sensitive detection of the highly toxic hydrazine in both environmental and biological science. In this work, under mild condition, a novel colorimetric and off-on fluorescent probe was synthesized for rapid recognition of hydrazine with excellent selectivity over other various species including some biological species, metal ions and anions. The limit of quantification (LOQ) value was 1.5 × 10- 4 M-3.2 × 10- 3 M (colorimetric method) and 1.5 × 10- 4 M - 3.2 × 10- 3 M (fluorescent method) with as low as detection limit of 46.2 μM.

Investigation of the Sensitivity, Selectivity, and Reversibility of the Chemically-Sensitive Field-Effect Transistor (CHEMFET) to Detect Nitrogen Dioxide, Dimethyl Methylphosphonate, and Boron Trifluoride

DTIC Science & Technology

1993-09-01

SENSITIVE FIELD- EFFECT TRANSISTOR (CHEMFET) TO DETECT NITROGEN DIOXIDE, DIMETHYL METHYLPHOSPHONATE, AND BORON TRIFLUORIDE CHAPTER 1 1 Introduction Our rapidly...AND REVERSIBILITY OF THE CHEMICALLY-SENSITIVE FIELD- EFFECT TRANSISTOR (CHEMFET) TO DETECT NITROGEN 3 E I1• DIOXIDE, DIMETHYL METHYLPHOSPHONATE, ELECTE...AND BORON TRIFLUORIDE Neal Terence Hauschild Second Lieutenant, USAF AFIT/GE/ENG/9 3S-10 93-23815I II11l11l11 l gll I 1i 1111 11 I DEPARTMENT OF THE

CdSe/ZnS Quantum Dot-Labeled Lateral Flow Strips for Rapid and Quantitative Detection of Gastric Cancer Carbohydrate Antigen 72-4

NASA Astrophysics Data System (ADS)

Yan, Xinyu; Wang, Kan; Lu, Wenting; Qin, Weijian; Cui, Daxiang; He, Jinghua

2016-03-01

Carbohydrate antigen 72-4 (CA72-4) is an important biomarker associated closely with diagnosis and prognosis of early gastric cancer. How to realize quick, sensitive, specific, and quantitative detection of CA72-4 in clinical specimens has become a great requirement. Herein, we reported a CdSe/ZnS quantum dot-labeled lateral flow test strip combined with a charge-coupled device (CCD)-based reader was developed for rapid, sensitive, and quantitative detection of CA72-4. Two mouse monoclonal antibodies (mAbs) against CA72-4 were employed. One of them was coated as a test line, while another mAb was labeled with quantum dots and coated onto conjugate pad. The goat anti-mouse IgG was immobilized as a control line. After sample was added, a sandwich structure was formed with CA72-4 and these two mAbs. The fluorescent signal from quantum dots (QD)-labeled mAb in sandwich structure was related to the amount of detected CA72-4. A CCD-based reader was used to realize quantitative detection of CA72-4. Results showed that developed QD-labeled lateral flow strips to detect CA72-4 biomarker with the sensitivity of 2 IU/mL and 10 min detection time. One hundred sera samples from clinical patients with gastric cancer and healthy people were used to confirm specificity of this strip method; results showed that established strip method own 100 % reproducibility and 100 % specificity compared with Roche electrochemiluminescence assay results. In conclusion, CdSe/ZnS quantum dot-labeled lateral flow strips for detection of CA72-4 could realize rapid, sensitive, and specific detection of clinical samples and could own great potential in clinical translation in near future.

Evaluation of wondfo rapid diagnostic kit (Pf-HRP2/PAN-pLDH) for diagnosis of malaria by using nano-gold immunochromatographic assay.

PubMed

Wu, Junlin; Peng, Yunping; Liu, Xiaoyun; Li, Wenmei; Tang, Shixing

2014-06-01

Prompt and accurate diagnosis is necessary to start adequate treatment for different affecting species including P. falciparum and P. vivax. Here we described the Wondfo Rapid diagnostic Kit (Pf-HRP2/PAN-pLDH) for the detection of P. falciparum and pan-plasmodium in patient specimen by using a nano-gold immunochromatographic assay. Our rapid assay adapted nano-gold labeling techniques and the monoclonal antibodies (mAbs) against both histidine rich protein-2 (Pf HRP-2) of P. falciparum and pan plasmodium-specific pLDH (pan pLDH). The established two-antibody sandwich immunochromatographic assay could detect P. falciparum and pan-plasmodium. The sensitivity and specificity of Wondfo rapid diagnostic kit were determined by comparing with the "gold standard" of microscopic examination of blood smears. In this study1023 blood samples were collected from outpatient clinics in China and Burma, and detected by both Wondfo kit and microscopic examination. The detection sensitivity and specificity of Wondfo rapid diagnostic kit were 96.46% and 99.67% for P. falciparum (HRP2), 95.03% and 99.24% for pLDH, 96.83% and 99.74% for non-falciparum species, 96.70% and 99.74% for P. vivax, respectively. These results indicate that Wondfo rapid diagnostic assay may be useful for detecting P. falciparum and non-P. falciparum (especially P.v.) in patient specimen.

Recent developments in detection and enumeration of waterborne bacteria: a retrospective minireview.

PubMed

Deshmukh, Rehan A; Joshi, Kopal; Bhand, Sunil; Roy, Utpal

2016-12-01

Waterborne diseases have emerged as global health problems and their rapid and sensitive detection in environmental water samples is of great importance. Bacterial identification and enumeration in water samples is significant as it helps to maintain safe drinking water for public consumption. Culture-based methods are laborious, time-consuming, and yield false-positive results, whereas viable but nonculturable (VBNCs) microorganisms cannot be recovered. Hence, numerous methods have been developed for rapid detection and quantification of waterborne pathogenic bacteria in water. These rapid methods can be classified into nucleic acid-based, immunology-based, and biosensor-based detection methods. This review summarizes the principle and current state of rapid methods for the monitoring and detection of waterborne bacterial pathogens. Rapid methods outlined are polymerase chain reaction (PCR), digital droplet PCR, real-time PCR, multiplex PCR, DNA microarray, Next-generation sequencing (pyrosequencing, Illumina technology and genomics), and fluorescence in situ hybridization that are categorized as nucleic acid-based methods. Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence are classified into immunology-based methods. Optical, electrochemical, and mass-based biosensors are grouped into biosensor-based methods. Overall, these methods are sensitive, specific, time-effective, and important in prevention and diagnosis of waterborne bacterial diseases. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Sensitive and specific detection of classical scrapie prions in the brain of goats by real-time quaking-induced conversion

USDA-ARS?s Scientific Manuscript database

The real-time quaking-induced conversion (RT-QuIC) is a rapid, specific, and sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect sub-infectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully us...

Evaluation of phage assay for rapid phenotypic detection of rifampicin resistance in Mycobacterium tuberculosis

PubMed Central

Yzquierdo, Sergio Luis; Lemus, Dihadenys; Echemendia, Miguel; Montoro, Ernesto; McNerney, Ruth; Martin, Anandi; Palomino, Juan Carlos

2006-01-01

Background Conventional methods for susceptibility testing require several months before results can be reported. However, rapid methods to determine drug susceptibility have been developed recently. Phage assay have been reported as a rapid useful tools for antimicrobial susceptibility testing. The aim of this study was to apply the Phage assay for rapid detection of resistance on Mycobacterium tuberculosis strains in Cuba. Methods Phage D29 assay was performed on 102 M. tuberculosis strains to detect rifampicin resistance. The results were compared with the proportion method (gold standard) to evaluate the sensitivity and specificity of Phage assay. Results Phage assay results were available in 2 days whereas Proportion Methods results were obtain in 42 days. A total of 44 strains were detected as rifampicin resistant by both methods. However, one strains deemed resistant by Proportion Methods was susceptible by Phage assay. The sensitivity and specificity of Phage assay were 97.8 % and 100% respectively. Conclusion Phage assay provides rapid and reliable results for susceptibility testing; it's easy to perform, requires no specialized equipment and is applicable to drug susceptibility testing in low income countries where tuberculosis is a major public health problem. PMID:16630356

Detection of shigella in lettuce by the use of a rapid molecular assay with increased sensitivity

PubMed Central

Jiménez, Kenia Barrantes; McCoy², Clyde B.; Achí, Rosario

2010-01-01

A Multiplex Polymerase Chain Reaction (PCR) assay to be used as an alternative to the conventional culture method in detecting Shigella and enteroinvasive Escherichia coli (EIEC) virulence genes ipaH and ial in lettuce was developed. Efficacy and rapidity of the molecular method were determined as compared to the conventional culture. Lettuce samples were inoculated with different Shigella flexneri concentrations (from 10 CFU/ml to 107 CFU/ml). DNA was extracted directly from lettuce after inoculation (direct-PCR) and after an enrichment step (enrichment PCR). Multiplex PCR detection limit was 104CFU/ml, diagnostic sensitivity and specificity were 100% accurate. An internal amplification control (IAC) of 100 bp was used in order to avoid false negative results. This method produced results in 1 to 2 days while the conventional culture method required 5 to 6 days. Also, the culture method detection limit was 106 CFU/ml, diagnostic sensitivity was 53% and diagnostic specificity was 100%. In this study a Multiplex PCR method for detection of virulence genes in Shigella and EIEC was shown to be effective in terms of diagnostic sensitivity, detection limit and amount of time as compared to Shigella conventional culture. PMID:24031579

A portable smart-phone device for rapid and sensitive detection of E. coli O157:H7 in Yoghurt and Egg.

PubMed

Zeinhom, Mohamed Maarouf Ali; Wang, Yijia; Song, Yang; Zhu, Mei-Jun; Lin, Yuehe; Du, Dan

2018-01-15

The detection of E. coli O157:H7 in foods has held the attention of many researchers because of the seriousness attributed to this pathogen. In this study, we present a simple, sensitive, rapid and portable smartphone based fluorescence device for E. coli O157:H7 detection. This field-portable fluorescent imager on the smartphone involves a compact laser-diode-based photosource, a long-pass (LP) thin-film interference filter and a high-quality insert lenses. The design of the device provided a low noise to background imaging system. Based on a sandwich ELISA and the specific recognition of antibody to E. coli O157:H7, the sensitive detection of E. coli O157:H7 were realized both in standard samples and real matrix in yoghurt and egg on our device. The detection limit are 1 CFU/mL and 10 CFU/mL correspondingly. Recovery percentages of spiked yogurt and egg samples with 10 3 , 10 4 and 10 5 CFU/mL E. coli O157:H7 were 106.98, 96.52 and 102.65 (in yogurt) and 107.37, 105.64 and 93.84 (in egg) samples using our device, respectively. Most importantly, the entire process could be quickly completed within two hours. This smartphone based device provides a simple, rapid, sensitive detection platform for fluorescent imaging which applied in pathogen detection for food safety monitoring. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid and highly sensitive detection of Enterovirus 71 by using nanogold-enhanced electrochemical impedance spectroscopy

NASA Astrophysics Data System (ADS)

Li, Hsing-Yuan; Tseng, Shing-Hua; Cheng, Tsai-Mu; Chu, Hsueh-Liang; Lu, Yu-Ning; Wang, Fang-Yu; Tsai, Li-Yun; Shieh, Juo-Yu; Yang, Jyh-Yuan; Juan, Chien-Chang; Tu, Lung-Chen; Chang, Chia-Ching

2013-07-01

Enterovirus 71 (EV71) infection is an emerging infectious disease causing neurological complications and/or death within two to three days after the development of fever and rash. A low viral titre in clinical specimens makes the detection of EV71 difficult. Conventional approaches for detecting EV71 are time consuming, poorly sensitive, or complicated, and cannot be used effectively for clinical diagnosis. Furthermore, EV71 and Coxsackie virus A16 (CA16) may cross react in conventional assays. Therefore, a rapid, highly sensitive, specific, and user-friendly test is needed. We developed an EV71-specific nanogold-modified working electrode for electrochemical impedance spectroscopy in the detection of EV71. Our results show that EV71 can be distinguished from CA16, Herpes simplex virus, and lysozyme, with the modified nanogold electrode being able to detect EV71 in concentrations as low as 1 copy number/50 μl reaction volume, and the duration between sample preparation and detection being 11 min. This detection platform may have the potential for use in point-of-care diagnostics.

Multiplex qPCR for reliable detection and differentiation of Burkholderia mallei and Burkholderia pseudomallei.

PubMed

Janse, Ingmar; Hamidjaja, Raditijo A; Hendriks, Amber C A; van Rotterdam, Bart J

2013-02-14

Burkholderia mallei and B. pseudomallei are two closely related species of highly virulent bacteria that can be difficult to detect. Pathogenic Burkholderia are endemic in many regions worldwide and cases of infection, sometimes brought by travelers from unsuspected regions, also occur elsewhere. Rapid, sensitive methods for identification of B. mallei and B. pseudomallei are urgently needed in the interests of patient treatment and epidemiological surveillance. Signature sequences for sensitive, specific detection of pathogenic Burkholderia based on published genomes were identified and a qPCR assay was designed and validated. A single-reaction quadruplex qPCR assay for the detection of pathogenic Burkholderia, which includes a marker for internal control of DNA extraction and amplification, was developed. The assay permits differentiation of B. mallei and B. pseudomallei strains, and probit analysis showed a very low detection limit. Use of a multicopy signature sequence permits detection of less than 1 genome equivalent per reaction. The new assay permits rapid detection of pathogenic Burkholderia and combines enhanced sensitivity, species differentiation, and inclusion of an internal control for both DNA extraction and PCR amplification.

Detection of HbsAg and hATIII genetically modified goats (Caprahircus) by loop-mediated isothermal amplification.

PubMed

Tao, Chenyu; Zhang, Qingde; Zhai, Shanli; Liu, Bang

2013-11-01

In this study, sensitive and rapid detection systems were designed using a loop-mediated isothermal amplification (LAMP) method to detect the genetically modified goats. A set of 4 primers were designed for each exogenous nucleic acids HBsAg and hATIII. The DNA samples were first amplified with the outer and inner primers and released a single-stranded DNA,of which both ends were stem-loop structure. Then one inner primer hybridized with the loop, and initiated displacement synthesis in less than 1 h. The result could be visualized by both agarose gel electrophoresis and unaided eyes directly after adding SYBR GREEN 1. The detection limit of LAMP was ten copies of target molecules, indicating that LAMP was tenfold more sensitive than the classical PCR. Furthermore, all the samples of genetically modified goats were tested positively by LAMP, and the results demonstrated that the LAMP was a rapid and sensitive method for detecting the genetically modified organism.

Rapid and Sensitive Enumeration of Viable Diluted Cells of Members of the Family Enterobacteriaceae in Freshwater and Drinking Water

PubMed Central

Baudart, Julia; Coallier, Josée; Laurent, Patrick; Prévost, Michèle

2002-01-01

Water quality assessment involves the specific, sensitive, and rapid detection of bacterial indicators and pathogens in water samples, including viable but nonculturable (VBNC) cells. This work evaluates the specificity and sensitivity of a new method which combines a fluorescent in situ hybridization (FISH) approach with a physiological assay (direct viable count [DVC]) for the direct enumeration, at the single-cell level, of highly diluted viable cells of members of the family Enterobacteriaceae in freshwater and drinking water after membrane filtration. The approach (DVC-FISH) uses a new direct detection device, the laser scanning cytometer (Scan RDI). Combining the DVC-FISH method on a membrane with Scan RDI detection makes it possible to detect as few as one targeted cell in approximately 108 nontargeted cells spread over the membrane. The ability of this new approach to detect and enumerate VBNC enterobacterial cells in freshwater and drinking water distribution systems was investigated and is discussed. PMID:12324357

Evaluation of a Cryptococcal antigen Lateral Flow Assay in serum and cerebrospinal fluid for rapid diagnosis of cryptococcosis in Colombia.

PubMed

Cáceres, Diego H; Zuluaga, Alejandra; Tabares, Ángela M; Chiller, Tom; González, Ángel; Gómez, Beatriz L

2017-12-21

A Lateral Flow Assay to detect cryptococcal antigen (CrAg® LFA) in serum and cerebrospinal fluid for the rapid diagnosis of cryptococcosis was evaluated. A retrospective validation was performed. Sensitivity and specificity of the CrAg® LFA was 100%. High concordance (kappa index=1.0) between Cryptococcal Antigen Latex Agglutination System (CALAS®) and CrAg® LFA was observed. CrAg® LFA showed higher analytical sensitivity for detecting low concentrations of cryptococcal antigen.

Evaluation of a Cryptococcal antigen Lateral Flow Assay in serum and cerebrospinal fluid for rapid diagnosis of cryptococcosis in Colombia

PubMed Central

Cáceres, Diego H.; Zuluaga, Alejandra; Tabares, Ángela M.; Chiller, Tom; González, Ángel; Gómez, Beatriz L.

2017-01-01

ABSTRACT A Lateral Flow Assay to detect cryptococcal antigen (CrAg® LFA) in serum and cerebrospinal fluid for the rapid diagnosis of cryptococcosis was evaluated. A retrospective validation was performed. Sensitivity and specificity of the CrAg® LFA was 100%. High concordance (kappa index=1.0) between Cryptococcal Antigen Latex Agglutination System (CALAS®) and CrAg® LFA was observed. CrAg® LFA showed higher analytical sensitivity for detecting low concentrations of cryptococcal antigen. PMID:29267584

Advancing the speed, sensitivity and accuracy of biomolecular detection using multi-length-scale engineering

PubMed Central

Kelley, Shana O.; Mirkin, Chad A.; Walt, David R.; Ismagilov, Rustem F.; Toner, Mehmet; Sargent, Edward H.

2015-01-01

Rapid progress in identifying disease biomarkers has increased the importance of creating high-performance detection technologies. Over the last decade, the design of many detection platforms has focused on either the nano or micro length scale. Here, we review recent strategies that combine nano- and microscale materials and devices to produce large improvements in detection sensitivity, speed and accuracy, allowing previously undetectable biomarkers to be identified in clinical samples. Microsensors that incorporate nanoscale features can now rapidly detect disease-related nucleic acids expressed in patient samples. New microdevices that separate large clinical samples into nanocompartments allow precise quantitation of analytes, and microfluidic systems that utilize nanoscale binding events can detect rare cancer cells in the bloodstream more accurately than before. These advances will lead to faster and more reliable clinical diagnostic devices. PMID:25466541

Advancing the speed, sensitivity and accuracy of biomolecular detection using multi-length-scale engineering

NASA Astrophysics Data System (ADS)

Kelley, Shana O.; Mirkin, Chad A.; Walt, David R.; Ismagilov, Rustem F.; Toner, Mehmet; Sargent, Edward H.

2014-12-01

Rapid progress in identifying disease biomarkers has increased the importance of creating high-performance detection technologies. Over the last decade, the design of many detection platforms has focused on either the nano or micro length scale. Here, we review recent strategies that combine nano- and microscale materials and devices to produce large improvements in detection sensitivity, speed and accuracy, allowing previously undetectable biomarkers to be identified in clinical samples. Microsensors that incorporate nanoscale features can now rapidly detect disease-related nucleic acids expressed in patient samples. New microdevices that separate large clinical samples into nanocompartments allow precise quantitation of analytes, and microfluidic systems that utilize nanoscale binding events can detect rare cancer cells in the bloodstream more accurately than before. These advances will lead to faster and more reliable clinical diagnostic devices.

Development of a loop-mediated isothermal amplification assay for rapid detection of Burkholderia mallei.

PubMed

Mirzai, S; Safi, S; Mossavari, N; Afshar, D; Bolourchian, M

2016-08-31

The present study was conducted to establish a Loop-mediated isothermal amplification (LAMP) technique for the rapid detection of B. mallei the etiologic agent of glanders, a highly contagious disease of equines. A set of six specific primers targeting integrase gene cluster were designed for the LAMP test. The reaction was optimized using different temperatures and time intervals. The specificity of the assay was evaluated using DNA from B.pseudomallei and Pseudomonas aeruginosa. The LAMP products were analyzed both visually and under UV light after electrophoresis. The optimized conditions were found to be at 63ºC for 60 min. The assay showed high specificity and sensitivity. It was concluded that the established LAMP assay is a rapid, sensitive and practical tool for detection of B. mallei and early diagnosis of glanders.

Black phosphorus nanosheets for rapid microRNA detection.

PubMed

Zhou, Jie; Li, Zhongjun; Ying, Ming; Liu, Maixian; Wang, Xiaomei; Wang, Xianyou; Cao, Liwei; Zhang, Han; Xu, Gaixia

2018-03-15

Herein, for the first time, a sensitive sensing platform for rapid detection of microRNA was developed by employing black phosphorus nanosheets as the fluorescence quenching material. The biosensor displayed a good linear response to microRNA ranging from 10 nM to 1000 nM. Moreover, the biosensor could distinguish triple nucleotide polymorphism.

A novel fluorescent probe for rapid and sensitive detection of hydrogen sulfide in living cells

NASA Astrophysics Data System (ADS)

Pan, Jian; Xu, Junchao; Zhang, Youlai; Wang, Liang; Qin, Caiqin; Zeng, Lintao; Zhang, Yue

2016-11-01

A novel fluorescent probe for H2S was developed based on a far-red emitting indole-BODIPY, which was decorated with morpholine and 2,4-dinitrobenzenesulfonyl (DNBS) group. This probe showed rapid response (t1/2 = 3 min), high selectivity and sensitivity for H2S with significant colorimetric and fluorescence OFF-ON signals, which was triggered by cleavage of 2,4-dinitrobenzenesulfonyl group. This probe could quantitatively detect the concentrations of H2S ranging from 0 to 60 μM, and the detection of limit was found to be as low as 26 nM. Cell imaging results indicated that the probe could detect and visualize H2S in the living cells.

Whole-bacterium SELEX of DNA aptamers for rapid detection of E.coli O157:H7 using a QCM sensor.

PubMed

Yu, Xiaofan; Chen, Fang; Wang, Ronghui; Li, Yanbin

2018-01-20

The rapid detection of foodborne pathogens is critical to ensure food safety. The objective of this study is to select aptamers specifically bound to Escherichia coli O157:H7 using the whole-bacterium SELEX (Systematic Evolution of Ligands by Exponential Enrichment) and apply the selected aptamer to a QCM (quartz crystal microbalance) sensor for rapid and sensitive detection of target bacteria. A total of 19 rounds of selection against live E. coli O157:H7 and 6 rounds of counter selection against a mixture of Staphylococcus aureus, Listeria monocytogenes, and Salmonella Typhimurium, were performed. The aptamer pool from the last round was cloned and sequenced. One sequence S1 that appeared 16 times was characterized and a dissociation constant (K d ) of 10.30nM was obtained. Subsequently, a QCM aptasensor was developed for the rapid detection of E. coli O157:H7. The limit of detection (LOD) and the detection time of the aptasensor was determined to be 1.46×10 3 CFU/ml and 50min, respectively. This study demonstrated that the ssDNA aptamer selected by the whole-bacterium SELEX possessed higher sensitivity than previous work and the potential use of the constructed QCM aptasensor in rapid screening of foodborne pathogens. Copyright © 2017 Elsevier B.V. All rights reserved.

Tag Array gene chip rapid diagnosis anti-tuberculosis drug resistance in pulmonary tuberculosis -a feasibility study.

PubMed

Wu, Wenjie; Cheng, Peng; Lyu, Jingtong; Zhang, Zehua; Xu, Jianzhong

2018-05-01

We developed a Tag Array chip for detecting first- and second-line anti tuberculosis drug resistance in pulmonary tuberculosis and compared the analytical performance of the gene chip to that of phenotypic drug susceptibility testing (DST). From November 2011 to April 2016.234 consecutive culture-confirmed, clinically and imaging diagnosed patients with pulmonary tuberculosis from Southwest Hospital, Chongqing were enrolled into the study. Specimens collected during sputum or bronchoalveolar lavage fluid from the pulmonary tuberculosis patients were subjected to M. tuberculosis species identification and drug-resistance detection by the Tag Array gene chip, and evaluate the sensitivity and specificity of chip. A total of 186 patients was diagnosed drug-resistant tuberculosis. The detection of rifampicin (RFP), isoniazid (INH), fluoroquinolones (FQS), streptomycin (SM) resistance genes was highly sensitive and specific: however, for detection of amikacin (AMK), capreomycin (CPM), Kanamycin (KM), specificity was higher, but sensitivity was lower. Sensitivity for the detection of a mutation in the eis promoter region could be improved. The detection sensitivity of the EMB resistance gene was low, therefore it is easy to miss a diagnosis of EMB drug resistance, but its specificity was high. Tag Array chip can achieve rapid, accurate and high-throughput detection of tuberculosis resistance in pulmonary tuberculosis, which has important clinical significance and feasibility. Copyright © 2018. Published by Elsevier Ltd.

Evaluation of a rapid immunochromatographic ODK-0901 test for detection of pneumococcal antigen in middle ear fluids and nasopharyngeal secretions.

PubMed

Hotomi, Muneki; Togawa, Akihisa; Takei, Shin; Sugita, Gen; Sugita, Rinya; Kono, Masamitsu; Fujimaki, Yutaka; Kamide, Yosuke; Uchizono, Akihiro; Kanesada, Keiko; Sawada, Shoichi; Okitsu, Naohiro; Tanaka, Yumi; Saijo, Yoko; Yamanaka, Noboru

2012-01-01

Since the incidence of penicillin-resistant Streptococcus pneumoniae has been increasing at an astonishing rate throughout the world, the need for accurate and rapid identification of pneumococci has become increasingly important to determine the appropriate antimicrobial treatment. We have evaluated an immunochromatographic test (ODK-0901) that detects pneumococcal antigens using 264 middle ear fluids (MEFs) and 268 nasopharyngeal secretions (NPSs). A sample was defined to contain S. pneumoniae when optochin and bile sensitive alpha hemolytic streptococcal colonies were isolated by culture. The sensitivity and specificity of the ODK-0901 test were 81.4% and 80.5%, respectively, for MEFs from patients with acute otitis media (AOM). In addition, the sensitivity and specificity were 75.2% and 88.8%, respectively, for NPSs from patients with acute rhinosinusitis. The ODK-0901 test may provide a rapid and highly sensitive evaluation of the presence of S. pneumoniae and thus may be a promising method of identifying pneumococci in MEFs and NPSs.

Evaluation of a Rapid Immunochromatographic ODK-0901 Test for Detection of Pneumococcal Antigen in Middle Ear Fluids and Nasopharyngeal Secretions

PubMed Central

Hotomi, Muneki; Togawa, Akihisa; Takei, Shin; Sugita, Gen; Sugita, Rinya; Kono, Masamitsu; Fujimaki, Yutaka; Kamide, Yosuke; Uchizono, Akihiro; Kanesada, Keiko; Sawada, Shoichi; Okitsu, Naohiro; Tanaka, Yumi; Saijo, Yoko; Yamanaka, Noboru

2012-01-01

Since the incidence of penicillin-resistant Streptococcus pneumoniae has been increasing at an astonishing rate throughout the world, the need for accurate and rapid identification of pneumococci has become increasingly important to determine the appropriate antimicrobial treatment. We have evaluated an immunochromatographic test (ODK-0901) that detects pneumococcal antigens using 264 middle ear fluids (MEFs) and 268 nasopharyngeal secretions (NPSs). A sample was defined to contain S. pneumoniae when optochin and bile sensitive alpha hemolytic streptococcal colonies were isolated by culture. The sensitivity and specificity of the ODK-0901 test were 81.4% and 80.5%, respectively, for MEFs from patients with acute otitis media (AOM). In addition, the sensitivity and specificity were 75.2% and 88.8%, respectively, for NPSs from patients with acute rhinosinusitis. The ODK-0901 test may provide a rapid and highly sensitive evaluation of the presence of S. pneumoniae and thus may be a promising method of identifying pneumococci in MEFs and NPSs. PMID:22448257

A Highly Sensitive Method for Quantitative Determination of Abscisic Acid 1

PubMed Central

Michler, Charles H.; Lineberger, R. Daniel; Chism, Grady W.

1986-01-01

An abscisic acid derivative was formed by reaction with pentafluorobenzyl bromide which allowed highly sensitive detection by gas-liquid chromatography with electron capture detection. In comparison to the methyl ester derivative, the pentafluorobenzyl derivative of abscisic acid was four times more sensitive to electron capture detection and was stable at room temperature in the presence of ultraviolet light. Derivatization was rapid and the molecular weight of the new compound was confirmed by gas-liquid chromatography-mass spectrometry. PMID:16665076

An ultrasensitive alloyed near-infrared quinternary quantum dot-molecular beacon nanodiagnostic bioprobe for influenza virus RNA.

PubMed

Adegoke, Oluwasesan; Kato, Tatsuya; Park, Enoch Y

2016-06-15

Conventional techniques used to diagnose influenza virus face several challenges, such as low sensitivity, slow detection, false positive results and misinterpreted data. Hence, diagnostic probes that can offer robust detection qualities, such as high sensitivity, rapid detection, elimination of false positive data, and specificity for influenza virus, are urgently needed. The near-infrared (NIR) range is an attractive spectral window due to low photon absorption by biological tissues, hence well-constructed fluorescent biosensors that emit within the NIR window can offer an improved limit of detection (LOD). Here, we demonstrate the use of a newly synthesized NIR quinternary alloyed CdZnSeTeS quantum dots (QDs) as an ultrasensitive fluorescence reporter in a conjugated molecular beacon (MB) assay to detect extremely low concentrations of influenza virus H1N1 RNA. Under optimum conditions, two different strains of influenza virus H1N1 RNA were detected based on fluorescence enhancement signal transduction. We successfully discriminated between two different strains of influenza virus H1N1 RNA based on the number of complementary nucleotide base pairs of the MB to the target RNA sequence. The merits of our bioprobe system are rapid detection, high sensitivity (detects H1N1 viral RNA down to 2 copies/mL), specificity and versatility (detects H1N1 viral RNA in human serum). For comparison, a conventional CdSe/ZnS-MB probe could not detect the extremely low concentrations of H1N1 viral RNA detected by our NIR alloyed CdZnSeTeS-MB probe. Our bioprobe detection system produced a LOD as low as ~1 copy/mL and is more sensitive than conventional molecular tests and rapid influenza detection tests (RIDTS) probes. Copyright © 2016 Elsevier B.V. All rights reserved.

Commercial lateral flow devices for rapid detection of peanut (Arachis hypogaea) and hazelnut (Corylus avellana) cross-contamination in the industrial production of cookies.

PubMed

Röder, Martin; Vieths, Stefan; Holzhauser, Thomas

2009-09-01

Lateral flow devices (LFDs) are qualitative immunochromatographic tests for the rapid and specific detection of target analytes. We investigated commercially available LFDs for their ability to detect potentially allergenic peanut and hazelnut in raw cookie dough and chocolate, two important food matrices in the industrial production of cookies. Each three commercial LFDs for the detection of hazelnut and peanut were performed according to the manufacturers' instructions. All LFDs had comparably satisfactory specificity that was investigated with a variety of characteristic foods and food ingredients used in the production of cookies. In concordance with hazelnut-specific enzyme-linked immunosorbent assays (ELISAs), walnut was the most cross-reactive food for hazelnut-specific LFD. The sensitivity was verified in raw cookie doughs and chocolates that were either spiked with peanut or hazelnut between 1 and 25 mg/kg, respectively. Two hazelnut-specific LFDs detected hazelnut at a level of 3.5 mg/kg in both matrices, whereas the third LFD detected hazelnut at a level of 3.9 mg/kg in dough and 12.5 mg/kg in chocolate. Two peanut-specific LFDs detected peanut at a level of 1 mg/kg in both matrices. The third LFD detected peanut at a level of 14.2 mg/kg in chocolate and 4 mg/kg in dough. In conclusion, specific and sensitive LFD were identified for each hazelnut and peanut, having a level of sensitivity that is comparable to commercial ELISA for the investigated matrices. Such sensitive, specific, and rapid tests are useful analytical tools for allergen screening and sanitation in the industrial manufacture of foods.

Aptamer-based microspheres for highly sensitive protein detection using fluorescently-labeled DNA nanostructures.

PubMed

Han, Daehoon; Hong, Jinkee; Kim, Hyun Cheol; Sung, Jong Hwan; Lee, Jong Bum

2013-11-01

Many highly sensitive protein detection techniques have been developed and have played an important role in the analysis of proteins. Herein, we report a novel technique that can detect proteins sensitively and effectively using aptamer-based DNA nanostructures. Thrombin was used as a target protein and aptamer was used to capture fluorescent dye-labeled DNA nanobarcodes or thrombin on a microsphere. The captured DNA nanobarcodes were replaced by a thrombin and aptamer interaction. The detection ability of this approach was confirmed by flow cytometry with different concentrations of thrombin. Our detection method has great potential for rapid and simple protein detection with a variety of aptamers.

Rapid, quantitative and sensitive immunochromatographic assay based on stripping voltammetric detection of a metal ion label

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Fang; Wang, Kaihua; Lin, Yuehe

2005-10-10

A novel, sensitive immunochromatographic electrochemical biosensor (IEB) which combines an immunochromatographic strip technique with an electrochemical detection technique is demonstrated. The IEB takes advantages of the speed and low-cost of the conventional immunochromatographic test kits and high-sensitivity of stripping voltammetry. Bismuth ions (Bi3+) have been coupled with the antibody through the bifunctional chelating agent diethylenetriamine pentaacetic acid (DTPA). After immunoreactions, Bi3+ was released and quantified by anodic stripping voltammetry at a built-in single-use screen-printed electrode. As an example for the applications of such novel device, the detection of human chorionic gonadotronphin (HCG) in a specimen was performed. This biosensor providesmore » a more user-friendly, rapid, clinically accurate, and less expensive immunoassay for such analysis in specimens than currently available test kits.« less

Detection of Delta9-tetrahydrocannabinol and amphetamine-type stimulants in oral fluid using the Rapid Stat point-of-collection drug-testing device.

PubMed

Röhrich, J; Zörntlein, S; Becker, J; Urban, R

2010-04-01

The Rapid Stat assay, a point-of-collection drug-testing device for detection of amphetamines, cannabinoids, cocaine, opiates, methadone, and benzodiazepines in oral fluid, was evaluated for cannabis and amphetamine-type stimulants. The Rapid Stat tests (n = 134) were applied by police officers in routine traffic checks. Oral fluid and blood samples were analyzed using gas chromatography-mass spectrometry (GC-MS) for Delta(9)-tetrahydrocannabinol, amphetamine, methamphetamine, methylenedioxymethamphetamine, methylenedioxyethylamphetamine, and methylenedioxyamphetamine. The comparison of GC-MS analysis of oral fluid with the Rapid Stat results for cannabis showed a sensitivity of 85%, a specificity of 87%, and a total confirmation rate of 87%. When compared with serum, the sensitivity of the cannabis assay decreased to 71%, the specificity to 60%, and the total confirmation rate to 66%. These findings were possibly caused by an incorrect reading of the THC test results. Comparison of the Rapid Stat amphetamine assay with GC-MS in oral fluid showed a sensitivity of 94%, a specificity of 97%, and a total confirmation rate of 97%. Compared with serum, a sensitivity of 100%, a specificity of 90%, and a total confirmation rate of 92% was found. The amphetamine assay must, therefore, be regarded as satisfactory.

Rapid, cost-effective, sensitive and quantitative detection of Acinetobacter baumannii from pneumonia patients

PubMed Central

Nomanpour, B; Ghodousi, A; Babaei, A; Abtahi, HR; Tabrizi, M; Feizabadi, MM

2011-01-01

Background and Objectives Pneumonia with Acinetobacter baumannii has a major therapeutic problem in health care settings. Decision to initiate correct antibiotic therapy requires rapid identification and quantification of organism. The aim of this study was to develop a rapid and sensitive method for direct detection of A. baumannii from respiratory specimens. Materials and Methods A Taqman real time PCR based on the sequence of bla oxa-51 was designed and used for direct detection of A. baumannii from 361 respiratory specimens of patients with pneumonia. All specimens were checked by conventional bacteriology in parallel. Results The new real time PCR could detect less than 200 cfu per ml of bacteria in specimens. There was agreement between the results of real time PCR and culture (Kappa value 1.0, p value<0.001). The sensitivity, specificity and predictive values of real time PCR were 100%. The prevalence of A. baumannii in pneumonia patients was 10.53 % (n=38). Poly-microbial infections were detected in 65.71% of specimens. Conclusion Acinetobacter baumannii is the third causative agent in nosocomial pneumonia after Pseudomonas aeroginosa (16%) and Staphylococcus aureus (13%) at Tehran hospitals. We recommend that 104 CFU be the threshold for definition of infection with A. baumannii using real time PCR. PMID:22530083

Rapid Detection of Ebola Virus with a Reagent-Free, Point-of-Care Biosensor

PubMed Central

Baca, Justin T.; Severns, Virginia; Lovato, Debbie; Branch, Darren W.; Larson, Richard S.

2015-01-01

Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. This preliminary study demonstrates SAW biosensor detection of the Ebola virus in a concentration-dependent manner. The detection limit with this methodology is below the average level of viremia detected on the first day of symptoms by PCR. We observe a log-linear sensor response for highly fragmented Ebola viral particles, with a detection limit corresponding to 1.9 × 104 PFU/mL prior to virus inactivation. We predict greatly improved sensitivity for intact, infectious Ebola virus. This point-of-care methodology has the potential to detect Ebola viremia prior to symptom onset, greatly enabling infection control and rapid treatment. This biosensor platform is powered by disposable AA batteries and can be rapidly adapted to detect other emerging diseases in austere conditions. PMID:25875186

Development of a double-antibody sandwich ELISA for rapid detection of Bacillus Cereus in food

PubMed Central

Zhu, Longjiao; He, Jing; Cao, Xiaohan; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

2016-01-01

Bacillus cereus is increasingly recognized as one of the major causes of food poisoning in the industrialized world. In this paper, we describe a sensitive double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) that was developed for rapid detection of B. cereus in food to minimize the risk of contamination. The polyclonal antibody (pAb) and monoclonal antibodies (mAbs) specific to B. cereus were generated from rabbit antiserum and mouse ascites, respectively, using the octanoic acid/saturated ammonium sulfate precipitation method and protein A-sepharose columns. IgG-isotype mAbs were specially developed to undergo a novel peripheral multiple sites immunization for rapid gain of hybridomas and a subtractive screen was used to eliminate cross reactivity with closely related species such as Bacillus thuringiensis, B. subtilis, B. licheniformis and B. perfringens. The linear detection range of the method was approximately 1 × 104–2.8 × 106 cells/mL with a detection limit (LOD) of 0.9 × 103 cells/mL. The assay was able to detect B. cereus when the samples were prepared in meat with various pathogens. The newly developed analytical method provides a rapid method to sensitively detect B. cereus in food specimens. PMID:26976753

Spatial and temporal processing in healthy aging: implications for perceptions of driving skills.

PubMed

Conlon, Elizabeth; Herkes, Kathleen

2008-07-01

Sensitivity to the attributes of a stimulus (form or motion) and accuracy when detecting rapidly presented stimulus information were measured in older (N = 36) and younger (N = 37) groups. Before and after practice, the older group was significantly less sensitive to global motion (but not to form) and less accurate on a rapid sequencing task when detecting the individual elements presented in long but not short sequences. These effect sizes produced power for the different analyses that ranged between 0.5 and 1.00. The reduced sensitivity found among older individuals to temporal but not spatial stimuli, adds support to previous findings of a selective age-related deficit in temporal processing. Older women were significantly less sensitive than older men, younger men and younger women on the global motion task. Gender effects were evident when, in response to global motion stimuli, complex extraction and integration processes needed to be undertaken rapidly. Significant moderate correlations were found between age, global motion sensitivity and reports of perceptions of other vehicles and road signs when driving. These associations suggest that reduced motion sensitivity may produce functional difficulties for the older adults when judging speeds or estimating gaps in traffic while driving.

Development and clinical evaluation of a rapid diagnostic kit for feline leukemia virus infection.

PubMed

Kim, Won-Shik; Chong, Chom-Kyu; Kim, Hak-Yong; Lee, Gyu-Cheol; Jeong, Wooseog; An, Dong-Jun; Jeoung, Hye-Young; Lee, Jae-In; Lee, Young-Ki

2014-01-01

Feline leukemia virus (FeLV) causes a range of neoplastic and degenerative diseases in cats. To obtain a more sensitive and convenient diagnosis of the disease, we prepared monoclonal antibodies specific for the FeLV p27 to develop a rapid diagnostic test with enhanced sensitivity and specificity. Among these antibodies, we identified two clones (hybridomas 8F8B5 and 8G7D1) that specifically bound to FeLV and were very suitable for a diagnostic kit. The affinity constants for 8F8B5 and 8G7D1 were 0.35 × 10⁸ and 0.86 × 10⁸, respectively. To investigate the diagnostic abilities of the rapid kit using these antibodies, we performed several clinical studies. Assessment of analytical sensitivity revealed that the detection threshold of the rapid diagnostic test was 2 ng/mL for recombinant p27 and 12.5 × 10⁴ IU/mL for FeLV. When evaluating 252 cat sera samples, the kit was found to have a kappa value of 0.88 compared to polymerase chain reaction (PCR), indicating a significant correlation between data from the rapid diagnostic test and PCR. Sensitivity and specificity of the kit were 95.2% (20/21) and 98.5% (257/261), respectively. Our results demonstrated that the rapid diagnostic test would be a suitable diagnostic tool for the rapid detection of FeLV infection in cats.

Rapid, highly sensitive detection of herpes simplex virus-1 using multiple antigenic peptide-coated superparamagnetic beads.

PubMed

Ran, Ying-Fen; Fields, Conor; Muzard, Julien; Liauchuk, Viktoryia; Carr, Michael; Hall, William; Lee, Gil U

2014-12-07

A sensitive, rapid, and label free magnetic bead aggregation (MBA) assay has been developed that employs superparamagnetic (SPM) beads to capture, purify, and detect model proteins and the herpes simplex virus (HSV). The MBA assay is based on monitoring the aggregation state of a population of SPM beads using light scattering of individual aggregates. A biotin-streptavidin MBA assay had a femtomolar (fM) level sensitivity for analysis times less than 10 minutes, but the response of the assay becomes nonlinear at high analyte concentrations. A MBA assay for the detection of HSV-1 based on a novel peptide probe resulted in the selective detection of the virus at concentrations as low as 200 viral particles (vp) per mL in less than 30 min. We define the parameters that determine the sensitivity and response of the MBA assay, and the mechanism of enhanced sensitivity of the assay for HSV. The speed, relatively low cost, and ease of application of the MBA assay promise to make it useful for the identification of viral load in resource-limited and point-of-care settings where molecular diagnostics cannot be easily implemented.

Urchin-like gold nanoparticle-based immunochromatographic strip test for rapid detection of fumonisin B1 in grains.

PubMed

Ren, Wenjie; Huang, Zhibing; Xu, Yang; Li, Yanping; Ji, Yanwei; Su, Baowei

2015-09-01

An immunochromatographic strip (ICS) using urchin-like gold nanoparticles (UGNs) for sensitive detection of fumonisin B1 (FB1) was developed to meet the requirement for rapidly monitoring FB1 in grain samples. The sensitivity of the ICS was 5.0 ng/mL, which represents a fourfold increase in sensitivity over conventional strip preparation using colloidal gold as the antibody-labeled probe. Analysis of FB1 in grain samples showed that data obtained from the strip tests were in a good agreement with those obtained from HPLC and enzyme-linked immunosorbent assays (ELISAs). This qualitative test did not require any specialized equipment, and the detection time was less than 5 min, which is suitable for on-site testing of FB1 in grain samples. Overall, to our knowledge, this is the first report of using a UGN as the antibody-labeled probe for sensitive detection of FB1 in grains using an ICS. Graphical Abstract Preparation of ICS using conventional colloidal gold and urchin-like gold nanoparticle, respectively.

Nucleic Acid-Based Approaches for Detection of Viral Hepatitis

PubMed Central

Behzadi, Payam; Ranjbar, Reza; Alavian, Seyed Moayed

2014-01-01

Context: To determining suitable nucleic acid diagnostics for individual viral hepatitis agent, an extensive search using related keywords was done in major medical library and data were collected, categorized, and summarized in different sections. Results: Various types of molecular biology tools can be used to detect and quantify viral genomic elements and analyze the sequences. These molecular assays are proper technologies for rapidly detecting viral agents with high accuracy, high sensitivity, and high specificity. Nonetheless, the application of each diagnostic method is completely dependent on viral agent. Conclusions: Despite rapidity, automation, accuracy, cost-effectiveness, high sensitivity, and high specificity of molecular techniques, each type of molecular technology has its own advantages and disadvantages. PMID:25789132

Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification

USDA-ARS?s Scientific Manuscript database

A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39 °C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in ...

Multi-colored immunochromatography using nanobeads for rapid and sensitive typing of seasonal influenza viruses.

PubMed

Sakurai, Akira; Takayama, Katsuyoshi; Nomura, Namiko; Yamamoto, Naoki; Sakoda, Yoshihiro; Kobayashi, Yukuharu; Kida, Hiroshi; Shibasaki, Futoshi

2014-12-01

Immunochromatography (IC) is an antigen-detection assay that plays an important role in the rapid diagnosis of influenza viruses because of its rapid turnaround and ease of use. Despite the usefulness of IC, the limit of detection of common IC kits is as high as 10(3)-10(4) plaque forming units (pfu) per reaction, resulting in their limited sensitivities. Early diagnosis within 24h would provide more appropriate timing of treatment. In this study, a multi-colored NanoAct™ bead IC was established to detect seasonal influenza viruses. This method has approximately 10-fold higher sensitivity than that of colloidal gold or colored latex bead IC assays, and does not require specific instruments. More notably, NanoAct™ bead IC can distinguish influenza A and B viruses from clinical samples with a straightforward readout composed of colored lines. Our results will provide new strategies for the diagnosis, treatment, and a chance to survey of influenza viruses in developing countries and in the field research. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid and sensitive multiplex single-tube nested PCR for the identification of five human Plasmodium species.

PubMed

Saito, Takahiro; Kikuchi, Aoi; Kaneko, Akira; Isozumi, Rie; Teramoto, Isao; Kimura, Masatsugu; Hirasawa, Noriyasu; Hiratsuka, Masahiro

2018-06-01

Malaria is caused by five species of Plasmodium in humans. Microscopy is currently used for pathogen detection, requiring considerable training and technical expertise as the parasites are often difficult to differentiate morphologically. Rapid diagnostic tests are as reliable as microscopy and offer faster diagnoses but possess lower detection limits and are incapable of distinguishing among the parasitic species. To improve global health efforts towards malaria control, a rapid, sensitive, species-specific, and economically viable diagnostic method is needed. In this study, we designed a malaria diagnostic method involving a multiplex single-tube nested PCR targeting Plasmodium mitochondrial cytochrome c oxidase III and single-stranded tag hybridization chromatographic printed-array strip. The detection sensitivity was found to be at least 40 times higher than that of agarose gel electrophoresis with ethidium bromide. This system also enables the identification of both single- and mixed-species malaria infections. The assay was validated with 152 Kenyan samples; using nested PCR as the standard, the assay's sensitivity and specificity were 88.7% and 100.0%, respectively. The turnaround time required, from PCR preparation to signal detection, is 90min. Our method should improve the diagnostic speed, treatment efficacy, and control of malaria, in addition to facilitating surveillance within global malaria eradication programs. Copyright © 2018 Elsevier B.V. All rights reserved.

Direct detection of Leishmania from clinical samples.

PubMed

Waitumbi, John N; Bast, Joshua; Nyakoe, Nancy; Magiri, Charles; Quintana, Miguel; Takhampunya, Ratree; Schuster, Anthony L; Van de Wyngaerde, Marshall T; McAvin, James C; Coleman, Russell E

2017-01-01

The ability to rapidly and accurately diagnose leishmaniasis is a military priority. Testing was conducted to evaluate diagnostic sensitivity and specificity of field-expedient Leishmania genus and visceral Leishmania specific dual-fluorogenic, hydrolysis probe (TaqMan), polymerase chain reaction assays previously established for use in vector surveillance. Blood samples of patients with confirmed visceral leishmaniasis and controls without the disease from Baringo District, Kenya, were tested. Leishmania genus assay sensitivity was 100% (14/14) and specificity was 84% (16/19). Visceral Leishmania assay sensitivity was 93% (13/14) and specificity 80% (4/5). Cutaneous leishmaniasis (CL) skin scrapes of patients from Honduras were also evaluated. Leishmania genus assay sensitivity was 100% (10/10). Visceral Leishmania assay specificity was 100% (10/10) from cutaneous leishmaniasis samples; no fluorescence above background was reported. These results show promise in a rapid, sensitive, and specific method for Leishmania direct detection from clinical samples.

Rapid single cell detection of Staphylococcus aureus by aptamer-conjugated gold nanoparticles

PubMed Central

Chang, Yi-Chung; Yang, Chia-Ying; Sun, Ruei-Lin; Cheng, Yi-Feng; Kao, Wei-Chen; Yang, Pan-Chyr

2013-01-01

Staphylococcus aureus is one of the most important human pathogens, causing more than 500,000 infections in the United States each year. Traditional methods for bacterial culture and identification take several days, wasting precious time for patients who are suffering severe bacterial infections. Numerous nucleic acid-based detection methods have been introduced to address this deficiency; however, the costs and requirement for expensive equipment may limit the widespread use of such technologies. Thus, there is an unmet demand of new platform technology to improve the bacterial detection and identification in clinical practice. In this study, we developed a rapid, ultra-sensitive, low cost, and non-polymerase chain reaction (PCR)-based method for bacterial identification. Using this method, which measures the resonance light-scattering signal of aptamer-conjugated gold nanoparticles, we successfully detected single S. aureus cell within 1.5 hours. This new platform technology may have potential to develop a rapid and sensitive bacterial testing at point-of-care. PMID:23689505

Rapid single cell detection of Staphylococcus aureus by aptamer-conjugated gold nanoparticles.

PubMed

Chang, Yi-Chung; Yang, Chia-Ying; Sun, Ruei-Lin; Cheng, Yi-Feng; Kao, Wei-Chen; Yang, Pan-Chyr

2013-01-01

Staphylococcus aureus is one of the most important human pathogens, causing more than 500,000 infections in the United States each year. Traditional methods for bacterial culture and identification take several days, wasting precious time for patients who are suffering severe bacterial infections. Numerous nucleic acid-based detection methods have been introduced to address this deficiency; however, the costs and requirement for expensive equipment may limit the widespread use of such technologies. Thus, there is an unmet demand of new platform technology to improve the bacterial detection and identification in clinical practice. In this study, we developed a rapid, ultra-sensitive, low cost, and non-polymerase chain reaction (PCR)-based method for bacterial identification. Using this method, which measures the resonance light-scattering signal of aptamer-conjugated gold nanoparticles, we successfully detected single S. aureus cell within 1.5 hours. This new platform technology may have potential to develop a rapid and sensitive bacterial testing at point-of-care.

Multiplex qRT-PCR for the Detection of Western Equine Encephalomyelitis, St. Louis Encephalitis, and West Nile Viral RNA in Mosquito Pools (Diptera: Culicidae)

PubMed Central

Brault, Aaron C.; Fang, Ying; Reisen, William K.

2015-01-01

Following the introduction of West Nile virus into California during the summer of 2003, public health and vector control programs expanded surveillance efforts and were in need of diagnostics capable of rapid, sensitive, and specific detection of arbovirus infections of mosquitoes to inform decision support for intervention. Development of a multiplex TaqMan or real-time semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay in which three virus specific primer–probe sets were used in the same reaction is described herein for the detection of western equine encephalomyelitis, St. Louis encephalitis and West Nile viral RNA. Laboratory validation and field data from 10 transmission seasons are reported. The comparative sensitivity and specificity of this multiplex assay to singleplex RT-PCR as well as an antigen detection (rapid analyte measurement platform) and standard plaque assays indicate this assay to be rapid and useful in providing mosquito infection data to estimate outbreak risk. PMID:26334826

Rapid ultrasensitive single particle surface-enhanced Raman spectroscopy using metallic nanopores.

PubMed

Cecchini, Michael P; Wiener, Aeneas; Turek, Vladimir A; Chon, Hyangh; Lee, Sangyeop; Ivanov, Aleksandar P; McComb, David W; Choo, Jaebum; Albrecht, Tim; Maier, Stefan A; Edel, Joshua B

2013-10-09

Nanopore sensors embedded within thin dielectric membranes have been gaining significant interest due to their single molecule sensitivity and compatibility of detecting a large range of analytes, from DNA and proteins, to small molecules and particles. Building on this concept we utilize a metallic Au solid-state membrane to translocate and rapidly detect single Au nanoparticles (NPs) functionalized with 589 dye molecules using surface-enhanced resonance Raman spectroscopy (SERRS). We show that, due to the plasmonic coupling between the Au metallic nanopore surface and the NP, signal intensities are enhanced when probing analyte molecules bound to the NP surface. Although not single molecule, this nanopore sensing scheme benefits from the ability of SERRS to provide rich vibrational information on the analyte, improving on current nanopore-based electrical and optical detection techniques. We show that the full vibrational spectrum of the analyte can be detected with ultrahigh spectral sensitivity and a rapid temporal resolution of 880 μs.

A self-pumping lab-on-a-chip for rapid detection of botulinum toxin.

PubMed

Lillehoj, Peter B; Wei, Fang; Ho, Chih-Ming

2010-09-07

A robust poly(dimethylsiloxane) (PDMS) surface treatment was utilized for the development of a self-pumping lab-on-a-chip (LOC) to rapidly detect minute quantities of toxic substances. One such toxin, botulinum neurotoxin (BoNT), is an extremely lethal substance, which has the potential to cause hundreds of thousands of fatalities if as little as a few grams are released into the environment. To prevent such an outcome, a quick (<45 min) and sensitive detection format is needed. We have developed a self-pumping LOC that can sense down to 1 pg of BoNT type A (in a 1 microL sample) within 15 min in an autonomous manner. The key technologies enabling for such a device are a sensitive electrochemical sensor, an optimized fluidic network and a robust hydrophilic PDMS coating, thereby facilitating autonomous delivery of liquid samples for rapid detection. The stability, simplicity and portability of this device make possible for a storable and distributable system for monitoring bioterrorist attacks.

Rapid detection of pandemic influenza in the presence of seasonal influenza

PubMed Central

2010-01-01

Background Key to the control of pandemic influenza are surveillance systems that raise alarms rapidly and sensitively. In addition, they must minimise false alarms during a normal influenza season. We develop a method that uses historical syndromic influenza data from the existing surveillance system 'SERVIS' (Scottish Enhanced Respiratory Virus Infection Surveillance) for influenza-like illness (ILI) in Scotland. Methods We develop an algorithm based on the weekly case ratio (WCR) of reported ILI cases to generate an alarm for pandemic influenza. From the seasonal influenza data from 13 Scottish health boards, we estimate the joint probability distribution of the country-level WCR and the number of health boards showing synchronous increases in reported influenza cases over the previous week. Pandemic cases are sampled with various case reporting rates from simulated pandemic influenza infections and overlaid with seasonal SERVIS data from 2001 to 2007. Using this combined time series we test our method for speed of detection, sensitivity and specificity. Also, the 2008-09 SERVIS ILI cases are used for testing detection performances of the three methods with a real pandemic data. Results We compare our method, based on our simulation study, to the moving-average Cumulative Sums (Mov-Avg Cusum) and ILI rate threshold methods and find it to be more sensitive and rapid. For 1% case reporting and detection specificity of 95%, our method is 100% sensitive and has median detection time (MDT) of 4 weeks while the Mov-Avg Cusum and ILI rate threshold methods are, respectively, 97% and 100% sensitive with MDT of 5 weeks. At 99% specificity, our method remains 100% sensitive with MDT of 5 weeks. Although the threshold method maintains its sensitivity of 100% with MDT of 5 weeks, sensitivity of Mov-Avg Cusum declines to 92% with increased MDT of 6 weeks. For a two-fold decrease in the case reporting rate (0.5%) and 99% specificity, the WCR and threshold methods, respectively, have MDT of 5 and 6 weeks with both having sensitivity close to 100% while the Mov-Avg Cusum method can only manage sensitivity of 77% with MDT of 6 weeks. However, the WCR and Mov-Avg Cusum methods outperform the ILI threshold method by 1 week in retrospective detection of the 2009 pandemic in Scotland. Conclusions While computationally and statistically simple to implement, the WCR algorithm is capable of raising alarms, rapidly and sensitively, for influenza pandemics against a background of seasonal influenza. Although the algorithm was developed using the SERVIS data, it has the capacity to be used at other geographic scales and for different disease systems where buying some early extra time is critical. PMID:21106071

Supersensitive fingerprinting of explosives by chemically modified nanosensors arrays.

PubMed

Lichtenstein, Amir; Havivi, Ehud; Shacham, Ronen; Hahamy, Ehud; Leibovich, Ronit; Pevzner, Alexander; Krivitsky, Vadim; Davivi, Guy; Presman, Igor; Elnathan, Roey; Engel, Yoni; Flaxer, Eli; Patolsky, Fernando

2014-06-24

The capability to detect traces of explosives sensitively, selectively and rapidly could be of great benefit for applications relating to civilian national security and military needs. Here, we show that, when chemically modified in a multiplexed mode, nanoelectrical devices arrays enable the supersensitive discriminative detection of explosive species. The fingerprinting of explosives is achieved by pattern recognizing the inherent kinetics, and thermodynamics, of interaction between the chemically modified nanosensors array and the molecular analytes under test. This platform allows for the rapid detection of explosives, from air collected samples, down to the parts-per-quadrillion concentration range, and represents the first nanotechnology-inspired demonstration on the selective supersensitive detection of explosives, including the nitro- and peroxide-derivatives, on a single electronic platform. Furthermore, the ultrahigh sensitivity displayed by our platform may allow the remote detection of various explosives, a task unachieved by existing detection technologies.

Supersensitive fingerprinting of explosives by chemically modified nanosensors arrays

NASA Astrophysics Data System (ADS)

Lichtenstein, Amir; Havivi, Ehud; Shacham, Ronen; Hahamy, Ehud; Leibovich, Ronit; Pevzner, Alexander; Krivitsky, Vadim; Davivi, Guy; Presman, Igor; Elnathan, Roey; Engel, Yoni; Flaxer, Eli; Patolsky, Fernando

2014-06-01

The capability to detect traces of explosives sensitively, selectively and rapidly could be of great benefit for applications relating to civilian national security and military needs. Here, we show that, when chemically modified in a multiplexed mode, nanoelectrical devices arrays enable the supersensitive discriminative detection of explosive species. The fingerprinting of explosives is achieved by pattern recognizing the inherent kinetics, and thermodynamics, of interaction between the chemically modified nanosensors array and the molecular analytes under test. This platform allows for the rapid detection of explosives, from air collected samples, down to the parts-per-quadrillion concentration range, and represents the first nanotechnology-inspired demonstration on the selective supersensitive detection of explosives, including the nitro- and peroxide-derivatives, on a single electronic platform. Furthermore, the ultrahigh sensitivity displayed by our platform may allow the remote detection of various explosives, a task unachieved by existing detection technologies.

Aptamer-mediated colorimetric method for rapid and sensitive detection of chloramphenicol in food.

PubMed

Yan, Chao; Zhang, Jing; Yao, Li; Xue, Feng; Lu, Jianfeng; Li, Baoguang; Chen, Wei

2018-09-15

We report an aptamer-mediated colorimetric method for sensitive detection of chloramphenicol (CAP). The aptamer of CAP is immobilized by the hybridization with pre-immobilized capture probe in the microtiter plate. The horseradish peroxidase (HRP) is covalently attached to the aptamer by the biotin-streptavidin system for signal production. CAP will preferably bind with aptamer due to the high binding affinity, which attributes to the release of aptamer and HRP and thus, affects the optical signal intensity. Quantitative determination of CAP is successfully achieved in the wide range from 0.001 to 1000 ng/mL with detection limit of 0.0031 ng/mL, which is more sensitive than traditional immunoassays. This method is further validated by measuring the recovery of CAP spiked in two different food matrices (honey and fish). The aptamer-mediated colorimetric method can be a useful protocol for rapid and sensitive screening of CAP, and may be used as an alternative means for traditional immunoassays. Copyright © 2018 Elsevier Ltd. All rights reserved.

Application of a SERS-based lateral flow immunoassay strip for the rapid and sensitive detection of staphylococcal enterotoxin B

NASA Astrophysics Data System (ADS)

Hwang, Joonki; Lee, Sangyeop; Choo, Jaebum

2016-06-01

A novel surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay (LFA) biosensor was developed to resolve problems associated with conventional LFA strips (e.g., limits in quantitative analysis and low sensitivity). In our SERS-based biosensor, Raman reporter-labeled hollow gold nanospheres (HGNs) were used as SERS detection probes instead of gold nanoparticles. With the proposed SERS-based LFA strip, the presence of a target antigen can be identified through a colour change in the test zone. Furthermore, highly sensitive quantitative evaluation is possible by measuring SERS signals from the test zone. To verify the feasibility of the SERS-based LFA strip platform, an immunoassay of staphylococcal enterotoxin B (SEB) was performed as a model reaction. The limit of detection (LOD) for SEB, as determined with the SERS-based LFA strip, was estimated to be 0.001 ng mL-1. This value is approximately three orders of magnitude more sensitive than that achieved with the corresponding ELISA-based method. The proposed SERS-based LFA strip sensor shows significant potential for the rapid and sensitive detection of target markers in a simplified manner.A novel surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay (LFA) biosensor was developed to resolve problems associated with conventional LFA strips (e.g., limits in quantitative analysis and low sensitivity). In our SERS-based biosensor, Raman reporter-labeled hollow gold nanospheres (HGNs) were used as SERS detection probes instead of gold nanoparticles. With the proposed SERS-based LFA strip, the presence of a target antigen can be identified through a colour change in the test zone. Furthermore, highly sensitive quantitative evaluation is possible by measuring SERS signals from the test zone. To verify the feasibility of the SERS-based LFA strip platform, an immunoassay of staphylococcal enterotoxin B (SEB) was performed as a model reaction. The limit of detection (LOD) for SEB, as determined with the SERS-based LFA strip, was estimated to be 0.001 ng mL-1. This value is approximately three orders of magnitude more sensitive than that achieved with the corresponding ELISA-based method. The proposed SERS-based LFA strip sensor shows significant potential for the rapid and sensitive detection of target markers in a simplified manner. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07243c

A sensitive and innovative detection method for rapid C-reactive proteins analysis based on a micro-fluxgate sensor system

PubMed Central

Yang, Zhen; Zhi, Shaotao; Feng, Zhu; Lei, Chong; Zhou, Yong

2018-01-01

A sensitive and innovative assay system based on a micro-MEMS-fluxgate sensor and immunomagnetic beads-labels was developed for the rapid analysis of C-reactive proteins (CRP). The fluxgate sensor presented in this study was fabricated through standard micro-electro-mechanical system technology. A multi-loop magnetic core made of Fe-based amorphous ribbon was employed as the sensing element, and 3-D solenoid copper coils were used to control the sensing core. Antibody-conjugated immunomagnetic microbeads were strategically utilized as signal tags to label the CRP via the specific conjugation of CRP to polyclonal CRP antibodies. Separate Au film substrates were applied as immunoplatforms to immobilize CRP-beads labels through classical sandwich assays. Detection and quantification of the CRP at different concentrations were implemented by detecting the stray field of CRP labeled magnetic beads using the newly-developed micro-fluxgate sensor. The resulting system exhibited the required sensitivity, stability, reproducibility, and selectivity. A detection limit as low as 0.002 μg/mL CRP with a linearity range from 0.002 μg/mL to 10 μg/mL was achieved, and this suggested that the proposed biosystem possesses high sensitivity. In addition to the extremely low detection limit, the proposed method can be easily manipulated and possesses a quick response time. The response time of our sensor was less than 5 s, and the entire detection period for CRP analysis can be completed in less than 30 min using the current method. Given the detection performance and other advantages such as miniaturization, excellent stability and specificity, the proposed biosensor can be considered as a potential candidate for the rapid analysis of CRP, especially for point-of-care platforms. PMID:29601593

Multiplexed lateral flow microarray assay for detection of citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis pv citri

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cary,; Bruce, R; Stubben, Christopher J

The invention provides highly sensitive and specific assays for the major citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis, including a field deployable multiplexed assay capable of rapidly assaying for both pathogens simultaneously. The assays are directed at particular gene targets derived from pathogenic strains that specifically cause the major citrus diseases of citrus variegated chlorosis (Xylella fastidiosa 9a5c) and citrus canker (Xanthomonas axonopodis pv citri). The citrus pathogen assays of the invention offer femtomole sensitivity, excellent linear dynamic range, and rapid and specific detection.

Ultra-Fast and Sensitive Detection of Non-Typhoidal Salmonella Using Microwave-Accelerated Metal-Enhanced Fluorescence (“MAMEF”)

PubMed Central

Galen, James E.; Geddes, Chris D.; Levine, Myron M.

2011-01-01

Certain serovars of Salmonella enterica subsp. enterica cause invasive disease (e.g., enteric fever, bacteremia, septicemia, meningitis, etc.) in humans and constitute a global public health problem. A rapid, sensitive diagnostic test is needed to allow prompt initiation of therapy in individual patients and for measuring disease burden at the population level. An innovative and promising new rapid diagnostic technique is microwave-accelerated metal-enhanced fluorescence (MAMEF). We have adapted this assay platform to detect the chromosomal oriC locus common to all Salmonella enterica subsp. enterica serovars. We have shown efficient lysis of biologically relevant concentrations of Salmonella spp. suspended in bacteriological media using microwave-induced lysis. Following lysis and DNA release, as little as 1 CFU of Salmonella in 1 ml of medium can be detected in <30 seconds. Furthermore the assay is sensitive and specific: it can detect oriC from Salmonella serovars Typhi, Paratyphi A, Paratyphi B, Paratyphi C, Typhimurium, Enteritidis and Choleraesuis but does not detect Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae or Acinetobacter baumanii. We have also performed preliminary experiments using a synthetic Salmonella oriC oligonucleotide suspended in whole human blood and observed rapid detection when the sample was diluted 1∶1 with PBS. These pre-clinical data encourage progress to the next step to detect Salmonella in blood (and other ordinarily sterile, clinically relevant body fluids). PMID:21494634

A Novel Surface Plasmon Resonance Biosensor for the Rapid Detection of Botulinum Neurotoxins

PubMed Central

Patel, Kruti; Halevi, Shmuel; Melman, Paul; Schwartz, John; Cai, Shuowei; Singh, Bal Ram

2017-01-01

Botulinum neurotoxins (BoNTs) are Category A agents on the NIAID (National Institute of Allergy and Infectious Diseases) priority pathogen list owing to their extreme toxicity and the relative ease of production. These deadly toxins, in minute quantities (estimated human i.v. lethal dose LD50 of 1–2 ng/kg body weight), cause fatal flaccid paralysis by blocking neurotransmitter release. The current gold standard detection method, the mouse-bioassay, often takes days to confirm botulism. Furthermore, there are no effective antidotes known to reverse the symptoms of botulism, and as a result, patients with severe botulism often require meticulous care during the prolonged paralytic illness. To combat potential bio-terrorism incidents of botulinum neurotoxins, their rapid detection is paramount. Surface plasmon resonance (SPR) is a very sensitive technique to examine bio-molecular interactions. The label-free, real-time analysis, with high sensitivity and low sample consumption makes this technology particularly suitable for detection of the toxin. In this study, we demonstrated the feasibility in an assay with a newly designed SPR instrument for the rapid detection of botulinum neurotoxins. The LOD (limit of detection) of the Newton Photonics (NP) SPR based assay is 6.76 pg/mL for Botulinum Neurotoxin type A Light Chain (BoNT/A LC). We established that the detection sensitivity of the system is comparable to the traditional mouse LD50 bioassay in BoNT/A using this SPR technology. PMID:28783115

Comparison of methods for the detection of coliphages in recreational water at two California, United States beaches.

PubMed

Rodríguez, Roberto A; Love, David C; Stewart, Jill R; Tajuba, Julianne; Knee, Jacqueline; Dickerson, Jerold W; Webster, Laura F; Sobsey, Mark D

2012-04-01

Methods for detection of two fecal indicator viruses, F+ and somatic coliphages, were evaluated for application to recreational marine water. Marine water samples were collected during the summer of 2007 in Southern California, United States from transects along Avalon Beach (n=186 samples) and Doheny Beach (n=101 samples). Coliphage detection methods included EPA method 1601 - two-step enrichment (ENR), EPA method 1602 - single agar layer (SAL), and variations of ENR. Variations included comparison of two incubation times (overnight and 5-h incubation) and two final detection steps (lysis zone assay and a rapid latex agglutination assay). A greater number of samples were positive for somatic and F+ coliphages by ENR than by SAL (p<0.01). The standard ENR with overnight incubation and detection by lysis zone assay was the most sensitive method for the detection of F+ and somatic coliphages from marine water, although the method takes up to three days to obtain results. A rapid 5-h enrichment version of ENR also performed well, with more positive samples than SAL, and could be performed in roughly 24h. Latex agglutination-based detection methods require the least amount of time to perform, although the sensitivity was less than lysis zone-based detection methods. Rapid culture-based enrichment of coliphages in marine water may be possible by further optimizing culture-based methods for saline water conditions to generate higher viral titers than currently available, as well as increasing the sensitivity of latex agglutination detection methods. Copyright © 2012 Elsevier B.V. All rights reserved.

A luminescent hybridoma-based biosensor for rapid detection of V. cholerae upon induction of calcium signaling pathway.

PubMed

Zamani, Parichehr; Sajedi, Reza H; Hosseinkhani, Saman; Zeinoddini, Mehdi; Bakhshi, Bita

2016-05-15

In this study, a hybridoma based biosensor was developed for rapid, sensitive and selective detection of Vibrio cholerae O1 which converts the antibody-antigen binding to bioluminescence light. After investigation on hybridoma performance, the biosensor was constructed by transfecting specific hybridoma cells with aequorin reporter gene and the bioluminescence activities of stable biosensor were measured. The sensitivity of biosensor was as few as 50 CFU/ml and it showed no responses to other entric bacteria. Moreover, the response time of biosensor was estimated in 7th second which means this method is considerably faster than many available detection assays. In addition, this biosensor was successfully applied to V. cholerae detection in environmental samples with no significant loss in sensitivity, demonstrating our proposed biosensor provides a sensitive and reliable method for detection of V. cholerae in natural samples. The application of whole hybridoma cell directly as a sensing element in biosensor construction which mentioned for the first time in present study suggests that hybridoma cells could provide a valuable tool for future studies in both basic and diagnostic sciences and could be considered as a fast and specific sensing element for detection of other pathogens in different applications. Copyright © 2015 Elsevier B.V. All rights reserved.

Improved Detection of Botulinum Neurotoxin Serotype A by Endopep-MS through Peptide Substrate Modification

PubMed Central

Wang, Dongxia; Baudys, Jakub; Ye, Yiming; Rees, Jon C.; Barr, John R.; Pirkle, James L.; Kalb, Suzanne R.

2015-01-01

Botulinum neurotoxins (BoNTs) are a family of seven toxin serotypes that are the most toxic substances known to man. Intoxication with BoNT causes flaccid paralysis and can lead to death if untreated with serotype specific antibodies. Supportive care, including ventilation, may be necessary. Rapid and sensitive detection of BoNT is necessary for timely clinical confirmation of clinical botulism. Previously, our laboratory developed a fast and sensitive mass spectrometry (MS) method termed the Endopep-MS assay. The BoNT serotypes are rapidly detected and differentiated by extracting the toxin with serotype specific antibodies and detecting the unique and serotype specific cleavage products of peptide substrates that mimic the sequence of the BoNT native targets. To further improve the sensitivity of the Endopep-MS assay, we report here the optimization of the substrate peptide for the detection of BoNT/A. Modifications on the terminal groups of the original peptide substrate with acetylation and amidation significantly improved the detection of BoNT/A cleavage products. The replacement of some internal amino acid residues with single or multiple substitutions led to further improvement. An optimized peptide increased assay sensitivity five fold with toxin spiked into buffer solution or different biological matrices. PMID:23017875

Comparison of two rapid biochemical tests and four chromogenic selective media for detection of carbapenemase-producing Gram-negative bacteria.

PubMed

Hinić, Vladimira; Amrein, Ivo; Stammler, Sabrina; Heckendorn, Judith; Meinel, Dominik; Frei, Reno; Egli, Adrian

2017-04-01

We evaluated RAPIDEC® CARBA NP, Neo-Rapid CARB, chromID® CARBA SMART (CARB/OXA), Brilliance™ CRE/ESBL, ChromArt CRE and BBL™ CHROMagar™ CPE for the detection of carbapenemase-producing bacteria. The analytical sensitivity of RAPIDEC® CARBA NP was better than that of Neo-Rapid CARB. A combination of carbapenemase and ESBL screening plates could be advantageous. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid and visual detection of Leptospira in urine by LigB-LAMP assay with pre-addition of dye.

PubMed

Ali, Syed Atif; Kaur, Gurpreet; Boby, Nongthombam; Sabarinath, T; Solanki, Khushal; Pal, Dheeraj; Chaudhuri, Pallab

2017-12-01

Leptospirosis is considered to be the most widespread zoonotic disease caused by pathogenic species of Leptospira. The present study reports a novel set of primers targeting LigB gene for visual detection of pathogenic Leptospira in urine samples through Loop-mediated isothermal amplification (LAMP). The results were recorded by using Hydroxyl napthol blue (HNB), SYBR GREEN I and calcein. Analytical sensitivity of LAMP was as few as 10 leptospiral organisms in spiked urine samples from cattle and dog. LigB gene based LAMP, termed as LigB-LAMP, was found 10 times more sensitive than conventional PCR. The diagnostic specificity of LAMP was 100% when compared to SYBR green qPCR for detection of Leptospira in urine samples. Though qPCR was found more sensitive, the rapidity and simplicity in setting LAMP test followed by visual detection of Leptospira infection in clinical samples makes LigB-LAMP an alternative and favourable diagnostic tool in resource poor setting. Copyright © 2017 Elsevier Ltd. All rights reserved.

Direct detection of methicillin-resistant Staphylococcus aureus from blood cultures using an immunochromatographic immunoassay-based MRSA rapid kit for the detection of penicillin-binding protein 2a.

PubMed

Shin, Kyeong Seob; Song, Hyung Geun; Kim, Haejung; Yoon, Sangsun; Hong, Seung Bok; Koo, Sun Hoe; Kim, Jimyung; Kim, Jongwan; Roh, Kyoung Ho

2010-07-01

Using an EZ-Step MRSA rapid kit, a novel screening test for methicillin-resistant Staphylococcus aureus (MRSA) that detects penicillin-binding protein 2a, 34 of 36 MRSA-positive clinical blood culture samples were positive on direct testing (sensitivity, 94.4%), whereas 21 of 21 methicillin-susceptible S. aureus-positive samples were negative (specificity, 100%).

A colloidal gold nanoparticle-based immunochromatographic test strip for rapid and convenient detection of Staphylococcus aureus.

PubMed

Niu, Kaili; Zheng, Xiaoping; Huang, Chusen; Xul, Kuan; Zhi, Yuan; Shen, Hebai; Jia, Nengqin

2014-07-01

An immunochromatographic test strip using gold nanoparticles-staphylococcus aureus monoclonal antibody conjugates was developed for the rapid and convenient detection of staphylococcus aureus based on a double-antibody sandwich format. The detection limit and the detection rate of this test strip is 10(3) CFU /mL and 98.7%, respectively. It could be used for the rapid detection of staphylococcus aureus in food and the results can be visually identified by the naked eye within 10 min. Compared with conventional bacterial detection methods, this developed immunochromatographic assay based test strip has several advantages including simple, fast, low-cost, favorable sensitivity and specificity, exhibiting a great potential for application in food safety control systems and clinical diagnosis.

Enzyme-Encapsulated Liposome-Linked Immunosorbent Assay Enabling Sensitive Personal Glucose Meter Readout for Portable Detection of Disease Biomarkers.

PubMed

Lin, Bingqian; Liu, Dan; Yan, Jinmao; Qiao, Zhi; Zhong, Yunxin; Yan, Jiawei; Zhu, Zhi; Ji, Tianhai; Yang, Chaoyong James

2016-03-23

There is considerable demand for sensitive, selective, and portable detection of disease-associated proteins, particularly in clinical practice and diagnostic applications. Portable devices are highly desired for detection of disease biomarkers in daily life due to the advantages of being simple, rapid, user-friendly, and low-cost. Herein we report an enzyme-encapsulated liposome-linked immunosorbent assay for sensitive detection of proteins using personal glucose meters (PGM) for portable quantitative readout. Liposomes encapsulating a large amount of amyloglucosidase or invertase are surface-coated with recognition elements such as aptamers or antibodies for target recognition. By translating molecular recognition signal into a large amount of glucose with the encapsulated enzyme, disease biomarkers such as thrombin or C-reactive protein (CRP) can be quantitatively detected by a PGM with a high detection limit of 1.8 or 0.30 nM, respectively. With the advantages of portability, ease of use, and low-cost, the method reported here has potential for portable and quantitative detection of various targets for different POC testing scenarios, such as rapid diagnosis in clinic offices, health monitoring at the bedside, and chemical/biochemical safety control in the field.

Rapid quantification of color vision: the cone contrast test.

PubMed

Rabin, Jeff; Gooch, John; Ivan, Douglas

2011-02-09

To describe the design, specificity, and sensitivity of the cone contrast test (CCT), a computer-based, cone-specific (L, M, S) contrast sensitivity test for diagnosing type and severity of color vision deficiency (CVD). The CCT presents a randomized series of colored letters visible only to L, M or S cones in decreasing steps of cone contrast to determine L, M, and S letter-recognition thresholds. Sensitivity and specificity were determined by retrospective comparison of CCT scores to anomaloscope and pseudoisochromatic plate (PIP) results in 1446 applicants for pilot training. CVD was detected in 49 (3.4%) of 1446 applicants with hereditary red-green (protan or deutan) CVD detected in 47 (3.5%) of 1359 men and blue-yellow (tritan) in 2 of 1446. In agreement with the anomaloscope, the CCT showed 100% sensitivity for detection and categorization of CVD (40 deutan, 7 protan, 2 tritan). PIP testing showed lower sensitivity (80% detected; 20% missed) due in part to the applicant's prior experience and/or pretest preparation. CCT specificity for confirming normal color vision was 100% for L and M cone tests and 99.8% for S cones. The CCT has sensitivity and specificity comparable to anomaloscope testing and exceeds PIP sensitivity in practiced observers. The CCT provides a rapid (6 minutes), clinically expedient, measure of color vision for quantifying normal color performance, diagnosing type and severity of hereditary deficiency, and detection of acquired sensitivity loss due to ocular, neurologic, and/or systemic disease, as well as injury and physiological stressors, such as altitude and fatigue.

Sensitive and rapid immunoassay for parathyroid hormone using magnetic particle labels and magnetic actuation.

PubMed

Dittmer, W U; de Kievit, P; Prins, M W J; Vissers, J L M; Mersch, M E C; Martens, M F W C

2008-09-30

A rapid method for the sensitive detection of proteins using actuated magnetic particle labels, which are measured with a giant magneto-resistive (GMR) biosensor, is described. The technique involves a 1-step sandwich immunoassay with no fluid replacement steps. The various assay binding reactions as well as the bound/free separation are entirely controlled by magnetic forces induced by electromagnets above and below the sensor chip. During the assay, particles conjugated with tracer antibodies are actuated through the sample for target capture, and rapidly brought to the sensor surface where they bind to immobilized capture antibodies. Weakly or unbound labels are removed with a magnetic force oriented away from the GMR sensor surface. For the measurement of parathyroid hormone (PTH), a detection limit in the 10 pM range is obtained with a total assay time of 15 min when 300 nm particles are used. The same sensitivity can be achieved in 5 min when 500 nm particles are used. If 500 nm particles are employed in a 15-minute assay, then 0.8 pM of PTH is detectable. The low sample volume, high analytical performance and high speed of the test coupled with the compact GMR biosensor make the system especially suitable for sensitive testing outside of laboratory environments.

Bead-based competitive fluorescence immunoassay for sensitive and rapid diagnosis of cyanotoxin risk in drinking water.

PubMed

Yu, Hye-Weon; Jang, Am; Kim, Lan Hee; Kim, Sung-Jo; Kim, In S

2011-09-15

Due to the increased occurrence of cyanobacterial blooms and their toxins in drinking water sources, effective management based on a sensitive and rapid analytical method is in high demand for security of safe water sources and environmental human health. Here, a competitive fluorescence immunoassay of microcystin-LR (MCYST-LR) is developed in an attempt to improve the sensitivity, analysis time, and ease-of-manipulation of analysis. To serve this aim, a bead-based suspension assay was introduced based on two major sensing elements: an antibody-conjugated quantum dot (QD) detection probe and an antigen-immobilized magnetic bead (MB) competitor. The assay was composed of three steps: the competitive immunological reaction of QD detection probes against analytes and MB competitors, magnetic separation and washing, and the optical signal generation of QDs. The fluorescence intensity was found to be inversely proportional to the MCYST-LR concentration. Under optimized conditions, the proposed assay performed well for the identification and quantitative analysis of MCYST-LR (within 30 min in the range of 0.42-25 μg/L, with a limit of detection of 0.03 μg/L). It is thus expected that this enhanced assay can contribute both to the sensitive and rapid diagnosis of cyanotoxin risk in drinking water and effective management procedures.

Evaluation of CHROMagar KPC for Rapid Detection of Carbapenem-Resistant Enterobacteriaceae▿

PubMed Central

Samra, Zmira; Bahar, Judi; Madar-Shapiro, Liora; Aziz, Nazi; Israel, Sara; Bishara, Jihad

2008-01-01

A new CHROMagar KPC medium was compared to MacConkey agar with carbapenem discs and PCR for the blaKPC gene for rapid detection of carbapenem-resistant Klebsiella pneumoniae. The sensitivity and specificity relative to PCR were 100% and 98.4%, respectively, for CHROMagar KPC and 92.7% and 95.9%, respectively, for MacConkey agar. PMID:18632915

Rapid, automated, parallel quantitative immunoassays using highly integrated microfluidics and AlphaLISA

PubMed Central

Tak For Yu, Zeta; Guan, Huijiao; Ki Cheung, Mei; McHugh, Walker M.; Cornell, Timothy T.; Shanley, Thomas P.; Kurabayashi, Katsuo; Fu, Jianping

2015-01-01

Immunoassays represent one of the most popular analytical methods for detection and quantification of biomolecules. However, conventional immunoassays such as ELISA and flow cytometry, even though providing high sensitivity and specificity and multiplexing capability, can be labor-intensive and prone to human error, making them unsuitable for standardized clinical diagnoses. Using a commercialized no-wash, homogeneous immunoassay technology (‘AlphaLISA’) in conjunction with integrated microfluidics, herein we developed a microfluidic immunoassay chip capable of rapid, automated, parallel immunoassays of microliter quantities of samples. Operation of the microfluidic immunoassay chip entailed rapid mixing and conjugation of AlphaLISA components with target analytes before quantitative imaging for analyte detections in up to eight samples simultaneously. Aspects such as fluid handling and operation, surface passivation, imaging uniformity, and detection sensitivity of the microfluidic immunoassay chip using AlphaLISA were investigated. The microfluidic immunoassay chip could detect one target analyte simultaneously for up to eight samples in 45 min with a limit of detection down to 10 pg mL−1. The microfluidic immunoassay chip was further utilized for functional immunophenotyping to examine cytokine secretion from human immune cells stimulated ex vivo. Together, the microfluidic immunoassay chip provides a promising high-throughput, high-content platform for rapid, automated, parallel quantitative immunosensing applications. PMID:26074253

Reactive chromophores for sensitive and selective detection of chemical warfare agents and toxic industrial chemicals

NASA Astrophysics Data System (ADS)

Frye-Mason, Greg; Leuschen, Martin; Wald, Lara; Paul, Kateri; Hancock, Lawrence F.

2005-05-01

A reactive chromophore developed at MIT exhibits sensitive and selective detection of surrogates for G-class nerve agents. This reporter acts by reacting with the agent to form an intermediate that goes through an internal cyclization reaction. The reaction locks the molecule into a form that provides a strong fluorescent signal. Using a fluorescent sensor platform, Nomadics has demonstrated rapid and sensitive detection of reactive simulants such as diethyl chloro-phosphate (simulant for sarin, soman, and related agents) and diethyl cyanophosphate (simulant for tabun). Since the unreacted chromophore does not fluoresce at the excitation wavelength used for the cyclized reporter, the onset of fluo-rescence can be easily detected. This fluorescence-based detection method provides very high sensitivity and could enable rapid detection at permissible exposure levels. Tests with potential interferents show that the reporter is very selective, with responses from only a few highly toxic, electrophilic chemicals such as phosgene, thionyl chloride, and strong acids such as HF, HCl, and nitric acid. Dimethyl methyl phosphonate (DMMP), a common and inactive simu-lant for other CW detectors, is not reactive enough to generate a signal. The unique selectivity to chemical reactivity means that a highly toxic and hazardous chemical is present when the reporter responds and illustrates that this sensor can provide very low false alarm rates. Current efforts focus on demonstrating the sensitivity and range of agents and toxic industrial chemicals detected with this reporter as well as developing additional fluorescent reporters for a range of chemical reactivity classes. The goal is to produce a hand-held sensor that can sensitively detect a broad range of chemical warfare agent and toxic industrial chemical threats.

Towards Detection and Diagnosis of Ebola Virus Disease at Point-of-Care

PubMed Central

Kaushik, Ajeet; Tiwari, Sneham; Jayant, Rahul Dev; Marty, Aileen; Nair, Madhavan

2015-01-01

Ebola outbreak-2014 (mainly Zaire strain related Ebola virus) has been declared most widely spread deadly persistent epidemic due to unavailability of rapid diagnostic, detection, and therapeutics. Ebola virus disease (EVD), a severe viral hemorrhagic fever syndrome caused by Ebola virus (EBOV) is transmitted by direct contact with the body fluids of infected person and objects contaminated with virus or infected animals. World Health Organization (WHO) has declared EVD epidemic as public health emergency of international concern with severe global economic burden. At fatal EBOV infection stage, patients usually die before the antibody response. Currently, rapid blood tests to diagnose EBOV infection include the antigen or antibodies capture using ELISA and RNA detection using RT/Q-PCR within 3–10 days after the onset of symptoms. Moreover, few nanotechnology-based colorimetric and paper-based immunoassay methods have been recently reported to detect Ebola virus. Unfortunately, these methods are limited to laboratory only. As state-of-the art (SoA) diagnostics time to confirm Ebola infection, varies from 6 hours to about 3 days, it causes delay in therapeutic approaches. Thus developing a cost-effective, rapid, sensitive, and selective sensor to detect EVD at point-of-care (POC) is certainly worth exploring to establish rapid diagnostics to decide therapeutics. This review highlights SoA of Ebola diagnostics and also a call to develop rapid, selective and sensitive POC detection of EBOV for global health care. We propose that adopting miniaturized electrochemical EBOV immunosensing can detect virus level at pM concentration within ~40 minute compared to 3 days of ELISA test at nM levels. PMID:26319169

Low sensitivity of the ImmunocardSTAT® Crypto/Giardia Rapid Assay test for the detection of Giardia and Cryptosporidium in fecal samples from children living in Libreville, Central Africa.

PubMed

Bouyou-Akotet, M K; Owono-Medang, M; Moussavou-Boussougou, M N; Mamfoumbi, M Mabika; Mintsa-Nguema, R; Mawili-Mboumba, D P; Kombila, M

2016-12-01

Giardiasis and cryptosporidiosis are now recognized as neglected tropical parasitic diseases. The risk of their dissemination  in developing countries, such as Gabon, is increasing, due to urban crowding and poor sanitation. Accurate, simple and rapid diagnosis tools are thus necessary for the estimation of their real burden. The aim of this study was to evaluate the performances of the ImmunocardSTAT ® Crypto/Giardia Rapid Assay test for the detection of Cryptosporidium ( C. ) spp. and Giardia ( G. ) duodenalis in children living in Libreville, Gabon. Stool samples of 173 healthy children were screened by routine microscopic using the merthiolate iodine formol concentration technique for Giardia , the modified Ziehl Neelsen (ZN) staining for Cryptosporidium and the ImmunocardSTAT ® Crypto/Giardia RDT for the detection of Giardia and Cryptosporidium parasite forms and antigens respectively. G. duodenalis was detected with microscopy and the ImmunocardSTAT ® Crypto/Giardia in 27 (15.6 %) and 22 (13.3 %) fecal samples respectively. C. spp. oocysts were found in 18 (10.4 %) ones, whereas only one sample was positive with the immunochromatographic assay. When microscopic examination was considered as the reference method, sensitivity and specificity of the ImmunocardSTAT ® Crypto/Giardia Rapid Assay were found to be 63.0 %, 96.6 and 5.5 %, 99.3 % for G. duodenalis and C. spp. respectively. The prevalence of G. duodenalis and C. spp. carriage is high in children from Libreville. A low sensitivity of the ImmunocardSTAT ® Crypto/Giardia for the detection of both parasites is observed. It is thus inappropriate as a diagnostic tool for detecting asymptomatic carriers.

Luciferase-Zinc-Finger System for the Rapid Detection of Pathogenic Bacteria.

PubMed

Shi, Chu; Xu, Qing; Ge, Yue; Jiang, Ling; Huang, He

2017-08-09

Rapid and reliable detection of pathogenic bacteria is crucial for food safety control. Here, we present a novel luciferase-zinc finger system for the detection of pathogens that offers rapid and specific profiling. The system, which uses a zinc-finger protein domain to probe zinc finger recognition sites, was designed to bind the amplified conserved regions of 16S rDNA, and the obtained products were detected using a modified luciferase. The luciferase-zinc finger system not only maintained luciferase activity but also allowed the specific detection of different bacterial species, with a sensitivity as low as 10 copies and a linear range from 10 to 10 4 copies per microliter of the specific PCR product. Moreover, the system is robust and rapid, enabling the simultaneous detection of 6 species of bacteria in artificially contaminated samples with excellent accuracy. Thus, we envision that our luciferase-zinc finger system will have far-reaching applications.

Facile and sensitive determination of N-nitrosamines in food samples by high-performance liquid chromatography via combining fluorescent labeling with dispersive liquid-liquid microextraction.

PubMed

Lu, Shuaimin; Wu, Di; Li, Guoliang; Lv, Zhengxian; Gong, Peiwei; Xia, Lian; Sun, Zhiwei; Chen, Guang; Chen, Xuefeng; You, Jinmao; Wu, Yongning

2017-11-01

The intake of N-nitrosamines (NAs) from foodstuffs is considered to be an important influence factor for several cancers. But the rapid and sensitive screening of NAs remains a challenge in the field of food safety. Inspired by that, a sensitive and rapid method was demonstrated for determination of five NAs (Nitrosopyrrolidine, Nitrosodimethylamine, Nitrosodiethylamine, Nitrosodipropylamine and Nitrosodibutylamine) using dispersive liquid-liquid microextraction (DLLME) followed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD). The NAs were firstly denitrosated and labeled by 2-(11H-benzo[a]carbazol-11-yl) ethyl carbonochloridate (BCEC-Cl) and finally enriched by DLLME. Furthermore, the main DLLME conditions were optimized systematically. Under the optimal conditions, satisfactory limits of detection (LODs) were obtained with a range of 0.01-0.07ngg -1 , which were significantly lower than the reported methods. The developed method showed many merits including rapidity, simplicity, high sensitivity and excellent selectivity, which shows a broad prospect in food safety analysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interleukin-6 Detection with a Plasmonic Chip

NASA Astrophysics Data System (ADS)

Tawa, Keiko; Sumiya, Masashi; Toma, Mana; Sasakawa, Chisato; Sujino, Takuma; Miyaki, Tatsuki; Nakazawa, Hikaru; Umetsu, Mitsuo

Interleukin-6, a cytokine relating inflammatory and autoimmune activity, was detected with three fluorescence assays using a plasmonic chip. In their assays, the way of surface modification, sample volume, incubation time and mixing solution, were found to influence the detection sensitivity. When the assay was revised in the point of a rapid and easy process, the detection sensitivity was not compromised compared to assays with sufficient sample volume and assay time. To suit the purpose of immunosensing, the assay conditions should be determined.

Label-Free Nanopore Biosensor for Rapid and Highly Sensitive Cocaine Detection in Complex Biological Fluids.

PubMed

Rauf, Sana; Zhang, Ling; Ali, Asghar; Liu, Yang; Li, Jinghong

2017-02-24

Detection of very low amounts of illicit drugs such as cocaine in clinical fluids like serum continues to be important for many areas in the fight against drug trafficking. Herein, we constructed a label-free nanopore biosensor for rapid and highly sensitive detection of cocaine in human serum and saliva samples based on target-induced strand release strategy. In this bioassay, an aptamer for cocaine was prehybridized with a short complementary DNA. Owing to cocaine specific binding with aptamer, the short DNA strand was displaced from aptamer and translocation of this output DNA through α-hemolysin nanopore generated distinct spike-like current blockages. When plotted in double-logarithmic scale, a linear relationship between target cocaine concentration and output DNA event frequency was obtained in a wide concentration range from 50 nM to 100 μM of cocaine, with the limit of detection down to 50 nM. In addition, this aptamer-based sensor method was successfully applied for cocaine detection in complex biological fluids like human saliva and serum samples with great selectivity. Simple preparation, low cost, rapid, label-free, and real sample detection are the motivating factors for practical application of the proposed biosensor.

Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification.

PubMed

Janse, Ingmar; Hamidjaja, Raditijo A; Bok, Jasper M; van Rotterdam, Bart J

2010-12-08

Several pathogens could seriously affect public health if not recognized timely. To reduce the impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools are needed for their detection in various samples, including environmental samples. Multiplex real-time PCRs were designed for rapid and reliable detection of three major pathogens that have the potential to cause high morbidity and mortality in humans: B. anthracis, F. tularensis and Y. pestis. The developed assays detect three pathogen-specific targets, including at least one chromosomal target, and one target from B. thuringiensis which is used as an internal control for nucleic acid extraction from refractory spores as well as successful DNA amplification. Validation of the PCRs showed a high analytical sensitivity, specificity and coverage of diverse pathogen strains. The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity. The application of B. thuringiensis spores as internal controls further reduces false negative results. This ensures highly reliable detection, while template consumption and laboratory effort are kept at a minimum.

Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification

PubMed Central

2010-01-01

Background Several pathogens could seriously affect public health if not recognized timely. To reduce the impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools are needed for their detection in various samples, including environmental samples. Results Multiplex real-time PCRs were designed for rapid and reliable detection of three major pathogens that have the potential to cause high morbidity and mortality in humans: B. anthracis, F. tularensis and Y. pestis. The developed assays detect three pathogen-specific targets, including at least one chromosomal target, and one target from B. thuringiensis which is used as an internal control for nucleic acid extraction from refractory spores as well as successful DNA amplification. Validation of the PCRs showed a high analytical sensitivity, specificity and coverage of diverse pathogen strains. Conclusions The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity. The application of B. thuringiensis spores as internal controls further reduces false negative results. This ensures highly reliable detection, while template consumption and laboratory effort are kept at a minimum PMID:21143837

Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6

PubMed Central

Gootenberg, Jonathan S.; Abudayyeh, Omar O.; Kellner, Max J.; Joung, Julia; Collins, James J.; Zhang, Feng

2018-01-01

Rapid detection of nucleic acids is integral for clinical diagnostics and biotechnological applications. We recently developed a platform termed SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) that combines isothermal pre-amplification with Cas13 to detect single molecules of RNA or DNA. Through characterization of CRISPR enzymology and application development, we report here four advances integrated into SHERLOCKv2: 1) 4-channel single reaction multiplexing using orthogonal CRISPR enzymes; 2) quantitative measurement of input down to 2 aM; 3) 3.5-fold increase in signal sensitivity by combining Cas13 with Csm6, an auxilary CRISPR-associated enzyme; and 4) lateral flow read-out. SHERLOCKv2 can detect Dengue or Zika virus ssRNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids. PMID:29449508

Development of real-time recombinase polymerase amplification assay for rapid and sensitive detection of canine parvovirus 2.

PubMed

Geng, Yunyun; Wang, Jianchang; Liu, Libing; Lu, Yan; Tan, Ke; Chang, Yan-Zhong

2017-11-06

Canine parvovirus 2, a linear single-stranded DNA virus belonging to the genus Parvovirus within the family Parvoviridae, is a highly contagious pathogen of domestic dogs and several wild canidae species. Early detection of canine parvovirus (CPV-2) is crucial to initiating appropriate outbreak control strategies. Recombinase polymerase amplification (RPA), a novel isothermal gene amplification technique, has been developed for the molecular detection of diverse pathogens. In this study, a real-time RPA assay was developed for the detection of CPV-2 using primers and an exo probe targeting the CPV-2 nucleocapsid protein gene. The real-time RPA assay was performed successfully at 38 °C, and the results were obtained within 4-12 min for 10 5 -10 1 molecules of template DNA. The assay only detected CPV-2, and did not show cross-detection of other viral pathogens, demonstrating a high level of specificity. The analytical sensitivity of the real-time RPA was 10 1 copies/reaction of a standard DNA template, which was 10 times more sensitive than the common RPA method. The clinical sensitivity of the real-time RPA assay matched 100% (n = 91) to the real-time PCR results. The real-time RPA assay is a simple, rapid, reliable and affordable method that can potentially be applied for the detection of CPV-2 in the research laboratory and point-of-care diagnosis.

Rapid determination of gizzerosine in fish meals using microchip capillary electrophoresis with laser-induced fluorescence detection.

PubMed

Xiao, Meng-Wei; Bai, Xiao-Lin; Xu, Pei-Li; Zhao, Yan; Yang, Li; Liu, Yi-Ming; Liao, Xun

2017-05-01

Sensitive detection of gizzerosine, a causative agent for deadly gizzard erosion in chicken feeds, is very important to the poultry industry. In this work, a new method was developed based on microchip capillary electrophoresis (MCE) with laser-induced fluorescence (LIF) detection for rapid analysis of gizzerosine, a biogenic amine in fish meals. The MCE separation was performed on a glass microchip using sodium dodecyl sulfate (SDS) as dynamic coating modifier. Separation conditions, including running buffer pH and concentration, SDS concentration, and the separation voltage were investigated to achieve fast and sensitive quantification of gizzerosine. The assay proposed was very quick and could be completed within 65 s. A linear calibration curve was obtained in the range from 0.04 to 1.8 μg ml -1 gizzerosine. The detection limit was 0.025 μg ml -1 (0.025 mg kg -1 ), which was far more sensitive than those previously reported. Gizzerosine was well separated from other endogenous components in fish meal samples. Recovery of gizzerosine from this sample matrix (n = 3) was determined to be 97.2-102.8%. The results from analysing fish meal samples indicated that the present MCE-LIF method might hold the potential for rapid detection of gizzerosine in poultry feeds.

Establishment of reverse transcription loop-mediated isothermal amplification for rapid detection and differentiation of canine distemper virus infected and vaccinated animals.

PubMed

Liu, Da-Fei; Liu, Chun-Guo; Tian, Jin; Jiang, Yi-Tong; Zhang, Xiao-Zhan; Chai, Hong-Liang; Yang, Tian-Kuo; Yin, Xiu-Chen; Zhang, Hong-Ying; Liu, Ming; Hua, Yu-Ping; Qu, Lian-Dong

2015-06-01

Although widespread vaccination against canine distemper virus (CDV) has been conducted for many decades, several canine distemper outbreaks in vaccinated animals have been reported frequently. In order to detect and differentiate the wild-type and vaccine strains of the CDV from the vaccinated animals, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed. A set of four primers-two internal and two external-were designed to target the H gene for the specific detection of wild-type CDV variants. The CDV-H RT-LAMP assay rapidly amplified the target gene, within 60 min, using a water bath held at a constant temperature of 65°C. The assay was 100-fold more sensitive than conventional RT-PCR, with a detection limit of 10(-1)TCID50ml(-1). The system showed a preference for wild-type CDV, and exhibited less sensitivity to canine parvovirus, canine adenovirus type 1 and type 2, canine coronavirus, and canine parainfluenza virus. The assay was validated using 102 clinical samples obtained from vaccinated dog farms, and the results were comparable to a multiplex nested RT-PCR assay. The specific CDV-H RT-LAMP assay provides a simple, rapid, and sensitive tool for the detection of canines infected with wild-type CDV from canines vaccinated with attenuated vaccine. Copyright © 2015 Elsevier B.V. All rights reserved.

Rapid Detection of Enterobacter Sakazakii in milk Powder using amino modified chitosan immunomagnetic beads.

PubMed

Zhu, Yinglian; Wang, Dongfeng

2016-12-01

Chitosan immunomagnetic beads (CIBs) were first prepared through converting hydroxyl groups of natural polymer material-chitosan into amino groups using epichlorohydrin and ethylenediamine as modification agent and then coupling with polyclonal antibodies of Enterobacter sakazakii using glutaraldehyde as cross-linking agent. The beads before coupling with antibodies were characterized by magnetic property measurement, FTIR, SEM and XRD technologies. In the assay a natural polysaccharide-chitosan, which has good biological and chemical properties such as non-toxicity, biocompatibility and high chemical reactivity was first used for synthesis of immunomagnetic beads. The detection method first established in this paper that combined the beads with chromogenic medium together to rapid detect E. sakazakii in milk powder could greatly improve the detection specificity and working efficiency. The beads exhibited a maximum capturing capacity of 1×10 6 cfu/g with the detection sensitivity of 4cfu/g. The results demonstrate that the assay is a straightforward, specific and sensitive alternative for rapid detection of E.sakazakii in food matrix. The total analysis time was as little as about 25h, which greatly shorten the detection time. The method can provides new ideas not only to preparation technique of immunomagnetic beads but to imunne detection technique in food safety. Copyright © 2016 Elsevier B.V. All rights reserved.

Sensitive Detection of Xanthomonas oryzae Pathovars oryzae and oryzicola by Loop-Mediated Isothermal Amplification

PubMed Central

Lang, Jillian M.; Langlois, Paul; Nguyen, Marian Hanna R.; Triplett, Lindsay R.; Purdie, Laura; Holton, Timothy A.; Djikeng, Appolinaire; Vera Cruz, Casiana M.; Verdier, Valérie

2014-01-01

Molecular diagnostics for crop diseases can enhance food security by enabling the rapid identification of threatening pathogens and providing critical information for the deployment of disease management strategies. Loop-mediated isothermal amplification (LAMP) is a PCR-based tool that allows the rapid, highly specific amplification of target DNA sequences at a single temperature and is thus ideal for field-level diagnosis of plant diseases. We developed primers highly specific for two globally important rice pathogens, Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight (BB) disease, and X. oryzae pv. oryzicola, the causal agent of bacterial leaf streak disease (BLS), for use in reliable, sensitive LAMP assays. In addition to pathovar distinction, two assays that differentiate X. oryzae pv. oryzae by African or Asian lineage were developed. Using these LAMP primer sets, the presence of each pathogen was detected from DNA and bacterial cells, as well as leaf and seed samples. Thresholds of detection for all assays were consistently 104 to 105 CFU ml−1, while genomic DNA thresholds were between 1 pg and 10 fg. Use of the unique sequences combined with the LAMP assay provides a sensitive, accurate, rapid, simple, and inexpensive protocol to detect both BB and BLS pathogens. PMID:24837384

Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thissen, James B.; McLoughlin, Kevin; Gardner, Shea

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. A multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated with minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition the performance of the array was tested when hybridization time was reduced from 17 h to 1 h. The LLMDA wasmore » able to detect unamplified vaccinia virus DNA at a concentration of 14 fM, or 100,000 genome copies in 12 μL of sample. With amplification, positive identification was made with only 100 genome copies of input material. When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. Accurate detection was found but with a 4-fold drop in sensitivity for a 1 h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1 h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain level resolution. Sensitivity improved by three orders of magnitude with random whole genome amplification prior to hybridization; for instance, the array detected a DNA virus with only 20 fg or 100 genome copies as input. This multiplexed microarray is an efficient tool to analyze clinical and environmental samples for the presence of multiple viral and bacterial pathogens rapidly.« less

Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray

DOE PAGES

Thissen, James B.; McLoughlin, Kevin; Gardner, Shea; ...

2014-06-01

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. A multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated with minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition the performance of the array was tested when hybridization time was reduced from 17 h to 1 h. The LLMDA wasmore » able to detect unamplified vaccinia virus DNA at a concentration of 14 fM, or 100,000 genome copies in 12 μL of sample. With amplification, positive identification was made with only 100 genome copies of input material. When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. Accurate detection was found but with a 4-fold drop in sensitivity for a 1 h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1 h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain level resolution. Sensitivity improved by three orders of magnitude with random whole genome amplification prior to hybridization; for instance, the array detected a DNA virus with only 20 fg or 100 genome copies as input. This multiplexed microarray is an efficient tool to analyze clinical and environmental samples for the presence of multiple viral and bacterial pathogens rapidly.« less

Short non-coding RNAs as bacteria species identifiers detected by surface plasmon resonance enhanced common path interferometry

NASA Astrophysics Data System (ADS)

Greef, Charles; Petropavlovskikh, Viatcheslav; Nilsen, Oyvind; Khattatov, Boris; Plam, Mikhail; Gardner, Patrick; Hall, John

2008-04-01

Small non-coding RNA sequences have recently been discovered as unique identifiers of certain bacterial species, raising the possibility that they can be used as highly specific Biowarfare Agent detection markers in automated field deployable integrated detection systems. Because they are present in high abundance they could allow genomic based bacterial species identification without the need for pre-assay amplification. Further, a direct detection method would obviate the need for chemical labeling, enabling a rapid, efficient, high sensitivity mechanism for bacterial detection. Surface Plasmon Resonance enhanced Common Path Interferometry (SPR-CPI) is a potentially market disruptive, high sensitivity dual technology that allows real-time direct multiplex measurement of biomolecule interactions, including small molecules, nucleic acids, proteins, and microbes. SPR-CPI measures differences in phase shift of reflected S and P polarized light under Total Internal Reflection (TIR) conditions at a surface, caused by changes in refractive index induced by biomolecular interactions within the evanescent field at the TIR interface. The measurement is performed on a microarray of discrete 2-dimensional areas functionalized with biomolecule capture reagents, allowing simultaneous measurement of up to 100 separate analytes. The optical beam encompasses the entire microarray, allowing a solid state detector system with no scanning requirement. Output consists of simultaneous voltage measurements proportional to the phase differences resulting from the refractive index changes from each microarray feature, and is automatically processed and displayed graphically or delivered to a decision making algorithm, enabling a fully automatic detection system capable of rapid detection and quantification of small nucleic acids at extremely sensitive levels. Proof-of-concept experiments on model systems and cell culture samples have demonstrated utility of the system, and efforts are in progress for full development and deployment of the device. The technology has broad applicability as a universal detection platform for BWA detection, medical diagnostics, and drug discovery research, and represents a new class of instrumentation as a rapid, high sensitivity, label-free methodology.

Evaluation of twenty-two rapid antigen detection tests in the diagnosis of Equine Influenza caused by viruses of H3N8 subtype.

PubMed

Yamanaka, Takashi; Nemoto, Manabu; Bannai, Hiroshi; Tsujimura, Koji; Kondo, Takashi; Matsumura, Tomio; Gildea, Sarah; Cullinane, Ann

2016-03-01

Equine influenza (EI) is a highly contagious disease caused by viruses of the H3N8 subtype. The rapid diagnosis of EI is essential to reduce the disease spread. Many rapid antigen detection (RAD) tests for diagnosing human influenza are available, but their ability to diagnose EI has not been systematically evaluated. The aim of this study was to compare the performance of 22 RAD tests in the diagnosis of EI. The 22 RAD tests were performed on fivefold serial dilutions of EI virus to determine their detection limits. The four most sensitive RAD tests (ImmunoAce Flu, BD Flu examan, Quick chaser Flu A, B and ESPLINE Influenza A&B-N) were further evaluated using nasopharyngeal samples collected from experimentally infected and naturally infected horses. The results were compared to those obtained using molecular tests. The detection limits of the 22 RAD tests varied hugely. Even the four RAD tests showing the best sensitivity were 125-fold less sensitive than the molecular techniques. The duration of virus detection in the experimentally infected horses was shorter using the RAD tests than using the molecular techniques. The RAD tests detected between 27% and 73% of real-time RT-PCR-positive samples from naturally infected horses. The study demonstrated the importance of choosing the right RAD tests as only three of 22 were fit for diagnosing EI. It was also indicated that even RAD tests with the highest sensitivity serve only as an adjunct to molecular tests because of the potential for false-negative results. © 2015 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

Development of a rapid loop-mediated isothermal amplification assay for diagnosis and assessment of cure of Leishmania infection.

PubMed

Verma, Sandeep; Singh, Ruchi; Sharma, Vanila; Bumb, Ram Avtar; Negi, Narendra Singh; Ramesh, V; Salotra, Poonam

2017-03-23

Leishmaniasis is a spectrum of diseases with great relevance to public health. Conventional diagnostic methods are time consuming, needing trained personnel. A robust, rapid and cost effective diagnostic test is warranted for on-time diagnosis and field application. We have developed a loop mediated isothermal amplification (LAMP) assay with primers (n = 6) based on Leishmania donovani kDNA for detection of Leishmania infection, using a closed tube to prevent cross-contamination. The assay was used to detect Leishmania infection in biological samples obtained from patients of visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL). The assay was positive for L. donovani, L. tropica and L. major parasites, with the highest sensitivity towards L. donovani (1 fg DNA). The high sensitivity of the assay for detection of L. donovani was reflected in its ability to detect parasite DNA within 30 min of amplification time with a threshold detection limit of ≥25 copies per reaction. The assay detected parasite in 64 of 66 VL blood samples (sensitivity, 96.9%; 95% CI: 89.6-99.2%), 15 of 15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI:79.6-100%), 65 of 67 PKDL tissue biopsy samples (sensitivity, 97%; 95% CI:89.7-99.2%). The assay was evaluated in a few cases of CL wherein it was found positive in 8 of 10 tissue biopsies (sensitivity, 80%; 95% CI: 49-94.3%). The assay was negative in all control blood (n = 76) and tissue biopsy (n = 24) samples (specificity, 100%; 95% CI: 96.3-100%). Further, the assay was evaluated for its utility in assessment of cure in treated VL and PKDL patients. The assay detected parasite DNA in 2 of 20VL blood samples and 2 of 21 PKDL tissue samples. Out of 4 cases that were positive for parasite DNA at post treatment stage, 2 patients (1VL and 1 PKDL) returned with relapse. The study demonstrated a Leishmania genus specific closed tube LAMP assay for reliable and rapid molecular diagnosis of VL and PKDL with potential for application in assessment of cure.

Rapid detection of EBOLA VP40 in microchip immunofiltration assay

NASA Astrophysics Data System (ADS)

Miethe, Peter; Gary, Dominik; Hlawatsch, Nadine; Gad, Anne-Marie

2015-05-01

In the spring of 2014, the Ebola virus (EBOV) strain Zaire caused a dramatic outbreak in several regions of West Africa. The RT-PCR and antigen capture diagnostic proved to be effective for detecting EBOV in blood and serum. In this paper, we present data of a rapid antigen capture test for the detection of VP40. The test was performed in a microfluidic chip for immunofiltration analysis. The chip integrates all necessary assay components. The analytical sensitivity of the rapid test was 8 ng/ml for recombinant VP40. In serum and whole blood samples spiked with virus culture material, the detection limit was 2.2 x 102 PFU/ml. The performance data of the rapid test (15 min) are comparable to that of the VP40 laboratory ELISA.

Clinical Application of Picodroplet Digital PCR Technology for Rapid Detection of EGFR T790M in Next-Generation Sequencing Libraries and DNA from Limited Tumor Samples.

PubMed

Borsu, Laetitia; Intrieri, Julie; Thampi, Linta; Yu, Helena; Riely, Gregory; Nafa, Khedoudja; Chandramohan, Raghu; Ladanyi, Marc; Arcila, Maria E

2016-11-01

Although next-generation sequencing (NGS) is a robust technology for comprehensive assessment of EGFR-mutant lung adenocarcinomas with acquired resistance to tyrosine kinase inhibitors, it may not provide sufficiently rapid and sensitive detection of the EGFR T790M mutation, the most clinically relevant resistance biomarker. Here, we describe a digital PCR (dPCR) assay for rapid T790M detection on aliquots of NGS libraries prepared for comprehensive profiling, fully maximizing broad genomic analysis on limited samples. Tumor DNAs from patients with EGFR-mutant lung adenocarcinomas and acquired resistance to epidermal growth factor receptor inhibitors were prepared for Memorial Sloan-Kettering-Integrated Mutation Profiling of Actionable Cancer Targets sequencing, a hybrid capture-based assay interrogating 410 cancer-related genes. Precapture library aliquots were used for rapid EGFR T790M testing by dPCR, and results were compared with NGS and locked nucleic acid-PCR Sanger sequencing (reference high sensitivity method). Seventy resistance samples showed 99% concordance with the reference high sensitivity method in accuracy studies. Input as low as 2.5 ng provided a sensitivity of 1% and improved further with increasing DNA input. dPCR on libraries required less DNA and showed better performance than direct genomic DNA. dPCR on NGS libraries is a robust and rapid approach to EGFR T790M testing, allowing most economical utilization of limited material for comprehensive assessment. The same assay can also be performed directly on any limited DNA source and cell-free DNA. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Rapid Detection of Urinary Tract Infections via Bacterial Nuclease Activity.

PubMed

Flenker, Katie S; Burghardt, Elliot L; Dutta, Nirmal; Burns, William J; Grover, Julia M; Kenkel, Elizabeth J; Weaver, Tyler M; Mills, James; Kim, Hyeon; Huang, Lingyan; Owczarzy, Richard; Musselman, Catherine A; Behlke, Mark A; Ford, Bradley; McNamara, James O

2017-06-07

Rapid and accurate bacterial detection methods are needed for clinical diagnostic, water, and food testing applications. The wide diversity of bacterial nucleases provides a rich source of enzymes that could be exploited as signal amplifying biomarkers to enable rapid, selective detection of bacterial species. With the exception of the use of micrococcal nuclease activity to detect Staphylococcus aureus, rapid methods that detect bacterial pathogens via their nuclease activities have not been developed. Here, we identify endonuclease I as a robust biomarker for E. coli and develop a rapid ultrasensitive assay that detects its activity. Comparison of nuclease activities of wild-type and nuclease-knockout E. coli clones revealed that endonuclease I is the predominant DNase in E. coli lysates. Endonuclease I is detectable by immunoblot and activity assays in uropathogenic E. coli strains. A rapid assay that detects endonuclease I activity in patient urine with an oligonucleotide probe exhibited substantially higher sensitivity for urinary tract infections than that reported for rapid urinalysis methods. The 3 hr turnaround time is much shorter than that of culture-based methods, thereby providing a means for expedited administration of appropriate antimicrobial therapy. We suggest this approach could address various unmet needs for rapid detection of E. coli. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Rapid detection of Ebola virus with a reagent-free, point-of-care biosensor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baca, Justin T.; Severns, Virginia; Lovato, Debbie

Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. This preliminary study demonstrates SAW biosensor detection of the Ebola virus in a concentration-dependent manner. The detection limit with this methodology is below the average level of viremia detected on the first day of symptoms by PCR. We observe a log-linear sensor response for highly fragmented Ebola viral particles, with a detection limit corresponding to 1.9 × 10⁴ PFU/mL prior to virus inactivation. We predict greatly improved sensitivity for intact, infectious Ebola virus. This point-of-care methodologymore » has the potential to detect Ebola viremia prior to symptom onset, greatly enabling infection control and rapid treatment. This biosensor platform is powered by disposable AA batteries and can be rapidly adapted to detect other emerging diseases in austere conditions.« less

Rapid detection of Ebola virus with a reagent-free, point-of-care biosensor

DOE PAGES

Baca, Justin T.; Severns, Virginia; Lovato, Debbie; ...

2015-04-14

Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. This preliminary study demonstrates SAW biosensor detection of the Ebola virus in a concentration-dependent manner. The detection limit with this methodology is below the average level of viremia detected on the first day of symptoms by PCR. We observe a log-linear sensor response for highly fragmented Ebola viral particles, with a detection limit corresponding to 1.9 × 10⁴ PFU/mL prior to virus inactivation. We predict greatly improved sensitivity for intact, infectious Ebola virus. This point-of-care methodologymore » has the potential to detect Ebola viremia prior to symptom onset, greatly enabling infection control and rapid treatment. This biosensor platform is powered by disposable AA batteries and can be rapidly adapted to detect other emerging diseases in austere conditions.« less

Development of a colloidal gold immunochromatographic strip assay for simple and fast detection of human α-lactalbumin in genetically modified cow milk.

PubMed

Tao, Chenyu; Zhang, Qingde; Feng, Na; Shi, Deshi; Liu, Bang

2016-03-01

The qualitative and quantitative declaration of food ingredients is important to consumers, especially for genetically modified food as it experiences a rapid increase in sales. In this study, we designed an accurate and rapid detection system using colloidal gold immunochromatographic strip assay (GICA) methods to detect genetically modified cow milk. First, we prepared 2 monoclonal antibodies for human α-lactalbumin (α-LA) and measured their antibody titers; the one with the higher titer was used for further experiments. Then, we found the optimal pH value and protein amount of GICA for detection of pure milk samples. The developed strips successfully detected genetically modified cow milk and non-modified cow milk. To determine the sensitivity of GICA, a quantitative ELISA system was used to determine the exact amount of α-LA, and then genetically modified milk was diluted at different rates to test the sensitivity of GICA; the sensitivity was 10 μg/mL. Our results demonstrated that the applied method was effective to detect human α-LA in cow milk. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Sensitive and selective determination of methylenedioxylated amphetamines by high-performance liquid chromatography with fluorimetric detection.

PubMed

Sadeghipour, F; Veuthey, J L

1997-11-07

A rapid, sensitive and selective liquid chromatographic method with fluorimetric detection was developed for the separation and quantification of four methylenedioxylated amphetamines without interference of other drugs of abuse and common substances found in illicit tablets. The method was validated by examining linearity, precision and accuracy as well as detection and quantification limits. Methylenedioxylated amphetamines were quantified in eight tablets from illicit drug seizures and results were quantitatively compared to HPLC-UV analyses. To demonstrate the better sensitivity of the fluorimetric detection, methylenedioxylated amphetamines were analyzed in serum after a liquid-liquid extraction procedure and results were also compared to HPLC-UV analyses.

A simple and highly sensitive colorimetric detection method for gaseous formaldehyde.

PubMed

Feng, Liang; Musto, Christopher J; Suslick, Kenneth S

2010-03-31

A colorimetric detection method using amine-functionalized polymer films doped with a pH indicator has been developed for the rapid, sensitive, and quantitative detection of gaseous formaldehyde at concentrations well below the immediately dangerous to life or health (IDLH) limit. In 1 min, visible color changes are easily observed, even down to the permissible exposure limit (PEL) at 750 ppb. The limit of detection is below 50 ppb (7% of the PEL) after 10 min of exposure. This sensor is essentially unaffected by changes in humidity or temperature (4 to 50 degrees C) and is not sensitive to common interferents.

An ultra-sensitive monoclonal antibody-based fluorescent microsphere immunochromatographic test strip assay for detecting aflatoxin M1 in milk

USDA-ARS?s Scientific Manuscript database

A rapid lateral flow fluorescent microspheres immunochromatography test strip (FMs-ICTS) has been developed for the detection of aflatoxin M1 (AFM1) residues in milk. For this purpose, an ultra-sensitive anti-AFM1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC50 value of the MA...

Liquid-phase exfoliated graphene as highly-sensitive sensor for simultaneous determination of endocrine disruptors: diethylstilbestrol and estradiol.

PubMed

Hu, Lintong; Cheng, Qin; Chen, Danchao; Ma, Ming; Wu, Kangbing

2015-01-01

It is quite important to develop convenient and rapid analytical methods for trace levels of endocrine disruptors because they heavily affect health and reproduction of humans and animals. Herein, graphene was easily prepared via one-step exfoliation using N-methyl-2-pyrrolidone as solvent, and then used to construct an electrochemical sensor for highly-sensitive detection of diethylstilbestrol (DES) and estradiol (E2). On the surface of prepared graphene film, two independent and greatly-increased oxidation waves were observed at 0.28V and 0.49V for DES and E2. The remarkable signal enlargements indicated that the detection sensitivity was improved significantly. The influences of pH value, amount of graphene and accumulation time on the oxidation signals of DES and E2 were discussed. As a result, a highly-sensitive and rapid electrochemical method was newly developed for simultaneous detection of DES and E2. The values of detection limit were evaluated to be 10.87 nM and 4.9 nM for DES and E2. Additionally, this new method was successfully used in lake water samples and the accuracy was satisfactory. Copyright © 2014 Elsevier B.V. All rights reserved.

Recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual detection of Schistosoma japonicum.

PubMed

Sun, Kui; Xing, Weiwei; Yu, Xinling; Fu, Wenliang; Wang, Yuanyuan; Zou, Minji; Luo, Zhihong; Xu, Donggang

2016-08-31

With the continuous decline in prevalence and intensity of Schistosoma japonicum infection in China, more accurate and sensitive methods suitable for field detection become much needed for schistosomiasis control. Here, a novel rapid and visual detection method based on the combination of recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) was developed to detect S. japonicum DNA in fecal samples. The LFD-RPA assay targeting SjR2 could detect 5 fg S. japonicum DNA, which was identical to qPCR and real-time RPA assay, and showed no cross-reaction with other parasites. The detection could be finished within 15-20 min at a wide temperature range (25-45 °C), and the results could be visualized by naked eye. The diagnostic validity of LFD-RPA assay was further assessed with 14 fecal samples of infected patients diagnosed by Kato-Katz method and 31 fecal samples of healthy persons, and compared with that of Enzyme-linked immunosorbent assay (ELSIA) and Indirect Hemagglutination Assay (IHA). The LFD-RPA assay showed 92.68 % sensitivity, 100 % specificity and excellent diagnostic agreement with the gold standard Kato-Katz test (k = 0.947, Z = 6.36, P < 0.001), whereas ELISA showed 85.71 % sensitivity, 93.55 % specificity, and substantial diagnostic agreement (k = 0.793, Z = 5.31, P < 0.001), and IHA showed 78.57 % sensitivity, 83.87 % specificity, and moderate diagnostic agreement (k = 0.600, Z = 4.05, P < 0.001), indicating that the LFD-RPA was much better than the traditional methods. The LFD-RPA assay established by us is a sensitive, specific, rapid and convenient method for the diagnosis of schistosomiasis, and shows a great potency in field application.

CGP stain: An inexpensive, odorless, rapid, sensitive, and in principle in vitro methylation-free Coomassie Brilliant Blue stain.

PubMed

Yasumitsu, Hidetaro; Ozeki, Yasuhiro; Kawsar, Sarkar M A; Toda, Tosifusa; Kanaly, Robert

2010-11-01

Coomassie Brilliant Blue (CBB) protein stains are inexpensive but detect proteins at only at microgram levels. Because of acetic acid and methanol, they cause skin irritation and reduce work motivation by malodor. Recent mass spectrometric (MS) analyses demonstrated that nanogram-sensitive colloidal CBB staining resulted in in vitro methylations of proteins. We propose a rapid, inexpensive, sensitive, odorless, less harsh, and in vitro methylation-free CBB stain. CGP uses three components: citric acid, CBB G-250, and polyvinylpyrrolidone. CGP detects proteins at 12ng within 45min, and because it is nonalcohol, in principle in vitro methylation would be eliminated. Indeed, MS analysis of CGP-stained bands confirmed a lack of methylation. 2010 Elsevier Inc. All rights reserved.

A Rapid and Sensitive Strip-Based Quick Test for Nerve Agents Tabun, Sarin, and Soman Using BODIPY-Modified Silica Materials.

PubMed

Climent, Estela; Biyikal, Mustafa; Gawlitza, Kornelia; Dropa, Tomáš; Urban, Martin; Costero, Ana M; Martínez-Máñez, Ramón; Rurack, Knut

2016-08-01

Test strips that in combination with a portable fluorescence reader or digital camera can rapidly and selectively detect chemical warfare agents (CWAs) such as Tabun (GA), Sarin (GB), and Soman (GD) and their simulants in the gas phase have been developed. The strips contain spots of a hybrid indicator material consisting of a fluorescent BODIPY indicator covalently anchored into the channels of mesoporous SBA silica microparticles. The fluorescence quenching response allows the sensitive detection of CWAs in the μg m(-3) range in a few seconds. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid detection of meticillin-resistant Staphylococcus aureus bacteraemia using combined three-hour short-incubation matrix-assisted laser desorption/ionization time-of-flight MS identification and Alere Culture Colony PBP2a detection test.

PubMed

Delport, Johannes Andries; Mohorovic, Ivor; Burn, Sandi; McCormick, John Kenneth; Schaus, David; Lannigan, Robert; John, Michael

2016-07-01

Meticillin-resistant Staphylococcus aureus (MRSA) bloodstream infection is responsible for significant morbidity, with mortality rates as high as 60 % if not treated appropriately. We describe a rapid method to detect MRSA in blood cultures using a combined three-hour short-incubation BRUKER matrix-assisted laser desorption/ionization time-of-flight MS BioTyper protocol and a qualitative immunochromatographic assay, the Alere Culture Colony Test PBP2a detection test. We compared this combined method with a molecular method detecting the nuc and mecA genes currently performed in our laboratory. One hundred and seventeen S. aureus blood cultures were tested of which 35 were MRSA and 82 were meticillin-sensitive S. aureus (MSSA). The rapid combined test correctly identified 100 % (82/82) of the MSSA and 85.7 % (30/35) of the MRSA after 3 h. There were five false negative results where the isolates were correctly identified as S. aureus, but PBP2a was not detected by the Culture Colony Test. The combined method has a sensitivity of 87.5 %, specificity of 100 %, a positive predictive value of 100 % and a negative predictive value of 94.3 % with the prevalence of MRSA in our S. aureus blood cultures. The combined rapid method offers a significant benefit to early detection of MRSA in positive blood cultures.

Real-time PCR detection of Listeria monocytogenes in infant formula and lettuce following macrophage-based isolation and enrichment.

PubMed

Day, J B; Basavanna, U

2015-01-01

To develop a rapid detection procedure for Listeria monocytogenes in infant formula and lettuce using a macrophage-based enrichment protocol and real-time PCR. A macrophage cell culture system was employed for the isolation and enrichment of L. monocytogenes from infant formula and lettuce for subsequent identification using real-time PCR. Macrophage monolayers were exposed to infant formula and lettuce contaminated with a serial dilution series of L. monocytogenes. As few as approx. 10 CFU ml(-1) or g(-1) of L. monocytogenes were detected in infant formula and lettuce after 16 h postinfection by real-time PCR. Internal positive PCR controls were utilized to eliminate the possibility of false-negative results. Co-inoculation with Listeria innocua did not reduce the L. monocytogenes detection sensitivity. Intracellular L. monocytogenes could also be isolated on Listeria selective media from infected macrophage lysates for subsequent confirmation. The detection method is highly sensitive and specific for L. monocytogenes in infant formula and lettuce and establishes a rapid identification time of 20 and 48 h for presumptive and confirmatory identification, respectively. The method is a promising alternative to many currently used q-PCR detection methods which employ traditional selective media for enrichment of contaminated food samples. Macrophage enrichment of L. monocytogenes eliminates PCR inhibitory food elements and contaminating food microflora which produce cleaner samples that increase the rapidity and sensitivity of detection. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

[Rapid Detection of Adenovirus in Fecal Samples by Capillary Electrophoresis-laser Induced Fluorescence and Microchip Capillary Electrophoresis-laser Induced Fluorescence].

PubMed

Ruan, Jia; Ren, Dong-xia; Yang, Dan-ni; Long, Pin-pin; Zhao, Hong-yue; Wang, Yi-qi; Li, Yong-xin

2015-07-01

To establish a rapid and sensitive method based on polymerase chain reaction (PCR) combined with capillary electrophoresis-laser induced fluorescence (CE-LIF) and microchip capillary electrophoresis-laser induced fluorescence (MCE-LIF) for detecting adenoviruses in fecal samples. The DNA of adenovirus in fecal samples were extracted by the commercial kits and the conserved region of hexon gene was selected as the target gene and amplified by PCR reaction. After labeling highly sensitive nucleic acid fluorescent dye SYBR Gold and SYBR Orange respectively, PCR amplification products were separated by CE and MCE under the optimized condition and detected by LIF detector. PCR amplification products could be detected within 9 min by CE-LIF and 6 min by MCE-LIF under the optimized separation condition. The sequenced PCR product showed good specificity in comparison with the prototype sequences from NCBI. The intraday and inter-day relative standard deviation (RSD) of the size (bp) of the target DNA was in the range of 1.14%-1.34% and 1.27%- 2.76%, respectively, for CE-LIF, and 1.18%-1.48% and 2.85%-4.06%, respectively, for MCE-LIF. The detection limits was 2.33 x 10(2) copies/mL for CE-LIF and 2.33 x 10(3) copies/mL for MCE-LIF. The two proposed methods were applied to detect fecal samples, both showing high accuracy. The two proposed methods of PCR-CE-LIF and PCR-MCE-LIF can detect adenovirus in fecal samples rapidly, sensitively and specifically.

A novel double recognition enzyme-linked immunosorbent assay based on the nucleocapsid protein for early detection of European porcine reproductive and respiratory syndrome virus infection.

PubMed

Venteo, A; Rebollo, B; Sarraseca, J; Rodriguez, M J; Sanz, A

2012-04-01

Precise and rapid detection of porcine reproductive respiratory syndrome virus (PRRSV) infection in swine farms is critical. Improvement of control procedures, such as testing incoming gilt and surveillance of seronegative herds requires more rapid and sensitive methods. However, standard serological techniques detect mainly IgG antibodies. A double recognition enzyme-linked immunosorbent assay (DR-ELISA) was developed for detection of antibodies specific to European PRRSV. This new assay can recognize both IgM and IgG antibodies to PRSSV which might be useful for detecting in routine surveillance assays pigs that are in the very early stages of infection and missed by conventional assays detecting only IgG antibodies. DR-ELISA is based on the double recognition of antigen by antibody. In this study, the recombinant nucleocapsid protein (N) of PRRSV was used both as the coating and the enzyme-conjugated antigen. To evaluate the sensitivity of the assay at early stages of the infection, sera from 69 pigs infected with PRRSV were collected during successive days post infection (pi) and tested. While standard methods showed low sensitivity rates before day 14 pi, DR-ELISA detected 88.4% seropositive samples at day 7 showing greater sensitivity at early stages of the infection. Further studies were carried out to assess the efficiency of the new assay, and the results showed DR-ELISA to be a sensitive and accurate method for early diagnosis of EU-PRRSV infection. Copyright © 2012 Elsevier B.V. All rights reserved.

Rapid and label-free detection of protein a by aptamer-tethered porous silicon nanostructures.

PubMed

Urmann, Katharina; Reich, Peggy; Walter, Johanna-Gabriela; Beckmann, Dieter; Segal, Ester; Scheper, Thomas

2017-09-10

Protein A, which is secreted by and displayed on the cell membrane of Staphylococcus aureus is an important biomarker for S. aureus. Thus, its rapid and specific detection may facilitate the pathogen identification and initiation of proper treatment. Herein, we present a simple, label-free and rapid optical biosensor enabling specific detection of protein A. Protein A-binding aptamer serves as the capture probe and is immobilized onto a nanostructured porous silicon thin film, which serves as the optical transducer element. We demonstrate high sensitivity of the biosensor with a linear detection range between 8 and 23μM. The apparent dissociation constant was determined as 13.98μM and the LoD is 3.17μM. Harnessing the affinity between protein A and antibodies, a sandwich assay format was developed to amplify the optical signal associated with protein A capture by the aptamer. Using this approach, we increase the sensitivity of the biosensor, resulting in a three times lower LoD. Copyright © 2017 Elsevier B.V. All rights reserved.

A real-time loop-mediated isothermal amplification assay for rapid detection of Shigella species.

PubMed

Liew, P S; Teh, C S J; Lau, Y L; Thong, K L

2014-12-01

Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health issue. The development of effective techniques for rapid detection of this pathogen is essential for breaking the chain of transmission. Therefore, we have developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the invasion plasmid antigen H (ipaH) gene to rapidly detect Shigella species. This assay could be performed in 90 min at an optimal temperature of 64ºC, with endpoint results visualized directly. Notably, the method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 10(5) CFU/ml, while PCR displayed a limit of 5.9 x 10(7) CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 10(4) CFU/g, whereas PCR was 3.6 x 10(5) CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative.

Comparison of Three Biochemical Tests for Rapid Detection of Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae.

PubMed

Poirel, Laurent; Fernández, Javier; Nordmann, Patrice

2016-02-01

Enterobacterial isolates producing clavulanic-inhibited extended-spectrum β-lactamases (ESBLs) are increasingly spreading in the community and are often responsible for nosocomial infections. Rapid biochemical tests have been developed recently for their detection. Three tests, namely, the Rapid ESBL NDP test, the β-Lacta test, and the Rapid ESBL Screen, have been evaluated with a collection of 108 well-characterized strains, including wild-type strains, strains producing ESBLs, overexpressed cephalosporinases, and carbapenemases. The ESBL NDP test and the Rapid ESBL Screen (a copy of the ESBL NDP test) are aimed at detecting ESBL producers, while the β-Lacta test is aimed at detecting not only ESBL producers but also cephalosporinase and carbapenemase producers. The sensitivity and specificity for detecting ESBL producers (n = 60) were 95% and 100% for the Rapid ESBL NDP test, 80% and 87% (after 30 min) and 92% and 83% (after 2 h) for the Rapid ESBL Screen, and 88% and 71% for the β-Lacta test, respectively. Varied and time-consuming detection (up to 2 h) of ESBLs by the Rapid ESBL Screen and concomitant and varied detection of producers of AmpC and several types of carbapenemases correspond to significant shortcomings of using the Rapid Screen ESBL and β-Lacta tests, respectively. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Horseradish peroxidase-labeled oligonucleotides and fluorescent tyramides for rapid detection of chromosome-specific repeat sequences.

PubMed

van Gijlswijk, R P; Wiegant, J; Vervenne, R; Lasan, R; Tanke, H J; Raap, A K

1996-01-01

We present a sensitive and rapid fluorescence in situ hybridization (FISH) strategy for detecting chromosome-specific repeat sequences. It uses horseradish peroxidase (HRP)-labeled oligonucleotide sequences in combination with fluorescent tyramide-based detection. After in situ hybridization, the HRP conjugated to the oligonucleotide probe is used to deposit fluorescently labeled tyramide molecules at the site of hybridization. The method features full chemical synthesis of probes, strong FISH signals, and short processing periods, as well as multicolor capabilities.

Development of an immunochromatographic test with anti-LipL32-coupled gold nanoparticles for Leptospira detection.

PubMed

Chirathaworn, Chintana; Janwitthayanan, Weena; Sereemaspun, Amornpun; Lertpocasombat, Kanchalee; Rungpanich, Utane; Ekpo, Pattama; Suwancharoen, Duangjai

2014-04-01

Detection of antibody specific to Leptospira by various immunological techniques has been used for leptospirosis diagnosis. However, the sensitivity of antibody detection during the first few days after infection is low. Molecular techniques are suggested to provide earlier diagnosis than antibody detection, but a rapid and easy to perform assay for Leptospira antigen detection would provide an additional useful tool for disease diagnosis. In this study, we coupled gold nanoparticles with antibody to LipL32, a protein commonly found in pathogenic Leptospira. This coupled gold reagent was used in the immunochromatographic strip for Leptospira detection. We demonstrated that the sensitivity of Leptospira detection by this strip was 10(3) ml(-1). There was no positive result detected when strips were tested with non-pathogenic Leptospira, Staphylococcus aureus, Streptococcus group B, Acinetobacter baumannii, Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, Enterococcus faecalis or Enterococcus faecium. These data suggest that gold nanoparticles coupled with antibody to LipL32 could be used for Leptospira detection by a rapid test based on an immunochromatographic technique.

Development and evaluation of a polydiacetylene based biosensor for the detection of H5 influenza virus.

PubMed

Jiang, Lixiang; Luo, Jing; Dong, Wenjie; Wang, Chengmin; Jin, Wen; Xia, Yuetong; Wang, Haijing; Ding, Hua; Jiang, Long; He, Hongxuan

2015-07-01

H5N1 avian influenza has caused serious economic losses as well as posed significant threats to public health, agriculture and wildlife. It is important to develop a rapid, sensitive and specific detection platform suitable for disease surveillance and control. In this study, a highly sensitive, specific and rapid biosensor based on polydiacetylene was developed for detecting H5 influenza virus. The polydiacetylene based biosensor was produced from an optimized ratio of 10,12-pentacosadiynoic acid and 1,2-dimyristoyl-sn-glycero-3-phosphocholine, with the anti-H5 influenza antibody embedded onto the vesicle surface. The optimized polydiacetylene vesicle could detect H5 influenza virus sensitively with a detection limit of 0.53 copies/μL, showing a dramatic blue-to-red color change that can be observed directly by the naked eye and recorded by a UV-vis spectrometer. The sensitivity, specificity and accuracy of the biosensor were also evaluated. The sensor could specifically differentiate H5 influenza virus from H3 influenza virus, Newcastle disease virus and porcine reproductive and respiratory syndrome virus. Detection using tracheal swabs was in accord with virus isolation results, and comparable to the RT-PCR method. These results offer the possibility and potential of simple polydiacetylene based bio-analytical method for influenza surveillance. Copyright © 2015 Elsevier B.V. All rights reserved.

Rapid and Sensitive Salmonella Typhi Detection in Blood and Fecal Samples Using Reverse Transcription Loop-Mediated Isothermal Amplification.

PubMed

Fan, Fenxia; Yan, Meiying; Du, Pengcheng; Chen, Chen; Kan, Biao

2015-09-01

Typhoid fever caused by Salmonella enterica serovar Typhi remains a significant public health problem in developing countries. Although the main method for diagnosing typhoid fever is blood culture, the test is time consuming and not always able to detect infections. Thus, it is very difficult to distinguish typhoid from other infections in patients with nonspecific symptoms. A simple and sensitive laboratory detection method remains necessary. The purpose of this study is to establish and evaluate a rapid and sensitive reverse transcription-based loop-mediated isothermal amplification (RT-LAMP) method to detect Salmonella Typhi infection. In this study, a new specific gene marker, STY1607, was selected to develop a STY1607-RT-LAMP assay; this is the first report of specific RT-LAMP detection assay for typhoid. Human-simulated and clinical blood/stool samples were used to evaluate the performance of STY1607-RT-LAMP for RNA detection; this method was compared with STY1607-LAMP, reverse transcription real-time polymerase chain reaction (rRT-PCR), and bacterial culture methods for Salmonella Typhi detection. Using mRNA as the template, STY1607-RT-LAMP exhibited 50-fold greater sensitivity than STY1607-LAMP for DNA detection. The STY1607-RT-LAMP detection limit is 3 colony-forming units (CFU)/mL for both the pure Salmonella Typhi samples and Salmonella Typhi-simulated blood samples and was 30 CFU/g for the simulated stool samples, all of which were 10-fold more sensitive than the rRT-PCR method. RT-LAMP exhibited improved Salmonella Typhi detection sensitivity compared to culture methods and to rRT-PCR of clinical blood and stool specimens from suspected typhoid fever patients. Because it can be performed without sophisticated equipment or skilled personnel, RT-LAMP is a valuable tool for clinical laboratories in developing countries. This method can be applied in the clinical diagnosis and care of typhoid fever patients as well as for a quick public health response.

Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations

PubMed Central

Law, Jodi Woan-Fei; Ab Mutalib, Nurul-Syakima; Chan, Kok-Gan; Lee, Learn-Han

2015-01-01

The incidence of foodborne diseases has increased over the years and resulted in major public health problem globally. Foodborne pathogens can be found in various foods and it is important to detect foodborne pathogens to provide safe food supply and to prevent foodborne diseases. The conventional methods used to detect foodborne pathogen are time consuming and laborious. Hence, a variety of methods have been developed for rapid detection of foodborne pathogens as it is required in many food analyses. Rapid detection methods can be categorized into nucleic acid-based, biosensor-based and immunological-based methods. This review emphasizes on the principles and application of recent rapid methods for the detection of foodborne bacterial pathogens. Detection methods included are simple polymerase chain reaction (PCR), multiplex PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP) and oligonucleotide DNA microarray which classified as nucleic acid-based methods; optical, electrochemical and mass-based biosensors which classified as biosensor-based methods; enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay which classified as immunological-based methods. In general, rapid detection methods are generally time-efficient, sensitive, specific and labor-saving. The developments of rapid detection methods are vital in prevention and treatment of foodborne diseases. PMID:25628612

Genetic diversity among pandemic 2009 influenza viruses isolated from a transmission chain

PubMed Central

2013-01-01

Background Influenza viruses such as swine-origin influenza A(H1N1) virus (A(H1N1)pdm09) generate genetic diversity due to the high error rate of their RNA polymerase, often resulting in mixed genotype populations (intra-host variants) within a single infection. This variation helps influenza to rapidly respond to selection pressures, such as those imposed by the immunological host response and antiviral therapy. We have applied deep sequencing to characterize influenza intra-host variation in a transmission chain consisting of three cases due to oseltamivir-sensitive viruses, and one derived oseltamivir-resistant case. Methods Following detection of the A(H1N1)pdm09 infections, we deep-sequenced the complete NA gene from two of the oseltamivir-sensitive virus-infected cases, and all eight gene segments of the viruses causing the remaining two cases. Results No evidence for the resistance-causing mutation (resulting in NA H275Y substitution) was observed in the oseltamivir-sensitive cases. Furthermore, deep sequencing revealed a subpopulation of oseltamivir-sensitive viruses in the case carrying resistant viruses. We detected higher levels of intra-host variation in the case carrying oseltamivir-resistant viruses than in those infected with oseltamivir-sensitive viruses. Conclusions Oseltamivir-resistance was only detected after prophylaxis with oseltamivir, suggesting that the mutation was selected for as a result of antiviral intervention. The persisting oseltamivir-sensitive virus population in the case carrying resistant viruses suggests either that a small proportion survive the treatment, or that the oseltamivir-sensitive virus rapidly re-establishes itself in the virus population after the bottleneck. Moreover, the increased intra-host variation in the oseltamivir-resistant case is consistent with the hypothesis that the population diversity of a RNA virus can increase rapidly following a population bottleneck. PMID:23587185

Rapid micromotor-based naked-eye immunoassay.

PubMed

de Ávila, Berta Esteban-Fernández; Zhao, Mingjiao; Campuzano, Susana; Ricci, Francesco; Pingarrón, José M; Mascini, Marcello; Wang, Joseph

2017-05-15

A dynamic micromotor-based immunoassay, exemplified by cortisol detection, based on the use of tubular micromotors functionalized with a specific antibody is described. The use of antibody-functionalized micromotors offers huge acceleration of both direct and competitive cortisol immunoassays, along with greatly enhanced sensitivity of direct and competitive immunoassays. The dramatically improved speed and sensitivity reflect the greatly increased likelihood of antibody-cortisol contacts and fluid mixing associated with the dynamic movement of these microtube motors and corresponding bubble generation that lead to a highly efficient and rapid recognition process. Rapid naked-eye detection of cortisol in the sample is achieved in connection to use of horseradish peroxidase (HRP) tag and TMB/H 2 O 2 system. Key parameters of the competitive immunoassay (e.g., incubation time and reaction volume) were optimized. This fast visual micromotor-based sensing approach enables "on the move" specific detection of the target cortisol down to 0.1μgmL -1 in just 2min, using ultrasmall (50µL) sample volumes. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid and Sensitive Identification of Ostrich Meat

PubMed Central

Abdulmawjood, Amir; Grabowski, Nils; Fohler, Svenja; Kittler, Sophie; Nagengast, Helga; Klein, Guenter

2014-01-01

Animal species identification is one of the primary duties of official food control. Since ostrich meat is difficult to be differentiated macroscopically from beef, therefore new analytical methods are needed. To enforce labeling regulations for the authentication of ostrich meat, it might be of importance to develop and evaluate a rapid and reliable assay. In the present study, a loop-mediated isothermal amplification (LAMP) assay based on the cytochrome b gene of the mitochondrial DNA of the species Struthio camelus was developed. The LAMP assay was used in combination with a real-time fluorometer. The developed system allowed the detection of 0.01% ostrich meat products. In parallel, a direct swab method without nucleic acid extraction using the HYPLEX LPTV buffer was also evaluated. This rapid processing method allowed detection of ostrich meat without major incubation steps. In summary, the LAMP assay had excellent sensitivity and specificity for detecting ostrich meat and could provide a sampling-to-result identification-time of 15 to 20 minutes. PMID:24963709

Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay.

PubMed

Zhou, Dinggang; Wang, Chunfeng; Li, Zhu; Chen, Yun; Gao, Shiwu; Guo, Jinlong; Lu, Wenying; Su, Yachun; Xu, Liping; Que, Youxiong

2016-01-01

Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg(2+), 6:1 ratio of inner vs. outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was 10-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100%) by LAMP and 97/100 cases (97%) by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable, and cost-effective for detection of the bar specific transgenic sugarcane.

Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay

PubMed Central

Zhou, Dinggang; Wang, Chunfeng; Li, Zhu; Chen, Yun; Gao, Shiwu; Guo, Jinlong; Lu, Wenying; Su, Yachun; Xu, Liping; Que, Youxiong

2016-01-01

Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg2+, 6:1 ratio of inner vs. outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was 10-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100%) by LAMP and 97/100 cases (97%) by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable, and cost-effective for detection of the bar specific transgenic sugarcane. PMID:27014303

Direct Application of the INNO-LiPA Rif.TB Line-Probe Assay for Rapid Identification of Mycobacterium tuberculosis Complex Strains and Detection of Rifampin Resistance in 360 Smear-Positive Respiratory Specimens from an Area of High Incidence of Multidrug-Resistant Tuberculosis

PubMed Central

Viveiros, Miguel; Leandro, Clara; Rodrigues, Liliana; Almeida, Josefina; Bettencourt, Rosário; Couto, Isabel; Carrilho, Lurdes; Diogo, José; Fonseca, Ana; Lito, Luís; Lopes, João; Pacheco, Teresa; Pessanha, Mariana; Quirim, Judite; Sancho, Luísa; Salfinger, Max; Amaral, Leonard

2005-01-01

The INNO-LiPA Rif.TB assay for the identification of Mycobacterium tuberculosis complex strains and the detection of rifampin (RIF) resistance has been evaluated with 360 smear-positive respiratory specimens from an area of high incidence of multidrug-resistant tuberculosis (MDR-TB). The sensitivity when compared to conventional identification/culture methods was 82.2%, and the specificity was 66.7%; the sensitivity and specificity were 100.0% and 96.9%, respectively, for the detection of RIF resistance. This assay has the potential to provide rapid information that is essential for the effective management of MDR-TB. PMID:16145166

A fluorescent immunochromatographic strip test using a quantum dot-antibody probe for rapid and quantitative detection of 1-aminohydantoin in edible animal tissues.

PubMed

Le, Tao; Zhang, Zhihao; Wu, Juan; Shi, Haixing; Cao, Xudong

2018-01-01

A rapid, simple, and sensitive fluorescent immunochromatographic strip test (ICST) based on quantum dots (QDs) has been developed to detect 1-aminohydantoin (AHD), a major metabolite of nitrofurantoin in animal tissues. To achieve this, QD-labeled antibody conjugates, which consist of CdSe/ZnS QDs and monoclonal antibodies, were prepared by an activated ester method. Under optimal conditions, with the nitrophenyl derivative of AHD as the target, the ICST had a linear range from 0.1 to 100 ng/mL, with a correlation coefficient of 0.9656 and a 50% inhibitory concentration of 4.51 ng/mL. The limit of detection was 0.14 ng/g, which was below the minimum required performance limit of 1 μg/kg for AHD established by the European Commission. The recoveries for AHD ranged from 81.5% to 108.2%, with coefficients of variation below 13%, based on intraday and interday analysis. Furthermore, for AHD in real samples, the ICST showed high reliability and high correlation with liquid chromatography-tandem mass spectrometry (correlation coefficient of 0.9916). To the best of our knowledge, this is the first report of a novel and sensitive method based on a fluorescent ICST to detect AHD below the minimum required performance limit. The ICST demonstrated high reliability, and could be ideally suited for rapid, simple, and on-site screening of AHD contamination in animal tissues. Graphical abstract A rapid, simple, and sensitive fluorescent immunochromatographic strip test that is based on quantum dots was developed to detect 1-aminohydantoin (AHD), a major metabolite of nitrofurantoin in animal tissues. 2-NBA 2-nitrobenzaldehyde, NP nitrophenyl.

Development of rapid immunochromatographic test for hemagglutinin antigen of H7 subtype in patients infected with novel avian influenza A (H7N9) virus.

PubMed

Kang, Keren; Chen, Li; Zhao, Xiang; Qin, Chengfeng; Zhan, Zanwu; Wang, Jihua; Li, Wenmei; Dzakah, Emmanuel E; Huang, Weijuang; Shu, Yuelong; Jiang, Tao; Cao, Wuchun; Xie, Mingquan; Luo, Xiaochun; Tang, Shixing

2014-01-01

Since human infection with the novel H7N9 avian influenza virus was identified in China in March 2013, the relatively high mortality rate and possibility of human-to-human transmission have highlighted the urgent need for sensitive and specific assays for diagnosis of H7N9 infection. We developed a rapid diagnostic test for the novel avian influenza A (H7N9) virus using anti-hemagglutinin (HA) monoclonal antibodies specifically targeting H7 in an immunochromatographic assay system. The assay limit of detection was 103.5 pfu/ml or 103TCID50 of H7N9 virus. The assay specifically detected H7N9 viral isolates and recombinant HA proteins of H7 subtypes including H7N7 and H7N9, but did not react with non-H7 subtypes including H1N1, H3N2, H5N1, H5N9, and H9N2. The detection sensitivity was 59.4% (19/32) for H7N9 patients confirmed by RT-PCR. Moreover, the highest sensitivity of 61.5% (16/26) was obtained when testing H7N9 positive sputum samples while 35.7% (5/14) of nasopharyngeal swabs and 20% (2/10) of fecal samples tested positive. No false positive detection was found when testing 180 H7N9 negative samples. Our novel rapid assay can specifically detect H7 HA antigen, facilitating rapid diagnosis for prevention and control of the on-going H7N9 epidemic.

Development of Rapid Immunochromatographic Test for Hemagglutinin Antigen of H7 Subtype in Patients Infected with Novel Avian Influenza A (H7N9) Virus

PubMed Central

Kang, Keren; Chen, Li; Zhao, Xiang; Qin, Chengfeng; Zhan, Zanwu; Wang, Jihua; Li, Wenmei; Dzakah, Emmanuel E.; Huang, Weijuang; Shu, Yuelong; Jiang, Tao; Cao, Wuchun; Xie, Mingquan; Luo, Xiaochun; Tang, Shixing

2014-01-01

Background Since human infection with the novel H7N9 avian influenza virus was identified in China in March 2013, the relatively high mortality rate and possibility of human-to-human transmission have highlighted the urgent need for sensitive and specific assays for diagnosis of H7N9 infection. Methodology/Principal Findings We developed a rapid diagnostic test for the novel avian influenza A (H7N9) virus using anti-hemagglutinin (HA) monoclonal antibodies specifically targeting H7 in an immunochromatographic assay system. The assay limit of detection was 103.5 pfu/ml or 103TCID50 of H7N9 virus. The assay specifically detected H7N9 viral isolates and recombinant HA proteins of H7 subtypes including H7N7 and H7N9, but did not react with non-H7 subtypes including H1N1, H3N2, H5N1, H5N9, and H9N2. The detection sensitivity was 59.4% (19/32) for H7N9 patients confirmed by RT-PCR. Moreover, the highest sensitivity of 61.5% (16/26) was obtained when testing H7N9 positive sputum samples while 35.7% (5/14) of nasopharyngeal swabs and 20% (2/10) of fecal samples tested positive. No false positive detection was found when testing 180 H7N9 negative samples. Conclusions/Significance Our novel rapid assay can specifically detect H7 HA antigen, facilitating rapid diagnosis for prevention and control of the on-going H7N9 epidemic. PMID:24647358

Field performance of malaria rapid diagnostic test for the detection of Plasmodium falciparum infection in Odisha State, India.

PubMed

Sahu, S S; Gunasekaran, K; Jambulingam, P

2015-12-01

Rapid diagnostic tests (RDTs) have become an essential surveillance tool in the malaria control programme in India. The current study aimed to assess the performance of ParaHIT-f, a rapid test in diagnosis of Plasmodium falciparum infection through detecting its specific antigen, histidine rich protein 2 (PfHRP-2), in Odisha State, India. The study was undertaken in eight falciparum malaria endemic southern districts of Odisha State. Febrile patients included through active case detection, were diagnosed by Accredited Social Health Activists (ASHAs) for P. falciparum infection using the RDT, ParaHIT-f. The performance of ParaHIT-f was evaluated using microscopy as the gold standard. A total of 1030 febrile patients were screened by both microscopy and the RDT for P. falciparum infection. The sensitivity of ParaHIT-f was 63.6% (95% CI: 56.0-70.6) and specificity was 98.9% (95% CI: 97.9-99.5), with positive and negative predictive values (PPV and NPV) of 92.6% (95% CI: 86.0-96.3) and 93.0% (95% CI: 91.0-94.5), respectively. When related to parasitaemia, the RDT sensitivity was 47.8% at the low parasitaemia of 4 to 40 parasites/µl of blood. The results showed that the performance of the RDT, ParaHIT-f, was not as sensitive as microscopy in detecting true falciparum infections; a high specificity presented a low frequency of false-positive RDT results. t0 he sensitivity of ParaHIT-f was around 60 per cent. It is, therefore, essential to improve the efficiency (sensitivity) of the kit so that the true falciparum infections will not be missed especially in areas where P. falciparum has been the predominant species causing cerebral malaria.

Multicenter Evaluation of the Solana Group A Streptococcus Assay: Comparison with Culture.

PubMed

Uphoff, Timothy S; Buchan, Blake W; Ledeboer, Nathan A; Granato, Paul A; Daly, Judy A; Marti, Tara N

2016-09-01

We compared group A Streptococcus (GAS) culture with a rapid helicase-dependent amplification (HDA) method using 1,082 throat swab specimens. The HDA method demonstrated 98.2% sensitivity and 97.2% specificity. GAS prevalence by culture was 20.7%, and it was 22.6% using the HDA method. In 35 min, the HDA method provided rapid, sensitive GAS detection, making culture confirmation unnecessary. Copyright © 2016 Uphoff et al.

In vitro detection and quantification of botulinum neurotoxin type E activity in avian blood

USGS Publications Warehouse

Piazza, T.M.; Blehert, D.S.; Dunning, F.M.; Berlowski-Zier, B. M.; Zeytin, F.N.; Samuel, M.D.; Tucker, W.C.

2011-01-01

Botulinum neurotoxin serotype E (BoNT/E) outbreaks in the Great Lakes region cause large annual avian mortality events, with an estimated 17,000 bird deaths reported in 2007 alone. During an outbreak investigation, blood collected from bird carcasses is tested for the presence of BoNT/E using the mouse lethality assay. While sensitive, this method is labor-intensive and low throughput and can take up to 7 days to complete. We developed a rapid and sensitive in vitro assay, the BoTest Matrix E assay, that combines immunoprecipitation with high-affinity endopeptidase activity detection by F??rster resonance energy transfer (FRET) to rapidly quantify BoNT/E activity in avian blood with detection limits comparable to those of the mouse lethality assay. On the basis of the analysis of archived blood samples (n = 87) collected from bird carcasses during avian mortality investigations, BoTest Matrix E detected picomolar quantities of BoNT/E following a 2-h incubation and femtomolar quantities of BoNT/E following extended incubation (24 h) with 100% diagnostic specificity and 91% diagnostic sensitivity. ?? 2011, American Society for Microbiology.

In vitro detection and quantification of botulinum neurotoxin type E activity in avian blood

USGS Publications Warehouse

Piazza, Timothy M.; Blehert, David S.; Dunning, F. Mark; Berlowski-Zier, Brenda M.; Zeytin, Fusun N.; Samuel, Michael D.; Tucker, Ward C.

2011-01-01

Botulinum neurotoxin serotype E (BoNT/E) outbreaks in the Great Lakes region cause large annual avian mortality events, with an estimated 17,000 bird deaths reported in 2007 alone. During an outbreak investigation, blood collected from bird carcasses is tested for the presence of BoNT/E using the mouse lethality assay. While sensitive, this method is labor-intensive and low throughput and can take up to 7 days to complete. We developed a rapid and sensitive in vitro assay, the BoTest Matrix E assay, that combines immunoprecipitation with high-affinity endopeptidase activity detection by Förster resonance energy transfer (FRET) to rapidly quantify BoNT/E activity in avian blood with detection limits comparable to those of the mouse lethality assay. On the basis of the analysis of archived blood samples (n = 87) collected from bird carcasses during avian mortality investigations, BoTest Matrix E detected picomolar quantities of BoNT/E following a 2-h incubation and femtomolar quantities of BoNT/E following extended incubation (24 h) with 100% diagnostic specificity and 91% diagnostic sensitivity.

In Vitro Detection and Quantification of Botulinum Neurotoxin Type E Activity in Avian Blood▿

PubMed Central

Piazza, Timothy M.; Blehert, David S.; Dunning, F. Mark; Berlowski-Zier, Brenda M.; Zeytin, Füsûn N.; Samuel, Michael D.; Tucker, Ward C.

2011-01-01

Botulinum neurotoxin serotype E (BoNT/E) outbreaks in the Great Lakes region cause large annual avian mortality events, with an estimated 17,000 bird deaths reported in 2007 alone. During an outbreak investigation, blood collected from bird carcasses is tested for the presence of BoNT/E using the mouse lethality assay. While sensitive, this method is labor-intensive and low throughput and can take up to 7 days to complete. We developed a rapid and sensitive in vitro assay, the BoTest Matrix E assay, that combines immunoprecipitation with high-affinity endopeptidase activity detection by Förster resonance energy transfer (FRET) to rapidly quantify BoNT/E activity in avian blood with detection limits comparable to those of the mouse lethality assay. On the basis of the analysis of archived blood samples (n = 87) collected from bird carcasses during avian mortality investigations, BoTest Matrix E detected picomolar quantities of BoNT/E following a 2-h incubation and femtomolar quantities of BoNT/E following extended incubation (24 h) with 100% diagnostic specificity and 91% diagnostic sensitivity. PMID:21908624

Specific, sensitive, and rapid assay for human immunodeficiency virus type 1 pol mutations associated with resistance to zidovudine and didanosine.

PubMed Central

Frenkel, L M; Wagner, L E; Atwood, S M; Cummins, T J; Dewhurst, S

1995-01-01

The effectiveness of antiretroviral therapy may be limited by the development of human immunodeficiency virus type 1 (HIV-1) resistance. Monitoring for resistance will perhaps allow changes in therapy prior to deterioration in the patient's clinical or immunologic status. Our objective was to develop a rapid, specific, and sensitive genotypic assay for HIV-1 resistance to zidovudine (ZDV) and didanosine (ddI) which is simple to perform. In our assay the DNA of HIV-1 pol was amplified by PCR using two sets of nested oligonucleotide primers. Mutations of reverse transcriptase (RT) encoding amino acids (aa) 74 and 41, 70, and 215 which have been associated with HIV-1 resistance to ddI and ZDV, respectively, were detected with a ligase detection reaction (LDR) and indicated colorimetrically. The RT genotypes of 35 patient specimens (140 codons) blindly assessed for these mutations were in agreement by PCR-LDR and by dideoxynucleotide sequencing. To evaluate the limits of the assay, other specimens with mutations close to the ligation site were evaluated by PCR-LDR. The assay was sensitive and specific for all specimens except when mutations occurred within 2 bases on either side of the ligation site. In summary, this PCR-LDR assay specifically, sensitively, and rapidly detected pol mutations (RT aa 74, 41, 70, and 215) associated with HIV-1 resistance to ddI and ZDV. PMID:7714190

Impedance biosensor for the rapid detection of Listeria spp. based on aptamer functionalized Pt-interdigitated microelectrodes array

NASA Astrophysics Data System (ADS)

Sidhu, R.; Rong, Y.; Vanegas, D. C.; Claussen, J.; McLamore, E. S.; Gomes, C.

2016-05-01

Listeria monocytogenes is one of the most common causes of food illness deaths worldwide, with multiple outbreaks in the United States alone. Current methods to detect foodborne pathogens are laborious and can take several hours to days to produce results. Thus, faster techniques are needed to detect bacteria within the same reliability level as traditional techniques. This study reports on a rapid, accurate, and sensitive aptamer biosensor device for Listeria spp. detection based on platinum interdigitated array microelectrodes (Pt-IDEs). Pt-IDEs with different geometric electrode gaps were fabricated by lithographic techniques and characterized by cyclic voltammetric (CV), electrochemical impedance spectroscopy (EIS), and potential amperometry (DCPA) measurements of reversible redox species. Based on these results, 50 μm Pt-IDE was chosen to further functionalize with a Listeria monocytogenes DNA aptamer selective to the cell surface protein internalin A, via metal-thiol self-assembly at the 5' end of the 47-mer's. EIS analysis was used to detect Listeria spp. without the need for label amplification and pre-concentration steps. The optimized aptamer concentration of 800 nM was selected to capture the bacteria through internalin A binding and the aptamer hairpin structure near the 3' end. The aptasensor was capable of detecting a wide range of bacteria concentration from 10 to 106 CFU/mL at lower detection limit of 5.39 +/- 0.21 CFU/mL with sensitivity of 268.1 +/- 25.40 (Ohms/log [CFU/mL]) in 17 min. The aptamer based biosensor offers a portable, rapid and sensitive alternative for food safety applications with one of the lowest detection limits reported to date.

Prevention of bacterial foodborne disease using nanobiotechnology.

PubMed

Billington, Craig; Hudson, J Andrew; D'Sa, Elaine

2014-01-01

Foodborne disease is an important source of expense, morbidity, and mortality for society. Detection and control constitute significant components of the overall management of foodborne bacterial pathogens, and this review focuses on the use of nanosized biological entities and molecules to achieve these goals. There is an emphasis on the use of organisms called bacteriophages (phages: viruses that infect bacteria), which are increasingly being used in pathogen detection and biocontrol applications. Detection of pathogens in foods by conventional techniques is time-consuming and expensive, although it can also be sensitive and accurate. Nanobiotechnology is being used to decrease detection times and cost through the development of biosensors, exploiting specific cell-recognition properties of antibodies and phage proteins. Although sensitivity per test can be excellent (eg, the detection of one cell), the very small volumes tested mean that sensitivity per sample is less compelling. An ideal detection method needs to be inexpensive, sensitive, and accurate, but no approach yet achieves all three. For nanobiotechnology to displace existing methods (culture-based, antibody-based rapid methods, or those that detect amplified nucleic acid) it will need to focus on improving sensitivity. Although manufactured nonbiological nanoparticles have been used to kill bacterial cells, nanosized organisms called phages are increasingly finding favor in food safety applications. Phages are amenable to protein and nucleic acid labeling, and can be very specific, and the typical large "burst size" resulting from phage amplification can be harnessed to produce a rapid increase in signal to facilitate detection. There are now several commercially available phages for pathogen control, and many reports in the literature demonstrate efficacy against a number of foodborne pathogens on diverse foods. As a method for control of pathogens, nanobiotechnology is therefore flourishing.

Rapid, highly sensitive detection of Gram-negative bacteria with lipopolysaccharide based disposable aptasensor.

PubMed

Zhang, Jian; Oueslati, Rania; Cheng, Cheng; Zhao, Ling; Chen, Jiangang; Almeida, Raul; Wu, Jayne

2018-07-30

Gram-negative bacteria are one of the most common microorganisms in the environment. Their differential detection and recognition from Gram-positive bacteria has been attracting much attention over the years. Using Escherichia coli (E. coli) as a model, we demonstrated on-site detection of Gram-negative bacteria by an AC electrokinetics-based capacitive sensing method using commercial microelectrodes functionalized with an aptamer specific to lipopolysaccharides. Dielectrophoresis effect was utilized to enrich viable bacteria to the microelectrodes rapidly, achieving a detection limit of 10 2 cells/mL within a 30 s' response time. The sensor showed a negligible response to Staphylococcus aureus (S. aureus), a Gram-positive species. The developed sensor showed significant advantages in sensitivity, selectivity, cost, operation simplicity, and response time. Therefore, this sensing method has shown great application potential for environmental monitoring, food safety, and real-time diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

A rapid, naked-eye detection of hypochlorite and bisulfite using a robust and highly-photostable indicator dye Quinaldine Red in aqueous medium

NASA Astrophysics Data System (ADS)

Dutta, Tanoy; Chandra, Falguni; Koner, Apurba L.

2018-02-01

A ;naked-eye; detection of health hazardous bisulfite (HSO3-) and hypochlorite (ClO-) using an indicator dye (Quinaldine Red, QR) in a wide range of pH is demonstrated. The molecule contains a quinoline moiety linked to an N,N-dimethylaniline moiety with a conjugated double bond. Treatment of QR with HSO3- and ClO-, in aqueous solution at near-neutral pH, resulted in a colorless product with high selectivity and sensitivity. The detection limit was 47.8 μM and 0.2 μM for HSO3- and ClO- respectively. However, ClO- was 50 times more sensitive and with 2 times faster response compared to HSO3-. The detail characterization and related analysis demonstrate the potential of QR for a rapid, robust and highly efficient colorimetric sensor for the practical applications to detect hypochlorite in water samples.

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of common genetically modified organisms (GMOs).

PubMed

Feng, Jiawang; Tang, Shiming; Liu, Lideng; Kuang, Xiaoshan; Wang, Xiaoyu; Hu, Songnan; You, Shuzhu

2015-03-01

Here, we developed a loop-mediated isothermal amplification (LAMP) assay for 11 common transgenic target DNA in GMOs. Six sets of LAMP primer candidates for each target were designed and their specificity, sensitivity, and reproductivity were evaluated. With the optimized LAMP primers, this LAMP assay was simply run within 45-60 min to detect all these targets in GMOs tested. The sensitivity, specificity, and reproductivity of the LAMP assay were further analyzed in comparison with those of Real-Time PCR. In consistent with real-time PCR, detection of 0.5% GMOs in equivalent background DNA was possible using this LAMP assay for all targets. In comparison with real-time PCR, the LAMP assay showed the same results with simple instruments. Hence, the LAMP assay developed can provide a rapid and simple approach for routine screening as well as specific events detection of many GMOs.

SERS-based pesticide detection by using nanofinger sensors

NASA Astrophysics Data System (ADS)

Kim, Ansoon; Barcelo, Steven J.; Li, Zhiyong

2015-01-01

Simple, sensitive, and rapid detection of trace levels of extensively used and highly toxic pesticides are in urgent demand for public health. Surface-enhanced Raman scattering (SERS)-based sensor was designed to achieve ultrasensitive and simple pesticide sensing. We developed a portable sensor system composed of high performance and reliable gold nanofinger sensor strips and a custom-built portable Raman spectrometer. Compared to the general procedure and previously reported studies that are limited to laboratory settings, our analytical method is simple, sensitive, rapid, and cost-effective. Based on the SERS results, the chemical interaction of two pesticides, chlorpyrifos (CPF) and thiabendazole (TBZ), with gold nanofingers was studied to determine a fingerprint for each pesticide. The portable SERS-sensor system was successfully demonstrated to detect CPF and TBZ pesticides within 15 min with a detection limit of 35 ppt in drinking water and 7 ppb on apple skin, respectively.

Establishment and application of cross-priming isothermal amplification coupled with lateral flow dipstick (CPA-LFD) for rapid and specific detection of red-spotted grouper nervous necrosis virus.

PubMed

Su, Zi Dan; Shi, Cheng Yin; Huang, Jie; Shen, Gui Ming; Li, Jin; Wang, Sheng Qiang; Fan, Chao

2015-09-26

Red-spotted grouper nervous necrosis virus (RGNNV) is an important pathogen that causes diseases in many species of fish in marine aquaculture. The larvae and juveniles are more easily infected by RGNNV and the cumulative mortality is as high as 100 % after being infected with RGNNV. This virus imposes a serious threat to aquaculture of grouper fry. This study aimed to establish a simple, accurate and highly sensitive method for rapid detection of RGNNV on the spot. In this study, the primers specifically targeting RGNNV were designed and cross-priming isothermal amplification (CPA) system was established. The product amplified by CPA was detected through visualization with lateral flow dipstick (LFD). Three important parameters, including the amplification temperature, the concentration of dNTPs and the concentration of Mg(2+) for the CPA system, were optimized. The sensitivity and specificity of this method for RGNNV were tested and compared with those of the conventional RT-PCR and real-time quantitative RT-PCR (qRT-PCR). The optimized conditions for the CPA amplification system were determined as follows: the optimal amplification temperature, the optimized concentration of dNTPs and the concentration for Mg(2+) were 69 °C, 1.2 mmol/L and 5 mmol/L, respectively. The lowest limit of detection (LLOD) of this method for RGNNV was 10(1) copies/μL of RNA sample, which was 10 times lower than that of conventional RT-PCR and comparable to that of RT-qPCR. This method was specific for RGNNV in combination with SJNNV and had no cross-reactions with 8 types of virus and bacterial strains tested. This method was successfully applied to detect RGNNV in fish samples. This study established a CPA-LFD method for detection of RGNNV. This method is simple and rapid with high sensitivity and good specificity and can be widely applied for rapid detection of this virus on the spot.

Surface scattering plasmon resonance fibre sensors: demonstration of rapid influenza A virus detection

NASA Astrophysics Data System (ADS)

Franςois, A.; Boehm, J.; Oh, S. Y.; Kok, T.; Monro, T. M.

2011-06-01

The management of threats such as pandemics and explosives, and of health and the environment requires the rapid deployment of highly sensitive detection tools. Sensors based on Surface Plasmon Resonance (SPR) allow rapid, labelfree, highly sensitive detection, and indeed this phenomenon underpins the only label-free optical biosensing technology that is available commercially. In these sensors, the existence of surface plasmons is inferred indirectly from absorption features that correspond to the coupling of light to the surface plasmon. Although SPR is not intrinsically a radiative process, under certain conditions the surface plasmon can itself couple to the local photon states, and emit light as first described byKretschmann. Here we show that by collecting and characterising this re-emitted light, it is possible to realise new SPR sensing architectures that are more compact, versatile and robust than existing approaches. This approach addresses existing practical limitations associated with current SPR technologies, including bulk, cost and calibration. It is applicable to a range of SPR geometries, including optical fibres, planar waveguides and prism configurations, and is in principle capable of detecting multiple analytes simultaneously. Moreover, this technique allows to combine SPR sensing and fluorescence sensing into a single platform which has never been demonstrated before and consequently use these two methods for a more reliable diagnostic. As an example, this approach has been used to demonstrate the rapid detection of the seasonal influenza virus.

Rapid visual detection of cyprinid herpesvirus 2 by recombinase polymerase amplification combined with a lateral flow dipstick.

PubMed

Wang, H; Sun, M; Xu, D; Podok, P; Xie, J; Jiang, Ys; Lu, Lq

2018-05-28

Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus 2 (CyHV-2), causes significant losses in crucian carp (Carassius carassius) aquaculture. Rapid and convenient DNA assay detection of CyHV-2 is useful for field diagnosis. Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that can amplify DNA within 30 min at ~37°C by simulating in vivo DNA recombination. Herein, a rapid and convenient detection assay based on RPA with a lateral flow dipstick (LFD) was developed for detecting CyHV-2. The highly conserved ORF72 of CyHV-2 was targeted by specific and sensitive primers and probes. The optimized assay takes only 15 min at 38°C using a water bath, with analysis of products by 2% agarose gel electrophoresis within 30 min. A simple lateral flow strip based on the unique probe in reaction buffer was developed for visualization. The entire RPA-LFD assay takes 50 min less than the routine PCR method, is 100 times more sensitive and displays no cross-reaction with other aquatic viruses. The combined isothermal RPA and lateral flow assay (RPA-LFD) provides a simple, rapid, reliable method that could improve field diagnosis of CyHV-2 when resources are limited. © 2018 John Wiley & Sons Ltd.

Population screening for coeliac disease in primary care by district nurses using a rapid antibody test: diagnostic accuracy and feasibility study

PubMed Central

2007-01-01

Objective To evaluate the feasibility and diagnostic accuracy of screening for coeliac disease by rapid detection of IgA antibodies to tissue transglutaminase performed in primary care. Design District nurses screened 6 year old children using rapid antibody testing of finger prick blood. They also collected capillary blood samples for laboratory determination of IgA and IgG antibodies to endomysium and IgA antibodies to tissue transglutaminase. Children with positive rapid test results were directly sent for biopsy of the small intestine. Setting Primary care in Jász-Nagykun-Szolnok county, Hungary. Participants 2690 children (77% of 6 year olds living in the county) and 120 nurses. Main outcome measures Positivity for antibodies to endomysium or transglutaminase in the laboratory and coeliac disease confirmed at biopsy. Results 37 children (1.4%, 95% confidence interval 0.9% to 1.8%) had biopsy confirmed coeliac disease. Only five of these children had been diagnosed clinically before screening. Rapid testing had a 78.1% sensitivity (70.0% to 89.3%) and 100% specificity (88.4% to 100%) for a final diagnosis of coeliac disease by biopsy. Sensitivity was 65.1% (50.2% to 77.6%) and specificity was 100% (99.8% to 100%) compared with combined results of IgA and IgG laboratory tests. Trained laboratory workers detected 30 of the 31 newly diagnosed IgA competent patients with the rapid test kit used blindly. Median time to biopsy after a positive rapid test result was significantly shorter (20 days, range 4-148) than after a positive laboratory result (142 days, 70-256; P<0.001). Children with coeliac disease detected at screening were smaller and had worse health status than their peers but they improved on a gluten-free diet. Conclusions A simple rapid antibody test enabled primary care nurses to detect patients with coeliac disease in the community who were not picked up in clinical care. Extra training is needed to improve sensitivity. PMID:18063612

Performance of the Directigen EZ Flu A+B rapid influenza diagnostic test to detect pandemic influenza A/H1N1 2009.

PubMed

Boyanton, Bobby L; Almradi, Amro; Mehta, Tejal; Robinson-Dunn, Barbara

2014-04-01

The Directigen EZ Flu A+B rapid influenza diagnostic test, as compared to real-time reverse transcriptase polymerase chain reaction, demonstrated suboptimal performance to detect pandemic influenza A/H1N1 2009. Age- and viral load-stratified test sensitivity ranged from 33.3 to 84.6% and 0 to 100%, respectively. © 2013.

Evaluation of OXA-48 K-Se T: an immunochromatographic assay for rapid detection of OXA-48-producing Enterobacteriaceae.

PubMed

Fernández, Javier; Fleites, Ana; Rodcio, María Rosario; Vazquez, Fernando

2016-05-01

The OXA-48 K-Se T, a new immunochromatographic assay for rapid detection of OXA-48-producing Enterobacteriaceae, has been evaluated in a Spanish Hospital during a 3-month period. A collection of 100 Enterobacteriaceae including 79 isolates producing OXA-48 has been tested. Sensitivity and specificity of 100% were obtained. Copyright © 2016 Elsevier Inc. All rights reserved.

Multiplex PCR for simultaneous identification of E. coli O157:H7, Salmonella spp. and L. monocytogenes in food.

PubMed

Nguyen, Thuy Trang; Van Giau, Vo; Vo, Tuong Kha

2016-12-01

The rapid detection of pathogens in food is becoming increasingly critical for ensuring the safety of consumers, since the majority of food-borne illnesses and deaths are caused by pathogenic bacteria. Hence, rapid, sensitive, inexpensive and convenient approaches to detect food-borne pathogenic bacteria is essential in controlling food safety. In this study, a multiplex PCR assay for the rapid and simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes was established. The invA, stx and hlyA genes specifically amplified DNA fragments of 284, 404 and 510 bp from Salmonella spp., L. monocytogenes and E. coli O157:H7, respectively. The 16S rRNA gene was targeted as an internal control gene in the presence of bacterial DNA. The specificity and sensitivity of the multiplex PCR were performed by testing different strains. The multiplex PCR assay was able to specifically simultaneously detect ten colony-forming unit/mL of each pathogen in artificially inoculated samples after enrichment for 12 h. The whole process took less than 24 h to complete, indicating that the assay is suitable for reliable and rapid identification of these three food-borne pathogens, which could be suitable in microbial epidemiology investigation.

Rapid direct methods for enumeration of specific, active bacteria in water and biofilms

NASA Technical Reports Server (NTRS)

McFeters, G. A.; Pyle, B. H.; Lisle, J. T.; Broadaway, S. C.

1999-01-01

Conventional methods for detecting indicator and pathogenic bacteria in water may underestimate the actual population due to sublethal environmental injury, inability of the target bacteria to take up nutrients and other physiological factors which reduce bacterial culturability. Rapid and direct methods are needed to more accurately detect and enumerate active bacteria. Such a methodological advance would provide greater sensitivity in assessing the microbiological safety of water and food. The principle goal of this presentation is to describe novel approaches we have formulated for the rapid and simultaneous detection of bacteria plus the determination of their physiological activity in water and other environmental samples. The present version of our method involves the concentration of organisms by membrane filtration or immunomagnetic separation and combines an intracellular fluorochrome (CTC) for assessment of respiratory activity plus fluorescent-labelled antibody detection of specific bacteria. This approach has also been successfully used to demonstrate spatial and temporal heterogeneities of physiological activities in biofilms when coupled with cryosectioning. Candidate physiological stains include those capable of determining respiratory activity, membrane potential, membrane integrity, growth rate and cellular enzymatic activities. Results obtained thus far indicate that immunomagnetic separation can provide a high degree of sensitivity in the recovery of seeded target bacteria (Escherichia coli O157:H7) in water and hamburger. The captured and stained target bacteria are then enumerated by either conventional fluorescence microscopy or ChemScan(R), a new instrument that is very sensitive and rapid. The ChemScan(R) laser scanning instrument (Chemunex, Paris, France) provides the detection of individual fluorescently labelled bacterial cells using three emission channels in less than 5 min. A high degree of correlation has been demonstrated between results obtained with the ChemScan and traditional plate counts of mixed natural bacterial populations in water. The continuing evolution of these methods will be valuable in the rapid and accurate analysis of environmental samples.

Rapid detection of multidrug resistant Gram-negative bacilli by Cica-Beta-Test strips.

PubMed

Lavigne, J-P; Pfeiffer, C; Vidal, L; Sotto, A

2011-02-01

This study aimed to evaluate the sensitivity and specificity of a new system (Cica-Beta-Test, Kanto Chemical, Japan) for rapid detection of AmpC-derepressed, extended-spectrum β-lactamases (ESβL) and metallo-β-lactamases (MβL). Two hundred Multi-Drug Resistant (MDR)-Gram-negative bacilli were studied: 170 Enterobacteriaceae and 30 Gram-negative non-fermentative bacteria. One hundred and eighteen strains produced an ESβL, seven MβL and 75 derepressed cephalosporinases. One drop of substrate was dispensed onto the filter pad of the Cica-Beta-Test strip. The bacterial colonies were spread on the filter pad of strip. The reading was performed after 2 to 15 min: turning chromogenic indicated the positive test. Three tests were used: Cica-Beta I for detection of MDR bacteria; Cica-Beta MβL for detection of MβL-producing bacteria and Cica-Beta CVA, which distinguish ESβL and AmpC-derepressed producers. Results were compared with molecular assays. Cica-Beta-Test I has detected 194 MDR (sensitivity 97%), Cica-Beta-Test MβL has shown the presence of six MβL tested (sensitivity 85.7%). Five strains were non-MβL false positive (specificity 97.3%). Cica-Beta-Test CVA allowed the differentiation of ESβL-producing strains (109/115) and AmpC-derepressed strains (56/67) (sensitivity 94.8%, specificity 83.8%). Because of their epidemic nature, the MDR strains are screened and require strict hygienic measures patients. The simultaneous use of three strips can quickly determine the presence of MDR including ESβL and MβL. Rapid screening of MDR avoids transmission and limits the use of broad-spectrum antibiotics. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Evaluation of a rapid diagnostic test for the detection of Burkholderia pseudomallei in the Lao People's Democratic Republic.

PubMed

Woods, Kate L; Boutthasavong, Latsaniphone; NicFhogartaigh, Caoimhe; Lee, Sue J; Davong, Viengmon; AuCoin, David P; Dance, David A B

2018-05-02

Burkholderia pseudomallei causes significant global morbidity and mortality, with the highest disease burden in parts of Asia where culture-based diagnosis is often not available. We prospectively evaluated the Active Melioidosis Detect (AMD, InBios International, USA) lateral flow immunoassay (LFI) for rapid detection of B. pseudomallei in turbid blood cultures, pus, sputum, sterile fluid, urine, and sera. Performance was compared to B. pseudomallei detection using monoclonal antibody latex agglutination (LA) and immunofluorescence assays (IFA), with culture as the gold standard. AMD was 99% (99/100; 94.6 - 100%) sensitive and 100% (308/308; 98.8-100%) specific on turbid blood culture bottles, with no difference to LA or IFA. AMD specificity was 100% on pus (122/122; 97.0-100%), sputum (20/20; 83.2-100%), and sterile fluid (44/44; 92 - 100%). Sensitivity on these samples was: pus 47.1% (8/17; 23.0 - 72.2%), sputum 33.3% (1/3; 0.84 - 90.6%), and sterile fluid 0% (0/2; 0 - 84.2%). Urine AMD had a positive predictive value of 94% (32/34; 79.7 - 98.5%) for diagnosing melioidosis in our cohort. AMD sensitivity on stored sera, collected prospectively from melioidosis cases during this study, was 13.9% (5/36; 4.7% - 29.5%) when compared to blood culture samples taken on the same day. In conclusion, the AMD is an excellent tool for rapid diagnosis of melioidosis from turbid blood cultures, and maintains specificity across all sample types. It is a promising tool for urinary antigen detection, which could revolutionise diagnosis of melioidosis in resource-limited settings. Further work is required to improve sensitivity on non-blood culture samples. Copyright © 2018 Woods et al.

A rapid, ratiometric, enzyme-free, and sensitive single-step miRNA detection using three-way junction based FRET probes

NASA Astrophysics Data System (ADS)

Luo, Qingying; Liu, Lin; Yang, Cai; Yuan, Jing; Feng, Hongtao; Chen, Yan; Zhao, Peng; Yu, Zhiqiang; Jin, Zongwen

2018-03-01

MicroRNAs (miRNAs) are single stranded endogenous molecules composed of only 18-24 nucleotides which are critical for gene expression regulating the translation of messenger RNAs. Conventional methods based on enzyme-assisted nucleic acid amplification techniques have many problems, such as easy contamination, high cost, susceptibility to false amplification, and tendency to have sequence mismatches. Here we report a rapid, ratiometric, enzyme-free, sensitive, and highly selective single-step miRNA detection using three-way junction assembled (or self-assembled) FRET probes. The developed strategy can be operated within the linear range from subnanomolar to hundred nanomolar concentrations of miRNAs. In comparison with the traditional approaches, our method showed high sensitivity for the miRNA detection and extreme selectivity for the efficient discrimination of single-base mismatches. The results reveal that the strategy paved a new avenue for the design of novel highly specific probes applicable in diagnostics and potentially in microscopic imaging of miRNAs in real biological environments.

A Multiplexed Diagnostic Platform for Point-of-Care Pathogen Detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Regan, J F; Letant, S E; Adams, K L

2008-02-04

We developed an automated point-of-care diagnostic instrument that is capable of analyzing nasal swab samples for the presence of respiratory diseases. This robust instrument, called FluIDx, performs autonomous multiplexed RT-PCR reactions that are analyzed by microsphere xMAP technology. We evaluated the performance of FluIDx, in comparison rapid tests specific for influenza and respiratory syncytial virus, in a clinical study performed at the UC Davis Medical Center. The clinical study included samples positive for RSV (n = 71), influenza A (n = 16), influenza B (n = 4), adenovirus (n = 5), parainfluenza virus (n = 2), and 44 negative samples,more » according to a composite reference method. FluIDx and the rapid tests detected 85.9% and 62.0% of the RSV positive samples, respectively. Similar sensitivities were recorded for the influenza B samples; whereas the influenza A samples were poorly detected, likely due to the utilization of an influenza A signature that did not accurately match currently circulating influenza A strains. Data for all pathogens were compiled and indicate that FluIDx is more sensitive than the rapid tests, detecting 74.2% (95% C.I. of 64.7-81.9%) of the positive samples in comparison to 53.6% (95% C.I. of 43.7-63.2%) for the rapid tests. The higher sensitivity of FluIDx was partially offset by a lower specificity, 77.3% versus 100.0%. Overall, these data suggest automated flow-through PCR-based instruments that perform multiplexed assays can successfully screen clinical samples for infectious diseases.« less

Rapid diagnosis of sepsis with TaqMan-Based multiplex real-time PCR.

PubMed

Liu, Chang-Feng; Shi, Xin-Ping; Chen, Yun; Jin, Ye; Zhang, Bing

2018-02-01

The survival rate of septic patients mainly depends on a rapid and reliable diagnosis. A rapid, broad range, specific and sensitive quantitative diagnostic test is the urgent need. Thus, we developed a TaqMan-Based Multiplex real-time PCR assays to identify bloodstream pathogens within a few hours. Primers and TaqMan probes were designed to be complementary to conserved regions in the 16S rDNA gene of different kinds of bacteria. To evaluate accurately, sensitively, and specifically, the known bacteria samples (Standard strains, whole blood samples) are determined by TaqMan-Based Multiplex real-time PCR. In addition, 30 blood samples taken from patients with clinical symptoms of sepsis were tested by TaqMan-Based Multiplex real-time PCR and blood culture. The mean frequency of positive for Multiplex real-time PCR was 96% at a concentration of 100 CFU/mL, and it was 100% at a concentration greater than 1000 CFU/mL. All the known blood samples and Standard strains were detected positively by TaqMan-Based Multiplex PCR, no PCR products were detected when DNAs from other bacterium were used in the multiplex assay. Among the 30 patients with clinical symptoms of sepsis, 18 patients were confirmed positive by Multiplex real-time PCR and seven patients were confirmed positive by blood culture. TaqMan-Based Multiplex real-time PCR assay with highly sensitivity, specificity and broad detection range, is a rapid and accurate method in the detection of bacterial pathogens of sepsis and should have a promising usage in the diagnosis of sepsis. © 2017 Wiley Periodicals, Inc.

Sensing the deadliest toxin: technologies for botulinum neurotoxin detection.

PubMed

Capek, Petr; Dickerson, Tobin J

2010-01-01

Sensitive and rapid detection of botulinum neurotoxins (BoNTs), the most poisonous substances known to date, is essential for studies of medical applications of BoNTs and detection of poisoned food, as well as for response to potential bioterrorist threats. Currently, the most common method of BoNT detection is the mouse bioassay. While this assay is sensitive, it is slow, quite expensive, has limited throughput and requires sacrificing animals. Herein, we discuss and compare recently developed alternative in vitro detection methods and assess their ability to supplement or replace the mouse bioassay in the analysis of complex matrix samples.

Multicenter Clinical Evaluation of the Novel Alere i Strep A Isothermal Nucleic Acid Amplification Test.

PubMed

Cohen, Daniel M; Russo, Michael E; Jaggi, Preeti; Kline, Jennifer; Gluckman, William; Parekh, Amisha

2015-07-01

Rapid detection of group A beta-hemolytic streptococcus (GAS) is used routinely to help diagnose and treat pharyngitis. However, available rapid antigen detection tests for GAS have relatively low sensitivity, and backup testing is recommended in children. Newer assays are more sensitive yet require excessive time for practical point-of-care use as well as laboratory personnel. The Alere i strep A test is an isothermal nucleic acid amplification test designed to offer highly sensitive results at the point of care within 8 min when performed by nonlaboratory personnel. The performance of the Alere i strep A test was evaluated in a multicenter prospective trial in a Clinical Laboratory Improvement Amendments (CLIA)-waived setting in comparison to bacterial culture in 481 children and adults. Compared to culture, the Aleri i strep A test had 96.0% sensitivity and 94.6% specificity. Discrepant results were adjudicated by PCR and found the Alere i strep A test to have 98.7% sensitivity and 98.5% specificity. Overall, the Alere i strep A test could provide a one-step, rapid, point-of-care testing method for GAS pharyngitis and obviate backup testing on negative results. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

WiFi-Based Real-Time Calibration-Free Passive Human Motion Detection.

PubMed

Gong, Liangyi; Yang, Wu; Man, Dapeng; Dong, Guozhong; Yu, Miao; Lv, Jiguang

2015-12-21

With the rapid development of WLAN technology, wireless device-free passive human detection becomes a newly-developing technique and holds more potential to worldwide and ubiquitous smart applications. Recently, indoor fine-grained device-free passive human motion detection based on the PHY layer information is rapidly developed. Previous wireless device-free passive human detection systems either rely on deploying specialized systems with dense transmitter-receiver links or elaborate off-line training process, which blocks rapid deployment and weakens system robustness. In the paper, we explore to research a novel fine-grained real-time calibration-free device-free passive human motion via physical layer information, which is independent of indoor scenarios and needs no prior-calibration and normal profile. We investigate sensitivities of amplitude and phase to human motion, and discover that phase feature is more sensitive to human motion, especially to slow human motion. Aiming at lightweight and robust device-free passive human motion detection, we develop two novel and practical schemes: short-term averaged variance ratio (SVR) and long-term averaged variance ratio (LVR). We realize system design with commercial WiFi devices and evaluate it in typical multipath-rich indoor scenarios. As demonstrated in the experiments, our approach can achieve a high detection rate and low false positive rate.

WiFi-Based Real-Time Calibration-Free Passive Human Motion Detection †

PubMed Central

Gong, Liangyi; Yang, Wu; Man, Dapeng; Dong, Guozhong; Yu, Miao; Lv, Jiguang

2015-01-01

With the rapid development of WLAN technology, wireless device-free passive human detection becomes a newly-developing technique and holds more potential to worldwide and ubiquitous smart applications. Recently, indoor fine-grained device-free passive human motion detection based on the PHY layer information is rapidly developed. Previous wireless device-free passive human detection systems either rely on deploying specialized systems with dense transmitter-receiver links or elaborate off-line training process, which blocks rapid deployment and weakens system robustness. In the paper, we explore to research a novel fine-grained real-time calibration-free device-free passive human motion via physical layer information, which is independent of indoor scenarios and needs no prior-calibration and normal profile. We investigate sensitivities of amplitude and phase to human motion, and discover that phase feature is more sensitive to human motion, especially to slow human motion. Aiming at lightweight and robust device-free passive human motion detection, we develop two novel and practical schemes: short-term averaged variance ratio (SVR) and long-term averaged variance ratio (LVR). We realize system design with commercial WiFi devices and evaluate it in typical multipath-rich indoor scenarios. As demonstrated in the experiments, our approach can achieve a high detection rate and low false positive rate. PMID:26703612

Point-Counterpoint: A Nucleic Acid Amplification Test for Streptococcus pyogenes Should Replace Antigen Detection and Culture for Detection of Bacterial Pharyngitis.

PubMed

Pritt, Bobbi S; Patel, Robin; Kirn, Thomas J; Thomson, Richard B

2016-10-01

Nucleic acid amplification tests (NAATs) have frequently been the standard diagnostic approach when specific infectious agents are sought in a clinic specimen. They can be applied for specific agents such as S. pyogenes, or commercial multiplex NAATs for detection of a variety of pathogens in gastrointestinal, bloodstream, and respiratory infections may be used. NAATs are both rapid and sensitive. For many years, S. pyogenes testing algorithms used a rapid and specific group A streptococcal antigen test to screen throat specimens, followed, in some clinical settings, by a throat culture for S. pyogenes to increase the sensitivity of its detection. Now S. pyogenes NAATs are being used with increasing frequency. Given their accuracy, rapidity, and ease of use, should they replace antigen detection and culture for the detection of bacterial pharyngitis? Bobbi Pritt and Robin Patel of the Mayo Clinic, where S. pyogenes NAATs have been used for well over a decade with great success, will explain the advantages of this approach, while Richard (Tom) Thomson and Tom Kirn of the NorthShore University HealthSystem will discuss their concerns about this approach to diagnosing bacterial pharyngitis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Development of a reverse transcription loop-mediated isothermal amplification method for the rapid detection of avian influenza virus subtype H7.

PubMed

Bao, Hongmei; Wang, Xiurong; Zhao, Yuhui; Sun, Xiaodong; Li, Yanbing; Xiong, Yongzhong; Chen, Hualan

2012-01-01

A rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of the H7 avian influenza virus (H7 AIV) isotype was developed. The minimum detection limit of the RT-LAMP assay was 0.1-0.01 PFU per reaction for H7 AIV RNA, making this assay 100-fold more sensitive than the conventional RT-PCR method. This RT-LAMP assay also has the capacity to detect both high- and low-pathogenic H7 AIV strains. Using a pool of RNAs extracted from influenza viruses corresponding to all 15 HA subtypes (in addition to other avian pathogenic viruses), the RT-LAMP system was confirmed to amplify only H7 AIV RNA. Furthermore, specific pathogen free (SPF) chickens were infected artificially with H7 AIV, throat and cloacal swabs were collected, and viral shedding was examined using viral isolation, RT-PCR and RT-LAMP. Shedding was detected following viral isolation and RT-LAMP one day after infection, whereas viral detection using RT-PCR was effective only on day 3 post-infection. These results indicate that the RT-LAMP method could facilitate epidemiological surveillance and the rapid diagnosis of the avian influenza subtype H7. Copyright © 2011 Elsevier B.V. All rights reserved.

Rapid detection of duck hepatitis A virus genotype C using reverse transcription loop-mediated isothermal amplification.

PubMed

Li, Chuanfeng; Chen, Zongyan; Meng, Chunchun; Liu, Guangqing

2014-02-01

A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was used and optimized to develop a rapid and sensitive detection system for duck hepatitis A virus genotype C (DHAV-C) RNA. A set of four specific primers was designed against highly conserved sequences located within the 3D gene from DHAV (strain GX1201). Under optimal reaction conditions, the sensitivity of DHAV-C-specific RT-LAMP was 100-fold higher than that of reverse transcriptase-polymerase chain reaction (RT-PCR), with a detection limit of 0.3pg (6.59×10(4) copies) per reaction. No cross-reactivity was observed from the samples of other duck viruses, which is in good accordance with RT-PCR. Furthermore, a positive reaction can be visually inspected by observing turbidity or color change after the addition of SYBR green I dye. The DHAV-C-specific RT-LAMP assay was applied to the samples and compared with RT-PCR. The positive-sample ratios were 26.7% (12 of 45) by RT-LAMP and 20% (9 of 45) by RT-PCR. Therefore, the newly developed RT-LAMP assay is a rapid, specific, sensitive, and cost-effective method of DHAV-C detection. This assay has potential applications in both clinical diagnosis and field surveillance of DHAV-C infection. Copyright © 2013. Published by Elsevier B.V.

Rapid and sensitive detection of foodborne pathogenic bacteria (Staphylococcus aureus) using an electrochemical DNA genomic biosensor and its application in fresh beef.

PubMed

Abdalhai, Mandour H; Fernandes, António Maximiano; Bashari, Mohand; Ji, Jian; He, Qian; Sun, Xiulan

2014-12-31

Rapid early detection of food contamination is the main key in food safety and quality control. Biosensors are emerging as a vibrant area of research, and the use of DNA biosensor recognition detectors is relatively new. In this study a genomic DNA biosensor system with a fixing and capture probe was modified by a sulfhydryl and amino group, respectively, as complementary with target DNA. After immobilization and hybridization, the following sandwich structure fixing DNA-target DNA-capture DNA-PbS NPs was formed to detect pathogenic bacteria (Staphylococuus aureus EF529607.1) by using GCE modified with (multiwalled carbon nanotubes-chitosan-bismuth) to increase the sensitivity of the electrode. The modification procedure was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. The sandwich structure was dissolved in 1 M nitric acid to become accessible to the electrode, and the PbS NPs was measured in solution by differential pulse voltammetry (DPV). The results showed that the detection limit of the DNA sensor was 3.17 × 10(-14) M S. aureus using PbS NPs, whereas the result for beef samples was 1.23 ng/mL. Thus, according to the experimental results presented, the DNA biosensor exhibited high sensitivity and rapid response, and it will be useful for the food matrix.

An isothermal based recombinase polymerase amplification assay for rapid, sensitive and robust indexing of citrus yellow mosaic virus.

PubMed

Kumar, P V; Sharma, S K; Rishi, N; Ghosh, D K; Baranwal, V K

Management of viral diseases relies on definite and sensitive detection methods. Citrus yellow mosaic virus (CYMV), a double stranded DNA virus of the genus Badnavirus, causes yellow mosaic disease in citrus plants. CYMV is transmitted through budwood and requires a robust and simplified indexing protocol for budwood certification programme. The present study reports development and standardization of an isothermal based recombinase polymerase amplification (RPA) assay for a sensitive, rapid, easy, and cost-effective method for detection and diagnosis of CYMV. Two different oligonucleotide primer sets were designed from ORF III (coding for polyprotein) and ORF II (coding for virion associated protein) regions of CYMV to perform amplification assays. Comparative evaluation of RPA, PCR and immuno-capture recombinase polymerase amplification (IC-RPA) based assays were done using purified DNA and plant crude sap. CYMV infection was efficiently detected from the crude sap in RPA and IC-RPA assays. The primer set used in RPA was specific and did not show any cross-amplification with banana streak MY virus (BSMYV), another Badnavirus species. The results from the present study indicated that RPA assay can be used easily in routine indexing of citrus planting material. To the best of our knowledge, this is the first report on development of a rapid and simplified isothermal detection assay for CYMV and can be utilized as an effective technique in quarantine and budwood certification process.

Preparation of immunochromatographic strips for rapid detection of early secreted protein ESAT-6 and culture filtrate protein CFP-10 from Mycobacterium tuberculosis

PubMed Central

Wu, Xiaoxin; Wang, Yeping; Weng, Tianhao; Hu, Chenyu; Wang, Frederick X.C.; Wu, Zhigang; Yu, Dongshan; Lu, Huoquan; Yao, Hangping

2017-01-01

Abstract The early secreted protein early secretory antigenic target 6(ESAT-6) and the culture filtrate protein 10 (CFP-10) are 2 antigens that are specific to Mycobacterium tuberculosis. These 2 antigens are good targets for tuberculosis (TB) detection. To rapidly diagnose TB across a variety of samples, we developed colloidal gold immunochromatographic strips (ICSs) based on ESAT-6 and CFP-10. The strips were evaluated using 233 samples, including sputum, plasma, and pleural effusion samples. The positive detection rates for ICSs for ESAT-6 and CFP-10 in sputum (culture-positive for M tuberculosis) were 100% and 91.2%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in plasma were 34.1% and 29.4%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in pleural effusion were 64.7% and 55.9%, respectively. Experimental analysis of culture supernatant showing that the ICS developed for ESAT-6 had a sensitivity of 100% and a specificity of 91.2%. While the ICS developed for CFP-10 had a sensitivity of 91.2% and a specificity of 88.2%. The validity of the test is limited by source of sample. The technique is sensitive and specific for samples in sputum and culture media but not for plasma or pleural effusion samples. Detection of M tuberculosis using ICSs is rapid, simple, and relatively effective; thus, ICSs are a potential screening tool for TB. PMID:29390519

Sensitive, Rapid Detection of Bacterial Spores

NASA Technical Reports Server (NTRS)

Kern, Roger G.; Venkateswaran, Kasthuri; Chen, Fei; Pickett, Molly; Matsuyama, Asahi

2009-01-01

A method of sensitive detection of bacterial spores within delays of no more than a few hours has been developed to provide an alternative to a prior three-day NASA standard culture-based assay. A capability for relatively rapid detection of bacterial spores would be beneficial for many endeavors, a few examples being agriculture, medicine, public health, defense against biowarfare, water supply, sanitation, hygiene, and the food-packaging and medical-equipment industries. The method involves the use of a commercial rapid microbial detection system (RMDS) that utilizes a combination of membrane filtration, adenosine triphosphate (ATP) bioluminescence chemistry, and analysis of luminescence images detected by a charge-coupled-device camera. This RMDS has been demonstrated to be highly sensitive in enumerating microbes (it can detect as little as one colony-forming unit per sample) and has been found to yield data in excellent correlation with those of culture-based methods. What makes the present method necessary is that the specific RMDS and the original protocols for its use are not designed for discriminating between bacterial spores and other microbes. In this method, a heat-shock procedure is added prior to an incubation procedure that is specified in the original RMDS protocols. In this heat-shock procedure (which was also described in a prior NASA Tech Briefs article on enumerating sporeforming bacteria), a sample is exposed to a temperature of 80 C for 15 minutes. Spores can survive the heat shock, but nonspore- forming bacteria and spore-forming bacteria that are not in spore form cannot survive. Therefore, any colonies that grow during incubation after the heat shock are deemed to have originated as spores.

Preparation of immunochromatographic strips for rapid detection of early secreted protein ESAT-6 and culture filtrate protein CFP-10 from Mycobacterium tuberculosis.

PubMed

Wu, Xiaoxin; Wang, Yeping; Weng, Tianhao; Hu, Chenyu; Wang, Frederick X C; Wu, Zhigang; Yu, Dongshan; Lu, Huoquan; Yao, Hangping

2017-12-01

The early secreted protein early secretory antigenic target 6(ESAT-6) and the culture filtrate protein 10 (CFP-10) are 2 antigens that are specific to Mycobacterium tuberculosis. These 2 antigens are good targets for tuberculosis (TB) detection.To rapidly diagnose TB across a variety of samples, we developed colloidal gold immunochromatographic strips (ICSs) based on ESAT-6 and CFP-10.The strips were evaluated using 233 samples, including sputum, plasma, and pleural effusion samples.The positive detection rates for ICSs for ESAT-6 and CFP-10 in sputum (culture-positive for M tuberculosis) were 100% and 91.2%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in plasma were 34.1% and 29.4%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in pleural effusion were 64.7% and 55.9%, respectively. Experimental analysis of culture supernatant showing that the ICS developed for ESAT-6 had a sensitivity of 100% and a specificity of 91.2%. While the ICS developed for CFP-10 had a sensitivity of 91.2% and a specificity of 88.2%.The validity of the test is limited by source of sample. The technique is sensitive and specific for samples in sputum and culture media but not for plasma or pleural effusion samples. Detection of M tuberculosis using ICSs is rapid, simple, and relatively effective; thus, ICSs are a potential screening tool for TB. Copyright © 2017 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.

Rapid specific and visible detection of porcine circovirus type 3 using loop-mediated isothermal amplification (LAMP).

PubMed

Zheng, S; Wu, X; Shi, J; Peng, Z; Gao, M; Xin, C; Liu, Y; Wang, S; Xu, S; Han, H; Yu, J; Sun, W; Cong, X; Li, J; Wang, J

2018-06-01

In this study, a rapid and specific assay for the detection of porcine circovirus type 3 (PCV3) was established using loop-mediated isothermal amplification (LAMP). Four primers were specifically designed to amplify PCV3. The LAMP assay was effectively optimized to amplify PCV3 by water bath at 60°C for 60 min. The detection limit was approximately 1 × 10 1 copy in this LAMP assay. Compared to porcine circovirus type 2 (PCV2), both gE and gD genes of pseudorabies virus (PRV) and porcine parvovirus (PPV), the LAMP assay showed a high specific detection of PCV3. A visible detection method was developed using SYBR Green I to recognize the results rapidly. Based on the detection of 20 clinical tissue samples, the LAMP assay was more practical and convenient than classical PCR due to its simplicity, high sensitivity, rapidity, specificity, visibility and cost efficiency. © 2018 Blackwell Verlag GmbH.

Porous silicon photoluminescence biosensor for rapid and sensitive detection of toxins

NASA Astrophysics Data System (ADS)

Melnyk, Yulia; Pavlova, Karyna; Myndrul, Valerii; Viter, Roman; Smyntyna, Valentyn; Iatsunskyi, Igor

2017-08-01

A rapid and low cost photoluminescence (PL) immunosensor for the determination of low concentrations of Ochratoxin A(OTA) and Aflatoxine B1 (AfB1) has been developed. This biosensor was based on porous silicon (PSi) fabricated by metal-assisted chemical etching (MACE) and modified by antibodies against OTA/AfB1 (anti-OTA/anti-AfB1). Biofunctionalization method of the PSi surface by anti-OTA/ anti-AfB1 was developed. The changes of the PL intensity after interaction of the immobilized anti-OTA/anti-AfB1with OTA/AfB1 antigens were used as biosensor signal, allowing sensitive and selective detection of OTA/AfB1 antigens in BSA solution. The sensitivity of the reported optical biosensor towards OTA/AfB1 antigens is in the range from 10-3 to 102 ng/ml.

Comparative study of efficacy, rapidity of detection, and cost-effectiveness of potassium hydroxide, calcofluor white, and Chicago sky blue stains in the diagnosis of dermatophytoses.

PubMed

Prakash, R; Prashanth, Hosakere Veerappa; Ragunatha, Shivanna; Kapoor, Meenakshi; Anitha, T K; Krishnamurthy, Veena

2016-04-01

The diagnosis of superficial mycosis such as dermatophytosis is often done clinically. However, in difficult cases, a rapid test with high sensitivity and specificity helps in the immediate confirmation and administration of treatment. The efficacy, rapidity of detection, and cost-effectiveness of KOH preparation, calcofluor white (CW) stain, and Chicago sky blue (CSB) stain in the identification of fungal elements were assessed in patients with dermatophytoses attending the dermatology clinic of a tertiary care hospital. All three tests were performed on each sample collected from 73 patients according to standard procedure. The slides were examined after 5 and 30 minutes in × 10 and × 40 magnifications. The sensitivity and specificity of CW and CSB at 5 and 30 minutes were calculated using KOH preparation as the standard test. CSB stain showed highest positivity (94.5%) within 5 minutes when compared to KOH (75.3%) and CW (83.5%). After 30 minutes, positivity increased in KOH (84.9%) and CW stains (89%), but it remained the same in CSB stain. Both CW and CSB stains when compared to 10% KOH are equally sensitive (100%), but CW was more specific (72.7%), particularly at 30 minutes. When cost of performing tests on 100 specimens is considered, KOH, CW, and CSB stains cost Rs 5, 100, and 15, respectively. CSB stain is a better stain for rapid diagnosis of dermatophytoses because of ease of performance, rapidity of detection, better appreciation of morphology of fungal elements, and cost effectiveness. © 2016 The International Society of Dermatology.

Validation of a Rapid Bacteria Endospore Enumeration System for Planetary Protection Application

NASA Astrophysics Data System (ADS)

Chen, Fei; Kern, Roger; Kazarians, Gayane; Venkateswaran, Kasthuri

NASA monitors spacecraft surfaces to assure that the presence of bacterial endospores meets strict criteria at launch, to minimize the risk of inadvertent contamination of the surface of Mars. Currently, the only approved method for enumerating the spores is a culture based assay that requires three days to produce results. In order to meet the demanding schedules of spacecraft assembly, a more rapid spore detection assay is being considered as an alternate method to the NASA standard culture-based assay. The Millipore Rapid Microbiology Detection System (RMDS) has been used successfully for rapid bioburden enumeration in the pharmaceutical and food industries. The RMDS is rapid and simple, shows high sensitivity (to 1 colony forming unit [CFU]/sample), and correlates well with traditional culture-based methods. It combines membrane filtration, adenosine triphosphate (ATP) bioluminescence chemistry, and image analysis based on photon detection with a Charge Coupled Device (CCD) camera. In this study, we have optimized the assay conditions and evaluated the use of the RMDS as a rapid spore detection tool for NASA applications. In order to select for spores, the samples were subjected to a heat shock step before proceeding with the RMDS incubation protocol. Seven species of Bacillus (nine strains) that have been repeatedly isolated from clean room environments were assayed. All strains were detected by the RMDS in 5 hours and these assay times were repeatedly demonstrated along with low image background noise. Validation experiments to compare the Rapid Sore Assay (RSA) and NASA standard assay (NSA) were also performed. The evaluation criteria were modeled after the FDA Guideline of Process Validation, and Analytical Test Methods. This body of research demonstrates that the Rapid Spore Assay (RSA) is quick, and of equivalent sensitivity to the NASA standard assay, potentially reducing the assay time for bacterial endospores from over 72 hours to less than 8 hours. Accordingly, JPL has produced a report recommending that NASA adopt the RSA method as a suitable alternative to the NASA standard assay.

Development of a real-time PCR for the detection of pathogenic Leptospira spp. in California sea lions

USDA-ARS?s Scientific Manuscript database

Rapid detection of pathogenic Leptospira spp. in marine mammals is challenging: microbiological culture can take 3-6 months and has low sensitivity, immunohistochemical staining of kidney to detect leptospires is invasive and time consuming, and serological methods, such as the microscopic agglutina...

Biomagnetic separation of Salmonella Typhimurium with high affine and specific ligand peptides isolated by phage display technique

NASA Astrophysics Data System (ADS)

Steingroewer, Juliane; Bley, Thomas; Bergemann, Christian; Boschke, Elke

2007-04-01

Analyses of food-borne pathogens are of great importance in order to minimize the health risk for customers. Thus, very sensitive and rapid detection methods are required. Current conventional culture techniques are very time consuming. Modern immunoassays and biochemical analysis also require pre-enrichment steps resulting in a turnaround time of at least 24 h. Biomagnetic separation (BMS) is a promising more rapid method. In this study we describe the isolation of high affine and specific peptides from a phage-peptide library, which combined with BMS allows the detection of Salmonella spp. with a similar sensitivity as that of immunomagnetic separation using antibodies.

Development and validation of a quantitative PCR for rapid and specific detection of California sea lion adenovirus 1 and prevalence in wild and managed populations.

PubMed

Cortés-Hinojosa, Galaxia; Gulland, Frances M D; Goldstein, Tracey; Venn-Watson, Stephanie; Rivera, Rebecca; Archer, Linda L; Waltzek, Thomas B; Gray, Gregory C; Wellehan, James F X

2017-03-01

California sea lion adenovirus 1 (CSLAdV-1) has been associated with hepatitis and enteritis in several wild and captive populations of diverse pinniped species. Currently available tests have been limited to pan-adenoviral polymerase chain reaction (PCR) followed by sequencing. We present the development of a quantitative probe-hybridization PCR (qPCR) assay for rapid, sensitive, and specific detection of this virus in California sea lions ( Zalophus californianus) and other pinnipeds. This assay did not amplify other mammalian adenoviruses and is able to detect consistently down to 10 viral copies per well. Compared with the gold standard conventional pan-adenovirus PCR/sequencing assay, diagnostic sensitivity and specificity of 100% and 88.2% were found, respectively. The lower diagnostic specificity of this qPCR assay may be the result of the lower limit of detection of this assay compared with the gold standard rather than the result of detection of true false-positives.

Sensitivity quantification of remote detection NMR and MRI

NASA Astrophysics Data System (ADS)

Granwehr, J.; Seeley, J. A.

2006-04-01

A sensitivity analysis is presented of the remote detection NMR technique, which facilitates the spatial separation of encoding and detection of spin magnetization. Three different cases are considered: remote detection of a transient signal that must be encoded point-by-point like a free induction decay, remote detection of an experiment where the transient dimension is reduced to one data point like phase encoding in an imaging experiment, and time-of-flight (TOF) flow visualization. For all cases, the sensitivity enhancement is proportional to the relative sensitivity between the remote detector and the circuit that is used for encoding. It is shown for the case of an encoded transient signal that the sensitivity does not scale unfavorably with the number of encoded points compared to direct detection. Remote enhancement scales as the square root of the ratio of corresponding relaxation times in the two detection environments. Thus, remote detection especially increases the sensitivity of imaging experiments of porous materials with large susceptibility gradients, which cause a rapid dephasing of transverse spin magnetization. Finally, TOF remote detection, in which the detection volume is smaller than the encoded fluid volume, allows partial images corresponding to different time intervals between encoding and detection to be recorded. These partial images, which contain information about the fluid displacement, can be recorded, in an ideal case, with the same sensitivity as the full image detected in a single step with a larger coil.

Detection of Shigella in Milk and Clinical Samples by Magnetic Immunocaptured-Loop-Mediated Isothermal Amplification Assay

PubMed Central

Zhang, Liding; Wei, Qiujiang; Han, Qinqin; Chen, Qiang; Tai, Wenlin; Zhang, Jinyang; Song, Yuzhu; Xia, Xueshan

2018-01-01

Shigella is an important human food-borne zoonosis bacterial pathogen, and can cause clinically severe diarrhea. There is an urgent need to develop a specific, sensitive, and rapid methodology for detection of this pathogen. In this study, loop-mediated isothermal amplification (LAMP) combined with magnetic immunocapture assay (IC-LAMP) was first developed for the detection of Shigella in pure culture, artificial milk, and clinical stool samples. This method exhibited a detection limit of 8.7 CFU/mL. Compared with polymerase chain reaction, IC-LAMP is sensitive, specific, and reliable for monitoring Shigella. Additionally, IC-LAMP is more convenient, efficient, and rapid than ordinary LAMP, as it is more efficiently enriches pathogen cells without extraction of genomic DNA. Under isothermal conditions, the amplification curves and the green fluorescence were detected within 30 min in the presence of genomic DNA template. The overall analysis time was approximately 1 h, including the enrichment and lysis of the bacterial cells, a significantly short detection time. Therefore, the IC-LAMP methodology described here is potentially useful for the efficient detection of Shigella in various samples. PMID:29467730

Detection of swine-origin influenza A (H1N1) viruses using a paired surface plasma waves biosensor

NASA Astrophysics Data System (ADS)

Su, Li-Chen; Chang, Ying-Feng; Li, Ying-Chang; Hsieh, Jo-Ping; Lee, Cheng-Chung; Chou, Chien

2010-08-01

In order to enhance the sensitivity of conventional rapid test technique for the detection of swine-origin influenza A (H1N1) viruses (S-OIVs), we used a paired surface plasma waves biosensor (PSPWB) based on SPR in conjunction with an optical heterodyne technique. Experimentally, PSPWB showed a 125-fold improvement at least in the S-OIV detection as compared to conventional enzyme linked immunosorbent assay. Moreover, the detection limit of the PSPWB for the S-OIV detection was enhanced 250-fold in buffer at least in comparison with that of conventional rapid influenza diagnostic test.

Direct PCR - A rapid method for multiplexed detection of different serotypes of Salmonella in enriched pork meat samples.

PubMed

Chin, Wai Hoe; Sun, Yi; Høgberg, Jonas; Quyen, Than Linh; Engelsmann, Pia; Wolff, Anders; Bang, Dang Duong

2017-04-01

Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture method is time consuming. In response to the demand for rapid on line or at site detection of pathogens, in this study, we developed a multiplex Direct PCR method for rapid detection of different Salmonella serotypes directly from pork meat samples without any DNA purification steps. An inhibitor-resistant Phusion Pfu DNA polymerase was used to overcome PCR inhibition. Four pairs of primers including a pair of newly designed primers targeting Salmonella spp. at subtype level were incorporated in the multiplex Direct PCR. To maximize the efficiency of the Direct PCR, the ratio between sample and dilution buffer was optimized. The sensitivity and specificity of the multiplex Direct PCR were tested using naturally contaminated pork meat samples for detecting and subtyping of Salmonella spp. Conventional bacterial culture methods were used as reference to evaluate the performance of the multiplex Direct PCR. Relative accuracy, sensitivity and specificity of 98.8%; 97.6% and 100%, respectively, were achieved by the method. Application of the multiplex Direct PCR to detect Salmonella in pork meat at slaughter reduces the time of detection from 5 to 6 days by conventional bacterial culture and serotyping methods to 14 h (including 12 h enrichment time). Furthermore, the method poses a possibility of miniaturization and integration into a point-of-need Lab-on-a-chip system for rapid online pathogen detection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Reverse transcription recombinase polymerase amplification assay for the rapid detection of type 2 porcine reproductive and respiratory syndrome virus.

PubMed

Wang, Jian-Chang; Yuan, Wan-Zhe; Han, Qing-An; Wang, Jin-Feng; Liu, Li-Bing

2017-05-01

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in pigs, and has tremendous negative economic impact on the swine industry worldwide. PRRSV is classified into the two distinct genotypes: type 1 and type 2, and most of the described PRRSV isolates in China are type 2. Rapid and sensitive detection of PRRSV is of great importance for the disease control and regional eradication programs. Recombinase polymerase amplification (RPA) has emerged as a novel isothermal amplification technology for the molecular diagnosis of infectious diseases. In this study, a fluorescence reverse transcription RPA (RT-RPA) assay was developed to detect the type 2 PRRSV using primers and exo probe specific for the viral nucleocapsid gene. The reaction was performed at 40°C within 20min. The RT-RPA assay could detect both the classical (C-PRRSV) and highly pathogenic PRRSV (HP-PRRSV), but there was no cross-reaction to other pathogens. Using the in vitro transcribed PRRSV RNA as template, the analytical sensitivity of RT-RPA was 690 copies. The assay performance was evaluated by testing 60 field samples and compared to real-time RT-PCR. The detection rate of RT-RPA was 86.6% (52/60), while the detection rate of real-time RT-PCR was 83.3% (50/60). This simple, rapid and reliable method could be potentially applied for rapid detection of PRRSV in point-of-care and rural areas. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of a rapid and visual nucleotide detection method for a Chinese epidemic strain of Orientia tsutsugamushi based on recombinase polymerase amplification assay and lateral flow test.

PubMed

Qi, Yong; Yin, Qiong; Shao, Yinxiu; Cao, Min; Li, Suqin; Chen, Hongxia; Shen, Wanpeng; Rao, Jixian; Li, Jiameng; Li, Xiaoling; Sun, Yu; Lin, Yu; Deng, Yi; Zeng, Wenwen; Zheng, Shulong; Liu, Suyun; Li, Yuexi

2018-05-01

Orientia tsutsugamushi is an obligate intracellular pathogen that causes scrub typhus. Diagnosing scrub typhus remains a challenge, and a sensitive, specific, simple, and rapid diagnostic test is still needed. A recombinase polymerase amplification (RPA) assay combined with a lateral flow (LF) test targeting the 56-kDa gene of a Karp-like strain of O. tsutsugamushi was developed and optimized. The detection limits, sensitivity, specificity, and simulative clinical performance were evaluated. Primers and probe were screened to establish the RPA assay, and the reaction conditions were optimized. The detection limit was 10 copies/reaction in detecting plasmid DNA and 12 copies/reaction in detecting genomic DNA. The RPA-LF method could differentiate O. tsutsugamushi from other phylogenetically related bacteria. The sensitivity was 100% and specificity was over 90%, when evaluated using infected animal samples or simulative clinical samples. Furthermore, the method was completed in 20min at 37°C followed by a 3-5min incubation at room temperature for the development of an immunochromatographic strip, and the results could be determined visually. This method is promising for wide-ranging use in basic medical units considering that it requires minimal instruments and infrastructure and is highly time-efficient, sensitive, and specific for diagnosing scrub typhus. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Sensitive and rapid detection of Chlamydia trachomatis by recombinase polymerase amplification directly from urine samples.

PubMed

Krõlov, Katrin; Frolova, Jekaterina; Tudoran, Oana; Suhorutsenko, Julia; Lehto, Taavi; Sibul, Hiljar; Mäger, Imre; Laanpere, Made; Tulp, Indrek; Langel, Ülo

2014-01-01

Chlamydia trachomatis is the most common sexually transmitted human pathogen. Infection results in minimal to no symptoms in approximately two-thirds of women and therefore often goes undiagnosed. C. trachomatis infections are a major public health concern because of the potential severe long-term consequences, including an increased risk of ectopic pregnancy, chronic pelvic pain, and infertility. To date, several point-of-care tests have been developed for C. trachomatis diagnostics. Although many of them are fast and specific, they lack the required sensitivity for large-scale application. We describe a rapid and sensitive form of detection directly from urine samples. The assay uses recombinase polymerase amplification and has a minimum detection limit of 5 to 12 pathogens per test. Furthermore, it enables detection within 20 minutes directly from urine samples without DNA purification before the amplification reaction. Initial analysis of the assay from clinical patient samples had a specificity of 100% (95% CI, 92%-100%) and a sensitivity of 83% (95% CI, 51%-97%). The whole procedure is fairly simple and does not require specific machinery, making it potentially applicable in point-of-care settings. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Development and performance evaluation of a novel immunofluorescence chromatographic assay for histidine-rich protein 2 of Plasmodium falciparum.

PubMed

Kang, Keren; Dzakah, Emmanuel E; Huang, Yongping; Xie, Mingquan; Luo, Xiaochun; Li, Wenmei; Wang, Jihua

2015-05-30

The low sensitivity and specificity of Plasmodium falciparum diagnostic tests pose a serious health threat to people living in endemic areas. The objective of the study was to develop a rapid assay for the detection of histidine-rich protein 2 (HRP2) of P. falciparum in whole blood by immunofluorescence chromatographic technology. A total of 1163 positive and negative blood samples were screened. The double-antibody sandwich assay was used to establish the kit and its performance was evaluated for sensitivity, specificity, accuracy, precision, stability, and clinical effectiveness. The cut-off level of detection of the kit was 25 parasites/μl. Common interfering substances in human blood specimens, such as bilirubin, triglyceride and cholesterol had no significant effect on HRP2 antigen detection. The precision of the kit was run with different concentration of standard calibrators and the values were less than 10 %. The performance of this diagnostic kit in the detection of the calibrators has shown that a shelf life of about 12 months gives a more reliable result. Among clinical samples tested, the HRP2 test kit and the reference products had good coincidence rate in a parallel experiment and this test kit had a more sensitive detecting level to the target protein than the reference kits used in this study. The specificity and sensitivity for this test were 99.6 % (800/803) and 99.7 % (1160/1163), respectively. A novel HRP2 immunofluorescence detection method was developed in this study. Overall performance evaluation indicated that the kit has a rapid, high sensitivity and on-spot method for detecting P. falciparum.

Sensitive Detection of Francisella tularensis Directly from Whole Blood by Use of the GeneXpert System.

PubMed

Banada, Padmapriya P; Deshpande, Srinidhi; Chakravorty, Soumitesh; Russo, Riccardo; Occi, James; Meister, Gabriel; Jones, Kelly J; Gelhaus, Carl H; Valderas, Michelle W; Jones, Martin; Connell, Nancy; Alland, David

2017-01-01

Francisella tularensis is a potential bioterrorism agent that is highly infectious at very low doses. Diagnosis of tularemia by blood culture and nucleic acid-based diagnostic tests is insufficiently sensitive. Here, we demonstrate a highly sensitive F. tularensis assay that incorporates sample processing and detection into a single cartridge suitable for point-of-care detection. The assay limit of detection (LOD) and dynamic range were determined in a filter-based cartridge run on the GeneXpert system. F. tularensis DNA in buffer or CFU of F. tularensis was spiked into human or macaque blood. To simulate detection in human disease, the assay was tested on blood drawn from macaques infected with F. tularensis Schu S4 at daily intervals. Assay detection was compared to that with a conventional quantitative PCR (qPCR) assay and blood culture. The assay LOD was 0.1 genome equivalents (GE) per reaction and 10 CFU/ml F. tularensis in both human and macaque blood. In infected macaques, the assay detected F. tularensis on days 1 to 4 postinfection in 21%, 17%, 60%, and 83% of macaques, respectively, compared to conventional qPCR positivity rates of 0%, 0%, 30%, and 100% and CFU detection of blood culture at 0%, 0%, 0%, and 10% positive, respectively. Assay specificity was 100%. The new cartridge-based assay can rapidly detect F. tularensis in bloodstream infections directly in whole blood at the early stages of infection with a sensitivity that is superior to that of other methods. The simplicity of the automated testing procedures may make this test suitable for rapid point-of-care detection. Copyright © 2016 American Society for Microbiology.

Comparison of nonstructural protein-1 antigen detection by rapid and enzyme-linked immunosorbent assay test and its correlation with polymerase chain reaction for early diagnosis of dengue

PubMed Central

Gaikwad, Seema; Sawant, Sandhya S.; Shastri, Jayanthi S.

2017-01-01

INTRODUCTION: Early diagnosis of dengue is important for appropriate clinical management and vector control. Different serological tests based on the principle of immunochromatography and enzyme-linked immunosorbent assay (ELISA) are commonly used for detection of antigen and antibodies of dengue virus. The performance of these tests depends on the sensitivity and specificity. Hence, the study was undertaken to compare nonstructural protein-1 (NS1) antigen detection by rapid and ELISA with real-time polymerase chain reaction (RT-PCR) for diagnosis of dengue. MATERIALS AND METHODS: Prospective laboratory study was carried out on sera samples (n = 200) from clinically suspected cases of dengue. The sera samples were subjected for NS1 antigen detection test by rapid test, NS1 ELISA, and RT-PCR. The results of rapid and ELISA tests were compared with real Time PCR. RESULTS: The sensitivity, specificity, positive, and negative predictive value of rapid dengue NS1 antigen test were 81.5%, 66.7%, 78.2%, and 71.1%, respectively whereas that of NS1 ELISA were 89.9%, 100%, 100%, and 94%, respectively. Concordance of Rapid NS1 and NS1 ELISA with PCR was 75.5% and 94%. DISCUSSION AND CONCLUSION: NS1 antigen ELISA can be implemented in diagnostic laboratories for diagnosis of dengue in the acute phase of illness. The test also has great potential value for use in epidemic situations, as it could facilitate the early screening of patients and limit disease expansion. PMID:28706387

Immunoliposome-based immunomagnetic concentration and separation assay for rapid detection of Cronobacter sakazakii.

PubMed

Shukla, Shruti; Lee, Gibaek; Song, Xinjie; Park, Sunhyun; Kim, Myunghee

2016-03-15

This study aimed to develop an immunoliposome-based immunomagnetic concentration and separation assay for the rapid detection of Cronobacter sakazakii (C. sakazakii), an acute opportunistic foodborne pathogenic bacterium, in both pure culture and infant formula. To develop the assay, magnetic nanoparticles (diameter 30 nm) were coated with immunoglobulin G (IgG), specifically anti-C. sakazakii IgG, and applied for the sensitive and efficient detection of C. sakazakii using immunoliposomes. The binding efficiency of anti-C. sakazakii IgG to the magnetic nanoparticles was 86.23 ± 0.59%. The assay developed in this study detected as few as 3.3 × 10(3) CFUmL(-1) of C. sakazakii in pure culture within 2h 30 min; in comparison, an indirect non-competitive enzyme-linked immunosorbent assay was able to detect 6.2 × 10(5) CFUmL(-1) of C. sakazakii in pure culture after 17 h. The developed assay did not show any cross-reactivity with other Cronobacter spp. or pathogens belonging to other genera. In addition, the method was able to detect 10(3) CFUmL(-1) of C. sakazakii in infant formula without any pre-incubation. These results confirm that the immunoliposome-based immunomagnetic concentration and separation assay may facilitate highly sensitive, efficient, and rapid detection of C. sakazakii. Copyright © 2015 Elsevier B.V. All rights reserved.

Gold nanoparticle-sensitized quartz crystal microbalance sensor for rapid and highly selective determination of Cu(II) ions.

PubMed

Jin, Yulong; Huang, Yanyan; Liu, Guoquan; Zhao, Rui

2013-09-21

A novel quartz crystal microbalance (QCM) sensor for rapid, highly selective and sensitive detection of copper ions was developed. As a signal amplifier, gold nanoparticles (Au NPs) were self-assembled onto the surface of the sensor. A simple dip-and-dry method enabled the whole detection procedure to be accomplished within 20 min. High selectivity of the sensor towards copper ions is demonstrated by both individual and coexisting assays with interference ions. This gold nanoparticle mediated amplification allowed a detection limit down to 3.1 μM. Together with good repeatability and regeneration, the QCM sensor was also applied to the analysis of copper contamination in drinking water. This work provides a flexible method for fabricating QCM sensors for the analysis of important small molecules in environmental and biological samples.

Serological Diagnosis of Acute Scrub Typhus in Southern India: Evaluation of InBios Scrub Typhus Detect IgM Rapid Test and Comparison with other Serological Tests.

PubMed

Anitharaj, Velmurugan; Stephen, Selvaraj; Pradeep, Jothimani; Park, Sungman; Kim, Seung-Han; Kim, Young Jin; Kim, Eun-Ye; Kim, Yoon-Won

2016-11-01

Scrub Typhus (ST) is being reported from different parts of India in the recent past. However, the diagnosis and confirmation of ST cases require specific serological and molecular diagnostic tests. Both rapid and conventional ELISA tests need to be properly evaluated. Evaluation of a new ST IgM Immunochromatography (ICT) test kit (InBios Scrub Typhus Detect IgM Rapid Test) and compare it with another rapid kit, conventional ELISA kit and Weil-Felix (WF) test. This prospective study was carried out in Mahatma Gandhi Medical College and Research Institute, Puducherry, during November 2015 to June 2016. Clinically suspected 220 ST patients were examined by a new kit, InBios Scrub Typhus Detect IgM Rapid Test, taking the conventional InBios Scrub Typhus Detect IgM ELISA as reference. Additional comparison was made with ImmuneMed Scrub Typhus Rapid, and WF test (single OXK titers ≥1:320). Statistical analysis was performed (Chi-square, Spearman's correlation and Kappa) using IBM SPSS Statistics 17 for Windows (SPSS Inc; Chicago, USA). Percentage Sensitivity, Specificity, Positive Predictive and Negative Predictive Values for InBios, ImmuneMed and WF were 99.25, 93.02, 95.68, 98.77; 94.87, 94.19, 96.21, 92.05 and 50.38, 95.51, 94.29, 56.67 respectively. A total of 134 patients were positive in reference standard InBios IgM ELISA. This new rapid ST IgM kit validated for the first time in India, showed good sensitivity and specificity. As a Point-of-Care (PoC) test, the kit would be helpful in both urban and remote rural parts of India.

Serological Diagnosis of Acute Scrub Typhus in Southern India: Evaluation of InBios Scrub Typhus Detect IgM Rapid Test and Comparison with other Serological Tests

PubMed Central

Anitharaj, Velmurugan; Pradeep, Jothimani; Park, Sungman; Kim, Seung-Han; Kim, Young Jin; Kim, Eun-Ye; Kim, Yoon-Won

2016-01-01

Introduction Scrub Typhus (ST) is being reported from different parts of India in the recent past. However, the diagnosis and confirmation of ST cases require specific serological and molecular diagnostic tests. Both rapid and conventional ELISA tests need to be properly evaluated. Aim Evaluation of a new ST IgM Immunochromatography (ICT) test kit (InBios Scrub Typhus Detect IgM Rapid Test) and compare it with another rapid kit, conventional ELISA kit and Weil-Felix (WF) test. Materials and Methods This prospective study was carried out in Mahatma Gandhi Medical College and Research Institute, Puducherry, during November 2015 to June 2016. Clinically suspected 220 ST patients were examined by a new kit, InBios Scrub Typhus Detect IgM Rapid Test, taking the conventional InBios Scrub Typhus Detect IgM ELISA as reference. Additional comparison was made with ImmuneMed Scrub Typhus Rapid, and WF test (single OXK titers ≥1:320). Statistical analysis was performed (Chi-square, Spearman’s correlation and Kappa) using IBM SPSS Statistics 17 for Windows (SPSS Inc; Chicago, USA). Results Percentage Sensitivity, Specificity, Positive Predictive and Negative Predictive Values for InBios, ImmuneMed and WF were 99.25, 93.02, 95.68, 98.77; 94.87, 94.19, 96.21, 92.05 and 50.38, 95.51, 94.29, 56.67 respectively. A total of 134 patients were positive in reference standard InBios IgM ELISA. Conclusion This new rapid ST IgM kit validated for the first time in India, showed good sensitivity and specificity. As a Point-of-Care (PoC) test, the kit would be helpful in both urban and remote rural parts of India. PMID:28050364

Development of a remote sensing network for time-sensitive detection of fine scale damage to transportation infrastructure : [final report].

DOT National Transportation Integrated Search

2015-09-23

This research project aimed to develop a remote sensing system capable of rapidly identifying fine-scale damage to critical transportation infrastructure following hazard events. Such a system must be pre-planned for rapid deployment, automate proces...

A novel, colorimetric method for biogenic amine detection based on arylalkylamine N-acetyltransferase.

PubMed

Leng, Pei-Qiang; Zhao, Feng-Lan; Yin, Bin-Cheng; Ye, Bang-Ce

2015-05-21

We developed a novel colorimetric method for rapid detection of biogenic amines based on arylalkylamine N-acetyltransferase (aaNAT). The proposed method offers distinct advantages including simple handling, high speed, low cost, good sensitivity and selectivity.

Comparative evaluation of two rapid Salmonella-IgM tests and blood culture in the diagnosis of enteric fever.

PubMed

Prasad, K J; Oberoi, J K; Goel, N; Wattal, C

2015-01-01

Enteric fever is a major public health problem in developing countries like India. An early and accurate diagnosis is necessary for a prompt and effective treatment. We have evaluated the diagnostic accuracy of two Rapid Salmonella-IgM tests (Typhidot-IgM and Enteroscreen-IgM) as compared to blood culture in rapid and early diagnosis of enteric fever. A total of 2,699 patients' serum samples were tested by Rapid Salmonella-IgM tests and blood culture. Patients were divided into two groups. Test group - patients with enteric fever and blood culture positives for Salmonella Typhi; and three types of Controls, i.e. patients with non-enteric fever illnesses, normal healthy controls and patients positive for S. Paratyphi- A. In addition to this we have also evaluated the significance of positive Salmonella-IgM tests among blood culture-negative cases. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the Typhidot-IgM test and Enteroscreen-IgM test considering blood culture as gold standard were 97.29% and 88.13%, 97.40% and 87.83%, 98.18% and 92.03%, 96.15% and 82.27%, respectively. Typhidot-IgM test was found to be significantly more sensitive and specific as compared to Enteroscreen-IgM. Among blood culture-negative patients, Rapid Salmonella-IgM tests detected 72.25% additional cases of enteric fever. Although the Rapid Salmonella-IgM tests are meant to diagnose S. Typhi only, but these tests detect S. Paratyphi- A also. Thirty-eight patients who were blood culture-positive for S. Paratyphi- A were also positive by Rapid Salmonella-IgM tests. Rapid Salmonella-IgM tests offer an advantage of increased sensitivity, rapidity, early diagnosis and simplicity over blood culture.

Mapping dominant negative mutation for triforine sensitivity in lettuce and its use as a selectable marker for detecting hybrids.

USDA-ARS?s Scientific Manuscript database

Some lettuce cultivars are highly sensitive to triforine, an inhibitor of sterol biosynthesis found in some commercial systemic fungicides. First symptoms of a sensitive reaction are usually observed within 24 to 48 hours after treatment and include severe wilting, necrosis and rapid plant death. We...

Rapid detection of highly pathogenic porcine reproductive and respiratory syndrome virus by a fluorescent probe-based isothermal recombinase polymerase amplification assay.

PubMed

Yang, Yang; Qin, Xiaodong; Sun, Yingjun; Chen, Ting; Zhang, Zhidong

2016-12-01

A novel fluorescent probe-based real-time reverse transcription recombinase polymerase amplification (real-time RT-RPA) assay was developed for rapid detection of highly pathogenic type 2 porcine reproductive and respiratory syndrome virus (HP-PRRSV). The sensitivity analysis showed that the detection limit of RPA was 70 copies of HP-PRRSV RNA/reaction. The real-time RT-RPA highly specific amplified HP-PRRSV with no cross-reaction with classic PRRSV, classic swine fever virus, pseudorabies virus, and foot-and-mouth disease virus. Assessment with 125 clinical samples showed that the developed real-time RT-RPA assay was well correlated with real-time RT-qPCR assays for detection of HP-PRRSV. These results suggest that the developed real-time RT-RPA assay is suitable for rapid detection of HP-PRRSV.

Rapid polymerase chain reaction diagnosis of white-nose syndrome in bats

USGS Publications Warehouse

Lorch, J.M.; Gargas, A.; Meteyer, C.U.; Berlowski-Zier, B. M.; Green, D.E.; Shearn-Bochsler, V.; Thomas, N.J.; Blehert, D.S.

2010-01-01

A newly developed polymerase chain reaction (PCR)-based method to rapidly and specifically detect Geomyces destructans on the wings of infected bats from small quantities (1-2 mg) of tissue is described in the current study (methods for culturing and isolating G. destructans from bat skin are also described). The lower limits of detection for PCR were 5 fg of purified fungal DNA or 100 conidia per 2 mg of wing tissue. By using histology as the standard, the PCR had a diagnostic specificity of 100% and a diagnostic sensitivity of 96%, whereas the diagnostic sensitivity of culture techniques was only 54%. The accuracy and fast turnaround time of PCR provides field biologists with valuable information on infection status more rapidly than traditional methods, and the small amount of tissue required for the test would allow diagnosis of white-nose syndrome in live animals.

[Efficacy of a rapid test to diagnose Plasmodium vivax in symptomatic patients of Chiapas, Mexico].

PubMed

González-Cerón, Lilia; Rodríguez, Mario H; Betanzos, Angel F; Abadía, Acatl

2005-01-01

To evaluate, under laboratory conditions, the sensitivity and specificity of a rapid diagnostic test (OptiMAL), based on immunoreactive strips, to detect Plasmodium vivax infection in febrile patients in Southern Chiapas, Mexico. The presence of parasites in blood samples of 893 patients was investigated by Giemsa-stained thick blood smear microscopic examination (gold standard). A blood drop from the same sample was smeared on immunoreactive strips to investigate the presence of the parasite pLDH. Discordant results were resolved by PCR amplification of the parasite's 18S SSU rRNA, to discard infection. OptiMAL had an overall sensitivity of 93.3% and its specificity was 99.5%. Its positive and negative predictive values were 96.5% and 98.9%, respectively. Signal intensity in OptiMAL strips correlated well with the parasitemia density in the blood samples (r = 0.601, p = 0.0001). This rapid test had acceptable sensitivity and specificity to detect P. vivax under laboratory conditions and could be useful for malaria diagnosis in field operations in Mexico.

Mind the Gap: Two Dissociable Mechanisms of Temporal Processing in the Auditory System

PubMed Central

Anderson, Lucy A.

2016-01-01

High temporal acuity of auditory processing underlies perception of speech and other rapidly varying sounds. A common measure of auditory temporal acuity in humans is the threshold for detection of brief gaps in noise. Gap-detection deficits, observed in developmental disorders, are considered evidence for “sluggish” auditory processing. Here we show, in a mouse model of gap-detection deficits, that auditory brain sensitivity to brief gaps in noise can be impaired even without a general loss of central auditory temporal acuity. Extracellular recordings in three different subdivisions of the auditory thalamus in anesthetized mice revealed a stimulus-specific, subdivision-specific deficit in thalamic sensitivity to brief gaps in noise in experimental animals relative to controls. Neural responses to brief gaps in noise were reduced, but responses to other rapidly changing stimuli unaffected, in lemniscal and nonlemniscal (but not polysensory) subdivisions of the medial geniculate body. Through experiments and modeling, we demonstrate that the observed deficits in thalamic sensitivity to brief gaps in noise arise from reduced neural population activity following noise offsets, but not onsets. These results reveal dissociable sound-onset-sensitive and sound-offset-sensitive channels underlying auditory temporal processing, and suggest that gap-detection deficits can arise from specific impairment of the sound-offset-sensitive channel. SIGNIFICANCE STATEMENT The experimental and modeling results reported here suggest a new hypothesis regarding the mechanisms of temporal processing in the auditory system. Using a mouse model of auditory temporal processing deficits, we demonstrate the existence of specific abnormalities in auditory thalamic activity following sound offsets, but not sound onsets. These results reveal dissociable sound-onset-sensitive and sound-offset-sensitive mechanisms underlying auditory processing of temporally varying sounds. Furthermore, the findings suggest that auditory temporal processing deficits, such as impairments in gap-in-noise detection, could arise from reduced brain sensitivity to sound offsets alone. PMID:26865621

Performance and usefulness of the Hexagon rapid diagnostic test in children with asymptomatic malaria living in the Mount Cameroon region.

PubMed

Wanji, Samuel; Kimbi, Helen K; Eyong, Joan E; Tendongfor, Nicholas; Ndamukong, Judith L

2008-05-22

Rapid and correct diagnosis of malaria is considered an important strategy in the control of the disease. However, it remains to be determined how well these tests can perform in those who harbour the parasite, but are asymptomatic, so that rapid diagnostic tests (RDTs) could be used in rapid mass surveillance in malaria control programmes. Microscopic and immunochromatographic diagnosis of malaria were performed on blood samples from the hyperendemic Mount Cameroon region. Thin and thick blood films were stained with Giemsa and examined under light microscopy for malaria parasites. The RDT was performed on the blood samples for the detection of Plasmodium species. In addition, the performance characteristics of the test were determined using microscopy as gold standard. Results revealed 40.32% to be positive for microscopy and 34.41% to be positive for the RDT. Parasites were detected in a greater proportion of samples as the parasite density increase. Plasmodium falciparum was the predominant Plasmodium species detected in the study population either by microscopy or by the RDT. Overall, the test recorded a sensitivity and specificity of 85.33% and 95.05% respectively, and an accuracy of 91.40%. The sensitivity and specificity of the RDT increased as parasite densities increased. The Hexagon Malaria Combi test showed a high sensitivity and specificity in diagnosing malaria in asymptomatic subjects and so could be suitable for use in mass surveillance programmes for the management and control of malaria.

An Estuarine Fish Bioassay for Sensitive Biomonitoring of Oil-related Contamination

EPA Science Inventory

An embryonic and larval bioassay using the estuarine fish, Fundulus heteroclitus, was modified for the rapid detection of bioavailable compounds that act through the aryl hydrocarbon receptor (AhR). The early development of fishes is particularly sensitive to AhR agonists, such ...

HIGHLY SENSITIVE ASSAY FOR ANTICHOLINESTERASE COMPOUNDS USING 96 WELL PLATE FORMAT

EPA Science Inventory

The rapid and sensitive detection of organophosphate insecticides using a 96 well plate format is reported. Several features of this assay make it attractive for development as a laboratory-based or field screening assay. Acetylcholinesterase (AChE) was stabilized in a gelati...

Multiplex qRT-PCR for the Detection of Western Equine Encephalomyelitis, St. Louis Encephalitis, and West Nile Viral RNA in Mosquito Pools (Diptera: Culicidae).

PubMed

Brault, Aaron C; Fang, Ying; Reisen, William K

2015-05-01

Following the introduction of West Nile virus into California during the summer of 2003, public health and vector control programs expanded surveillance efforts and were in need of diagnostics capable of rapid, sensitive, and specific detection of arbovirus infections of mosquitoes to inform decision support for intervention. Development of a multiplex TaqMan or real-time semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay in which three virus specific primer-probe sets were used in the same reaction is described herein for the detection of western equine encephalomyelitis, St. Louis encephalitis and West Nile viral RNA. Laboratory validation and field data from 10 transmission seasons are reported. The comparative sensitivity and specificity of this multiplex assay to singleplex RT-PCR as well as an antigen detection (rapid analyte measurement platform) and standard plaque assays indicate this assay to be rapid and useful in providing mosquito infection data to estimate outbreak risk. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Bioconjugated fluorescent silica nanoparticles for the rapid detection of Entamoeba histolytica.

PubMed

Hemadi, Ahmad; Ekrami, Alireza; Oormazdi, Hormozd; Meamar, Ahmad Reza; Akhlaghi, Lame; Samarbaf-Zadeh, Ali Reza; Razmjou, Elham

2015-05-01

Rapid detection of Entamoeba histolytica based on fluorescent silica nanoparticle (FSNP) indirect immunofluorescence microscopy was evaluated. Silica nanoparticles were synthesized using Stöber's method, with their surface activated to covalently bind to, and immobilize, protein A. For biolabeling, FSNP was added to conjugated E. histolytica trophozoites with monoclonal anti-E. histolytica IgG1 for microscopic observation of fluorescence. Fluorescent silica nanoparticle sensitivity was determined with axenically cultured E. histolytica serially diluted to seven concentrations. Specificity was evaluated using other intestinal protozoa. Fluorescent silica nanoparticles detected E. histolytica at the lowest tested concentration with no cross-reaction with Entamoeba dispar, Entamoeba moshkovskii, Blastocystis sp., or Giardia lamblia. Visualization of E. histolytica trophozoites with anti-E. histolytica antibody labeled with fluorescein isothiocyanate (FITC) was compared with that using anti-E. histolytica antibody bioconjugated FSNP. Although FITC and FSNP produced similar results, the amount of specific antibody required for FITC to induce fluorescence of similar intensity was fivefold that for FSNP. Fluorescent silica nanoparticles delivered a rapid, simple, cost-effective, and highly sensitive and specific method of detecting E. histolytica. Further study is needed before introducing FSNP for laboratory diagnosis of amoebiasis. Copyright © 2015 Elsevier B.V. All rights reserved.

Clinical profiling and use of loop-mediated isothermal amplification assay for rapid detection of Mycobacterium tuberculosis from sputum.

PubMed

Poudel, A; Pandey, B D; Lekhak, B; Rijal, B; Sapkota, B R; Suzuki, Y

2009-01-01

Tuberculosis is a global health problem and the situation is worsening with newer incidences of drug resistance and HIV association. Diagnosis of tuberculosis can be done by many methods and test, culture of sputum being the ideal one. Nucleic acid amplification (NAA) assay are more time efficient one, that amplify and detect specific nucleic acid sequences allows rapid, sensitive and specific detection of M. tuberculosis in sputum samples. The present study intends to compile the clinical presentations of the pulmonary tuberculosis (PTB) patients and to evaluate the efficacy of in-house loop-mediated isothermal amplification (LAMP) in detecting Mycobacterium tuberculosis in sputum samples by comparing with microscopy and culture. Two hundred two sputum samples were collected from 202 patients at National Tuberculosis Center, Bhaktapur, Nepal. Complete clinical profiling, epidemiological data and record on BCG vaccination were noted and the samples were subjected for microscopy, culture and in-house LAMP with six primers specific for 16S RNA gene of Mycobacterium tuberculosis. Of the 176 cases of clinical profiling, productive cough was most common symptom in 147 (83.52%), followed by chest pain 136 (77.27%), fever 133 (75.56%) and haemoptysis 61 (34.66%). There was a statistically significant association between BCG vaccination and development of TB (chi(2)=5.33, P=0.02). Of 202 cases, 115 (56.93%) were chest X-ray positive, 101(50%) were direct smear-positive and 100 (49.51%) were culture positive. LAMP had a sensitivity of 97% and specificity of 94.12% while comparing with culture. In addition, its sensitivity and specificity were 91.09% and 89.11% respectively with reference to microscopy. As in our previous study, overall, the result of present study further confirms that the in-house LAMP is a simple, rapid, sensitive and specific DNA amplification technique for PTB diagnosis. Because of rapidity of microscopy and specificity of culture, in-house LAMP assay can be used as a very powerful and useful supplementary tool with complete clinical profiling of the patients for rapid diagnosis of TB in both AFB-positive and negative cases who are suspected as PTB in disease endemic country like Nepal.

Advances in Anthrax Detection: Overview of Bioprobes and Biosensors.

PubMed

Kim, Joungmok; Gedi, Vinayakumar; Lee, Sang-Choon; Cho, Jun-Haeng; Moon, Ji-Young; Yoon, Moon-Young

2015-06-01

Anthrax is an infectious disease caused by Bacillus anthracis. Although anthrax commonly affects domestic and wild animals, it causes a rare but lethal infection in humans. A variety of techniques have been introduced and evaluated to detect anthrax using cultures, polymerase chain reaction, and immunoassays to address the potential threat of anthrax being used as a bioweapon. The high-potential harm of anthrax in bioterrorism requires sensitive and specific detection systems that are rapid, field-ready, and real-time monitoring. Here, we provide a systematic overview of anthrax detection probes with their potential applications in various ultra-sensitive diagnostic systems.

A highly sensitive and selective aptasensor based on graphene oxide fluorescence resonance energy transfer for the rapid determination of oncoprotein PDGF-BB.

PubMed

Liang, Junfei; Wei, Ran; He, Shuai; Liu, Yikan; Guo, Lin; Li, Lidong

2013-03-21

Oncoprotein platelet derived growth factor-BB (PDGF-BB) is one of the most critical growth factors that regulates tumor growth and division. In this work, a highly sensitive and selective fluorescence resonance energy transfer (FRET) aptasensor for PDGF-BB detection based on the assembly of dye-labeled aptamer and graphene oxide (GO) is developed for the first time. Due to the non-covalent assembly between aptamer and GO, fluorescence quenching of the dye takes place because of FRET. In the presence of PDGF-BB, the binding between aptamer and PDGF-BB will disturb the interaction between aptamer and GO, and release the dye-labeled aptamer from the GO surface, resulting in restoration of the fluorophore fluorescence. Because of the high fluorescence quenching efficiency, unique structure, and electronic properties of GO, the GO aptasensor exhibits extraordinarily high sensitivity. We also demonstrate that two highly related molecular variants of PDGF (AA, AB) can be distinguished from PDGF-BB, which indicates the aptasensor has excellent selectivity. Such an aptasensor opens a rapid, selective and sensitive route for the detection of PDGF-BB and provides a promising strategy for other cancer-related proteins detections.

Endpoint visual detection of three genetically modified rice events by loop-mediated isothermal amplification.

PubMed

Chen, Xiaoyun; Wang, Xiaofu; Jin, Nuo; Zhou, Yu; Huang, Sainan; Miao, Qingmei; Zhu, Qing; Xu, Junfeng

2012-11-07

Genetically modified (GM) rice KMD1, TT51-1, and KF6 are three of the most well known transgenic Bt rice lines in China. A rapid and sensitive molecular assay for risk assessment of GM rice is needed. Polymerase chain reaction (PCR), currently the most common method for detecting genetically modified organisms, requires temperature cycling and relatively complex procedures. Here we developed a visual and rapid loop-mediated isothermal amplification (LAMP) method to amplify three GM rice event-specific junction sequences. Target DNA was amplified and visualized by two indicators (SYBR green or hydroxy naphthol blue [HNB]) within 60 min at an isothermal temperature of 63 °C. Different kinds of plants were selected to ensure the specificity of detection and the results of the non-target samples were negative, indicating that the primer sets for the three GM rice varieties had good levels of specificity. The sensitivity of LAMP, with detection limits at low concentration levels (0.01%−0.005% GM), was 10- to 100-fold greater than that of conventional PCR. Additionally, the LAMP assay coupled with an indicator (SYBR green or HNB) facilitated analysis. These findings revealed that the rapid detection method was suitable as a simple field-based test to determine the status of GM crops.

Recombinase polymerase amplification assay for rapid detection of lumpy skin disease virus.

PubMed

Shalaby, Mohamed A; El-Deeb, Ayman; El-Tholoth, Mohamed; Hoffmann, Donata; Czerny, Claus-Peter; Hufert, Frank T; Weidmann, Manfred; Abd El Wahed, Ahmed

2016-11-02

Lumpy skin disease virus (LSDV) is a Capripoxvirus infecting cattle and Buffalos. Lumpy skin disease (LSD) leads to significant economic losses due to hide damage, reduction of milk production, mastitis, infertility and mortalities (10 %). Early detection of the virus is crucial to start appropriate outbreak control measures. Veterinarians rely on the presence of the characteristic clinical signs of LSD. Laboratory diagnostics including virus isolation, sequencing and real-time polymerase chain reaction (PCR) are performed at well-equipped laboratories. In this study, a portable, simple, and rapid recombinase polymerase amplification (RPA) assay for the detection of LSDV-genome for the use on farms was developed. The LSDV RPA assay was performed at 42 °C and detected down to 179 DNA copies/reaction in a maximum of 15 min. Unspecific amplification was observed with neither LSDV-negative samples (n = 12) nor nucleic acid preparations from orf virus, bovine papular stomatitis virus, cowpoxvirus, Peste des petits ruminants and Blue tongue virus (serotypes 1, 6 and 8). The clinical sensitivity of the LSDV RPA assay matched 100 % (n = 22) to real-time PCR results. In addition, the LSDV RPA assay detected sheep and goat poxviruses. The LSDV RPA assay is a rapid and sensitive test that could be implemented in field or at quarantine stations for the identification of LSDV infected case.

A biolayer interferometry-based enzyme-linked aptamer sorbent assay for real-time and highly sensitive detection of PDGF-BB.

PubMed

Gao, Shunxiang; Zheng, Xin; Wu, Jihong

2018-04-15

Accurate, fast and sensitive detection of disease-specific protein biomarkers, especially in blood, urine, or other bodily fluids, is an important approach to achieve early disease diagnosis. Platelet-derived growth factor-BB (PDGF-BB), a widely used biomarker, is involved in a substantial number of serious diseases, such as hepatic fibrosis, atherosclerosis, age-related macular degeneration and diabetic eye disease and is often over-expressed in human malignant tumors. Therefore, the development of sensitive and specific detection methods for PDGF-BB is of great importance for the early diagnosis of disease and assessments of patient recovery. In the current study, a biolayer interferometry-based enzyme-linked aptamer sorbent assay (BLI-ELASA) was successfully established for rapid (20-25min), high-throughput (8 or 16 samples) and real-time monitoring of PDGF-BB in clinical samples. The method exhibited a broad detection range from 0.5 to 1000ng/mL of PDGF-BB (good linear range from 0.5 to 10ng/mL), with a low detection limit of 0.08ng/mL. Moreover, BLI-ELASA was applied to the detection of PDGF-BB in spiked serum and urine samples and showed a high degree of selectivity for PDGF-BB, good reproducibility, and stability. We believe that the methodology in this work can be easily adapted to detect other biomolecules in clinical samples, including viruses, pathogens and toxins, in a rapid, sensitive, high-throughput and real-time manner. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of a High Throughput Assay for Rapid and Accurate 10-Plex Detection of Citrus Pathogens

USDA-ARS?s Scientific Manuscript database

The need to reliably detect and identify multiple plant pathogens simultaneously, especially in woody perennial hosts, has led to development of new molecular diagnostic approaches. In this study, a Luminex-based system was developed that provided a robust and sensitive test for simultaneous detect...

Detection of Legume Protease Inhibitors by the Gel-X-ray Film Contact Print Technique

ERIC Educational Resources Information Center

Mulimani, Veerappa H.; Sudheendra, Kulkarni; Giri, Ashok P.

2002-01-01

Redgram (Cajanus cajan L.) extracts have been analyzed for the protease inhibitors using a new, sensitive, simple, and rapid method for detection of electrophoretically separated protease inhibitors. The detection involves equilibrating the gel successively in the protease assay buffer and protease solution, rinsing the gel in assay buffer, and…

Fabrication of a novel transparent SERS substrate comprised of Ag-nanoparticle arrays and its application in rapid detection of ractopamine on meat

USDA-ARS?s Scientific Manuscript database

Surface-enhanced Raman spectroscopy (SERS) is an emerging analytical tool that boasts the feature of high detection sensitivity and molecular fingerprint specificity attracting increased attention and showing promise in applications including detecting residues of veterinary drugs. In practice, spec...

Rapid detection of ssDNA and RNA using multi-walled carbon nanotubes modified screen-printed carbon electrode.

PubMed

Ye, Yongkang; Ju, Huangxian

2005-11-15

A method for rapid sensitive detection of DNA or RNA was designed using a composite screen-printed carbon electrode modified with multi-walled carbon nanotubes (MWNTs). MWNTs showed catalytic characteristics for the direct electrochemical oxidation of guanine or adenine residues of signal strand DNA (ssDNA) and adenine residues of RNA, leading to indicator-free detection of ssDNA and RNA concentrations. With an accumulation time of 5 min, the proposed method could be used for detection of calf thymus ssDNA ranging from 17.0 to 345 microg ml(-1) with a detection limit of 2.0 microg ml(-1) at 3 sigma and yeast tRNA ranging from 8.2 microg ml(-1) to 4.1 mg ml(-1). AC impedance was employed to characterize the surface of modified electrodes. The advantages of convenient fabrication, low-cost detection, short analysis time and combination with nanotechnology for increasing the sensitivity made the subject worthy of special emphasis in the research programs and sources of new commercial products.

Graphene nano-ink biosensor arrays on a microfluidic paper for multiplexed detection of metabolites.

PubMed

Labroo, Pratima; Cui, Yue

2014-02-27

The development of a miniaturized and low-cost platform for the highly sensitive, selective and rapid detection of multiplexed metabolites is of great interest for healthcare, pharmaceuticals, food science, and environmental monitoring. Graphene is a delicate single-layer, two-dimensional network of carbon atoms with extraordinary electrical sensing capability. Microfluidic paper with printing technique is a low cost matrix. Here, we demonstrated the development of graphene-ink based biosensor arrays on a microfluidic paper for the multiplexed detection of different metabolites, such as glucose, lactate, xanthine and cholesterol. Our results show that the graphene biosensor arrays can detect multiple metabolites on a microfluidic paper sensitively, rapidly and simultaneously. The device exhibits a fast measuring time of less than 2 min, a low detection limit of 0.3 μM, and a dynamic detection range of 0.3-15 μM. The process is simple and inexpensive to operate and requires a low consumption of sample volume. We anticipate that these results could open exciting opportunities for a variety of applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Label-Free Biological and Chemical Sensing Using Whispering Gallery Mode Optical Resonators: Past, Present, and Future

PubMed Central

Su, Judith

2017-01-01

Sensitive and rapid label-free biological and chemical sensors are needed for a wide variety of applications including early disease diagnosis and prognosis, the monitoring of food and water quality, as well as the detection of bacteria and viruses for public health concerns and chemical threat sensing. Whispering gallery mode optical resonator based sensing is a rapidly developing field due to the high sensitivity and speed of these devices as well as their label-free nature. Here, we describe the history of whispering gallery mode optical resonator sensors, the principles behind detection, the latest developments in the fields of biological and chemical sensing, current challenges toward widespread adoption of these devices, and an outlook for the future. In addition, we evaluate the performance capabilities of these sensors across three key parameters: sensitivity, selectivity, and speed. PMID:28282881

Exciton-controlled fluorescence: application to hybridization-sensitive fluorescent DNA probe.

PubMed

Okamoto, Akimitsu; Ikeda, Shuji; Kubota, Takeshi; Yuki, Mizue; Yanagisawa, Hiroyuki

2009-01-01

A hybridization-sensitive fluorescent probe has been designed for nucleic acid detection, using the concept of fluorescence quenching caused by the intramolecular excitonic interaction of fluorescence dyes. We synthesized a doubly thiazole orange-labeled nucleotide showing high fluorescence intensity for a hybrid with the target nucleic acid and effective quenching for the single-stranded state. This exciton-controlled fluorescent probe was applied to living HeLa cells using microinjection to visualize intracellular mRNA localization. Immediately after injection of the probe into the cell, fluorescence was observed from the probe hybridizing with the target RNA. This fluorescence rapidly decreased upon addition of a competitor DNA. Multicoloring of this probe resulted in the simple simultaneous detection of plural target nucleic acid sequences. This probe realized a large, rapid, reversible change in fluorescence intensity in sensitive response to the amount of target nucleic acid, and facilitated spatiotemporal monitoring of the behavior of intracellular RNA.

Advanced defect classification by smart sampling, based on sub-wavelength anisotropic scatterometry

NASA Astrophysics Data System (ADS)

van der Walle, Peter; Kramer, Esther; Ebeling, Rob; Spruit, Helma; Alkemade, Paul; Pereira, Silvania; van der Donck, Jacques; Maas, Diederik

2018-03-01

We report on advanced defect classification using TNO's RapidNano particle scanner. RapidNano was originally designed for defect detection on blank substrates. In detection-mode, the RapidNano signal from nine azimuth angles is added for sensitivity. In review-mode signals from individual angles are analyzed to derive additional defect properties. We define the Fourier coefficient parameter space that is useful to study the statistical variation in defect types on a sample. By selecting defects from each defect type for further review by SEM, information on all defects can be obtained efficiently.

Rapid and Sensitive Detection of Norovirus Genomes in Oysters by a Two-Step Isothermal Amplification Assay System Combining Nucleic Acid Sequence-Based Amplification and Reverse Transcription-Loop-Mediated Isothermal Amplification Assays▿

PubMed Central

Fukuda, Shinji; Sasaki, Yukie; Seno, Masato

2008-01-01

We developed a two-step isothermal amplification assay system, which achieved the detection of norovirus (NoV) genomes in oysters with a sensitivity similar to that of reverse transcription-seminested PCR. The time taken for the amplification of NoV genomes from RNA extracts was shortened to about 3 h. PMID:18456857

Multicentre evaluations of two new rapid IgG4 tests (WB rapid and panLF rapid) for detection of lymphatic filariasis

PubMed Central

Noordin, Rahmah; Itoh, Makoto; Kimura, Eisaku; Abdul Rahman, Rohana; Ravindran, Balachandran; Mahmud, Rohela; Supali, Taniawati; Weerasooriya, Mirani

2007-01-01

In the global effort to eliminate lymphatic filariasis (LF), rapid field-applicable tests are useful tools that will allow on-site testing to be performed in remote places and the results to be obtained rapidly. Exclusive reliance on the few existing tests may jeopardize the progress of the LF elimination program, thus the introduction of other rapid tests would be useful to address this issue. Two new rapid immunochromatographic IgG4 cassette tests have been produced, namely WB rapid and panLF rapid, for detection of bancroftian filariasis and all three species of lymphatic filaria respectively. WB rapid was developed using BmSXP recombinant antigen, while PanLF rapid was developed using BmR1 and BmSXP recombinant antigens. A total of 165 WB rapid and 276 panLF rapid tests respectively were evaluated at USM and the rest were couriered to another university in Malaysia (98 WB rapid, 129 panLF rapid) and to universities in Indonesia (56 WB rapid, 62 panLF rapid), Japan (152 of each test) and India (18 of each test) where each of the tests underwent independent evaluations in a blinded manner. The average sensitivities of WB rapid and panLF rapid were found to be 97.6% (94%–100%) and 96.5% (94%–100%) respectively; while their average specificities were both 99.6% (99%–100%). Thus this study demonstrated that both the IgG4 rapid tests were highly sensitive and specific, and would be useful additional tests to facilitate the global drive to eliminate this disease. PMID:17961262

Rapid detection of fungal keratitis with DNA-stabilizing FTA filter paper.

PubMed

Menassa, Nardine; Bosshard, Philipp P; Kaufmann, Claude; Grimm, Christian; Auffarth, Gerd U; Thiel, Michael A

2010-04-01

Purpose. Polymerase chain reaction (PCR) is increasingly important for the rapid detection of fungal keratitis. However, techniques of specimen collection and DNA extraction before PCR may interfere with test sensitivity. The purpose of this study was to investigate the use of DNA-stabilizing FTA filter paper (Indicating FTA filter paper; Whatman International, Ltd., Maidstone, UK) for specimen collection without DNA extraction in a single-step, nonnested PCR for fungal keratitis. Methods. Specimens were collected from ocular surfaces with FTA filter discs, which automatically lyse collected cells and stabilize nucleic acids. Filter discs were directly used in single-step PCR reactions to detect fungal DNA. Test sensitivity was evaluated with serial dilutions of Candida albicans, Fusarium oxysporum, and Aspergillus fumigatus cultures. Test specificity was analyzed by comparing 196 and 155 healthy individuals from Switzerland and Egypt, respectively, with 15 patients with a diagnosis of microbial keratitis. Results. PCR with filter discs detected 3 C. albicans, 25 F. oxysporum, and 125 A. fumigatus organisms. In healthy volunteers, fungal PCR was positive in 1.0% and 8.4% of eyes from Switzerland and Egypt, respectively. Fungal PCR remained negative in 10 cases of culture-proven bacterial keratitis, became positive in 4 cases of fungal keratitis, but missed 1 case of culture-proven A. fumigatus keratitis. Conclusions. FTA filter paper for specimen collection together with direct PCR is a promising method of detecting fungal keratitis. The analytical sensitivity is high without the need for a semi-nested or nested second PCR, the clinical specificity is 91.7% to 99.0%, and the method is rapid and inexpensive.

A rapid, highly accurate method for quantifying CALR mutant allele burden in persons with myeloproliferative neoplasms.

PubMed

Yao, Qiu-Mei; Zhou, Jiao; Gale, Robert Peter; Li, Jin-Lan; Li, Ling-Di; Li, Ning; Chen, Shan-Shan; Ruan, Guo-Rui

2015-10-01

Calreticulin (CALR) mutations were recently identified in a substantial proportion of persons with essential thrombocythemia (ET) and with primary myelofibrosis (PMF) without JAK2(V617F). Consequently rapid, sensitive, and specific methods to detect and quantify these mutations are needed. We studied samples from 1088 persons with myeloproliferative neoplasms (MPNs) including 421 JAK2(V617F) negative subjects with ET, PMF, polycythemia vera (PV), chronic myeloid leukemia (CML) and hyper-eosinophilic syndrome (HES). Detection of CALR exon 9 mutations was done by PCR amplification followed by fragment length analysis and direct sequencing. Dilution assays were used to determine CALR mutant allele burden. We detected CALR mutations in blood and bone marrow samples from 152 subjects with ET and with PMF but not in samples from normal or persons with PV, CML, or HES. CALR mutant peaks were distinct from wild-type peaks and dilution experiments indicated a sensitivity level of 0.5-5% for a CALR mutant allele in a wild-type background. Diverse types of mutations were detected including deletions, insertions, and complex indels. All mutations were confirmed by direct sequencing. We also used dilution experiments to quantify mutant allele burden. We were able to reproducibly detect mutant allele levels as low 5% (0.5-5%) in a wild-type background. PCR amplification followed by fragment length analysis is a rapid, sensitive, and specific method for screening persons with MPNs for CALR mutations, especially those with ET and PMF and for estimating mutant allele burden.

Development of recombinase polymerase amplification assays for the rapid detection of peste des petits ruminants virus.

PubMed

Zhang, Yongning; Wang, Jianchang; Zhang, Zhou; Mei, Lin; Wang, Jinfeng; Wu, Shaoqiang; Lin, Xiangmei

2018-04-01

Peste des petits ruminants (PPR) is a severe infectious disease of small ruminants caused by PPR virus (PPRV). Rapid and sensitive detection of PPRV is critical for controlling PPR. This report describes the development and evaluation of a conventional reverse transcription recombinase polymerase amplification (RT-RPA) assay and a real-time RT-RPA assay, targeting the PPRV N gene. Sensitivity analysis revealed that the conventional RT-RPA assay could detect 852 copies of standard PPRV RNA per reaction at 95% probability within 20 min at 41 °C, and the real-time RT-RPA assay could detect 103 copies of RNA molecules per reaction at 95% probability. Specificity analysis showed that both assays have no cross-reactivity with nucleic acid templates prepared from other selected viruses or common pathogens. Clinical evaluation using 162 ovine and hircine serum and nasal swab samples showed that the performance of both the real-time RT-RPA assay and the conventional RT-RPA assay were comparable to that of real-time RT-PCR. The overall agreements between real-time RT-PCR and real-time RT-RPA, and conventional RT-RPA were 99.4% (161/162) and 98.8% (160/162), respectively. The R 2 value of real-time RT-RPA and real-time RT-PCR was 0.900 by linear regression analysis. Our results suggest that both RT-RPA assays have a potential application in the rapid, sensitive and specific detection of PPRV. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of a serotype colloidal gold strip using monoclonal antibody for rapid detection type Asia1 foot-and-mouth disease

PubMed Central

2011-01-01

Background In this study, we developed a rapid, one step colloid gold strip (CGS) capable of specifically detecting type Asia1 foot-and-mouth disease virus (FMDV). We have produced two monoclonal antibodies (mAb) to type Asia1 FMD (named 1B8 and 5E2). On the test strip, the purified 1B8 labelled with the colloidal gold was used as the detector, and the purified 5E2 and goat anti-mouse antibodies were wrapped onto nitrocellulose (NC) membranes as the test and the control line, respectively. The rapid colloidal gold stereotype diagnostic strip was housed in a plastic case. Results In specificity and sensitivity assay, there was no cross-reaction of the antigen with the other type of FMD and SVDV. The detection sensitivity was found to be as high as 10-5 dilution of Asia1/JSL/05 (1 × 107.2TCID50/50 μL). There was excellent agreement between the results obtained by CGS and reverse indirect hemagglutination assay (RIHA), and the agreement can reach to 98.75%. Conclusion We developed colloidal gold strips that have good qualities and does not require specialized equipment or technicians. This method provided a feasible, convenient, rapid, and effective for detecting type Asia1 FMDV in the fields. PMID:21880157

Multifunctional Au NPs-polydopamine-polyvinylidene fluoride membrane chips as probe for enrichment and rapid detection of organic contaminants.

PubMed

Wang, Saihua; Niu, Hongyun; Cai, Yaqi; Cao, Dong

2018-05-01

High-throughput and rapid detection of hazardous compounds in complicated samples is essential for the solution of environmental problems. We have prepared a "pH-paper-like" chip which can rapidly "indicate" the occurrence of organic contaminants just through dipping the chip in water samples for short time followed by fast analysis with surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF MS). The chips are composed of polyvinylidene fluoride membrane (PVDFM), polydopamine (PDA) film and Au nanoparticles (Au NPs), which are layer-by-layer assembled according to the adhesion, self-polymerization and reduction property of dopamine. In the Au NPs loaded polydopamine-polyvinylidene fluoride membrane (Au NPs-PDA-PVDFM) chips, PVDFM combined with PDA film are responsible for the enrichment of organic analyte through hydrophobic interactions and π-π stacking; Au NPs serve as effective SALDI matrix for the rapid detection of target analyte. After dipping into water solution for minutes, the Au-PDA-PVDFM chips with enriched organic analytes can be detected directly with SALDI-TOF MS. The good solid-phase extraction performance of the PDA-PVDFM components, remarkable matrix effect of the loaded AuNPs, and sensitivity of the SALDI-TOF MS technique ensure excellent sensitivity and reproducibility for the quantification of trace levels of organic contaminants in environmental water samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of a serotype colloidal gold strip using monoclonal antibody for rapid detection type Asia1 foot-and-mouth disease.

PubMed

Lin, Tong; Shao, Jun-jun; Du, Jun-zheng; Cong, Guo-zheng; Gao, Shan-dian; Chang, Huiyun

2011-09-01

In this study, we developed a rapid, one step colloid gold strip (CGS) capable of specifically detecting type Asia1 foot-and-mouth disease virus (FMDV). We have produced two monoclonal antibodies (mAb) to type Asia1 FMD (named 1B8 and 5E2). On the test strip, the purified 1B8 labelled with the colloidal gold was used as the detector, and the purified 5E2 and goat anti-mouse antibodies were wrapped onto nitrocellulose (NC) membranes as the test and the control line, respectively. The rapid colloidal gold stereotype diagnostic strip was housed in a plastic case. In specificity and sensitivity assay, there was no cross-reaction of the antigen with the other type of FMD and SVDV. The detection sensitivity was found to be as high as 10(-5) dilution of Asia1/JSL/05 (1 × 10(7.2)TCID(50)/50 μL). There was excellent agreement between the results obtained by CGS and reverse indirect hemagglutination assay (RIHA), and the agreement can reach to 98.75%. We developed colloidal gold strips that have good qualities and does not require specialized equipment or technicians. This method provided a feasible, convenient, rapid, and effective for detecting type Asia1 FMDV in the fields.

Rapid bacteriological screening of cosmetic raw materials by using bioluminescence.

PubMed

Nielsen, P; Van Dellen, E

1989-01-01

Incoming cosmetic raw materials are routinely tested for microbial content. Standard plate count methods require up to 72 h. A rapid, sensitive, and inexpensive raw material screening method was developed that detects the presence of bacteria by means of ATP (bioluminescence). With a 24-h broth enrichment, the minimum bacterial ATP detection threshold of 1 cfu/g sample can be achieved using purified firefly luciferin-luciferase and an ATP releasing reagent. By using this rapid screen, microbiologically free material may be released for production within 24 h, while contaminated material undergoes further quantitative and identification testing. In order for a raw material to be validated for this method it must be evaluated for (1) a potential nonmicrobial light-contributing reaction resulting in a false positive or, (2) degradation of the ATP giving a false negative, and (3) confirmation that the raw material has not overwhelmed the buffering capacity of the enrichment broth. The key criteria for a rapid screen was the sensitivity to detect less than one colony forming unit per g product, the speed to do this within 24 h, and cost efficiency. Bioluminescence meets these criteria. With an enrichment step, it can detect less than one cfu/g sample. After the enrichment step, analysis time per sample is approximately 2 min and the cost for material and reagents is less than one dollar per sample.

Chromogenic culture media or rapid immunochromatographic test: Which is better for detecting Klebsiella pneumoniae that produce OXA-48 and can they be used in blood and urine specimens.

PubMed

Genc, Ozlem; Aksu, Evrim

2018-05-01

Our goal was to compare a rapid test (OXA-48K-SeT) and four different chromogenic media (CHROMagar KPC, CHROMagar mSuperCARBA, ChromID Carba and ChromID OXA-48) for the detection of OXA-48 producing Klebsiella pneumoniae isolates and spiked urine/blood samples with these bacteria. In total 100 K.pneumoniae isolates, including 60 OXA-48 positive, 15 other carbapenemase producing, 15 Extended spectrum betalactamases (ESBL) positive and 10 carbapenem sensitive K.pneumoniae were included in the study. After all samples were inoculated into all chromogenic media, temocillin discs were placed onto the media. OXA-48K-SeT was studied according to the manufacturer's instructions and the lower detection limit was determined. Sensitivities and specificities of all chromogenic media and rapid test were detected as 100%. All of the OXA-48 producers were found resistant to temocillin on all chromogenic media. The lower detection limit of the rapid assay was determined as 10 6 in both direct bacterial samples and in spiked urine/blood samples. As a result, four chromogenic culture media and OXA-48 K-SeT can be used safely for detection of OXA-48 positive K.pneumoniae isolates. Although direct clinical specimens were not used, our study suggests that this media and OXA-48 K-SeT may be used in patient samples like blood and urine. Further studies are needed to assess this suggestion. Copyright © 2018 Elsevier B.V. All rights reserved.

Rapid colorimetric detection of Zika virus from serum and urine specimens by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

PubMed Central

Biggerstaff, Brad J.; Tanner, Nathan A.; Lauterbach, Molly; Lanciotti, Robert S.

2017-01-01

Zika virus (ZIKV) has emerged as a major global public health concern in the last two years due to its link as a causative agent of human birth defects. Its rapid expansion into the Western Hemisphere as well as the ability to be transmitted from mother to fetus, through sexual transmission and possibly through blood transfusions has increased the need for a rapid and expansive public health response to this unprecedented epidemic. A non-invasive and rapid ZIKV diagnostic screening assay that can be performed in a clinical setting throughout pregnancy is vital for prenatal care of women living in areas of the world where exposure to the virus is possible. To meet this need we have developed a sensitive and specific reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay to detect ZIKV RNA in urine and serum with a simple visual detection. RT-LAMP results were shown to have a limit of detection 10-fold higher than qRT-PCR. As little as 1.2 RNA copies/μl was detected by RT-LAMP from a panel of 178 diagnostic specimens. The assay was shown to be highly specific for ZIKV RNA when tested with diagnostic specimens positive for dengue virus (DENV) and chikungunya virus (CHIKV). The assay described here illustrates the potential for a fast, reliable, sensitive and specific assay for the detection of ZIKV from urine or serum that can be performed in a clinical or field setting with minimal equipment and technological expertise. PMID:28945787

Rapid colorimetric detection of Zika virus from serum and urine specimens by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

PubMed

Calvert, Amanda E; Biggerstaff, Brad J; Tanner, Nathan A; Lauterbach, Molly; Lanciotti, Robert S

2017-01-01

Zika virus (ZIKV) has emerged as a major global public health concern in the last two years due to its link as a causative agent of human birth defects. Its rapid expansion into the Western Hemisphere as well as the ability to be transmitted from mother to fetus, through sexual transmission and possibly through blood transfusions has increased the need for a rapid and expansive public health response to this unprecedented epidemic. A non-invasive and rapid ZIKV diagnostic screening assay that can be performed in a clinical setting throughout pregnancy is vital for prenatal care of women living in areas of the world where exposure to the virus is possible. To meet this need we have developed a sensitive and specific reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay to detect ZIKV RNA in urine and serum with a simple visual detection. RT-LAMP results were shown to have a limit of detection 10-fold higher than qRT-PCR. As little as 1.2 RNA copies/μl was detected by RT-LAMP from a panel of 178 diagnostic specimens. The assay was shown to be highly specific for ZIKV RNA when tested with diagnostic specimens positive for dengue virus (DENV) and chikungunya virus (CHIKV). The assay described here illustrates the potential for a fast, reliable, sensitive and specific assay for the detection of ZIKV from urine or serum that can be performed in a clinical or field setting with minimal equipment and technological expertise.

3D metal-organic framework as highly efficient biosensing platform for ultrasensitive and rapid detection of bisphenol A.

PubMed

Wang, Xue; Lu, Xianbo; Wu, Lidong; Chen, Jiping

2015-03-15

As is well known, bisphenol A (BPA), usually exists in daily plastic products, is one of the most important endocrine disrupting chemicals. In this work, copper-centered metal-organic framework (Cu-MOF) was synthesized, which was characterized by SEM, TEM, XRD, FTIR and electrochemical method. The resultant Cu-MOF was explored as a robust electrochemical biosensing platform by choosing tyrosinase (Tyr) as a model enzyme for ultrasensitive and rapid detection of BPA. The Cu-MOF provided a 3D structure with a large specific surface area, which was beneficial for enzyme and BPA absorption, and thus improved the sensitivity of the biosensor. Furthermore, Cu-MOF as a novel sorbent could increase the available BPA concentration to react with tyrosinase through π-π stacking interactions between BPA and Cu-MOF. The Tyr biosensor exhibited a high sensitivity of 0.2242A M(-1) for BPA, a wide linear range from 5.0×10(-8) to 3.0×10-6moll(-1), and a low detection limit of 13nmoll(-1). The response time for detection of BPA is less than 11s. The proposed method was successfully applied to rapid and selective detection of BPA in plastic products with satisfactory results. The recoveries are in the range of 94.0-101.6% for practical applications. With those remarkable advantages, MOFs-based 3D structures show great prospect as robust biosensing platform for ultrasensitive and rapid detection of BPA. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

Performance of the CareStart Glucose-6-Phosphate Dehydrogenase (G6PD) Rapid Diagnostic Test in Gressier, Haiti

PubMed Central

von Fricken, Michael E.; Weppelmann, Thomas A.; Eaton, Will T.; Masse, Roseline; Beau de Rochars, Madsen V. E.; Okech, Bernard A.

2014-01-01

Administering primaquine (PQ) to treat malaria patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency can pose a serious risk of drug-induced hemolysis (DIH). New easy to use point-of-care rapid diagnostic tests are being developed as an alternative to labor-intensive spectrophotometric methods, but they require field testing before they can be used at scale. This study screened 456 participants in Gressier, Haiti using the Access Bio CareStart qualitative G6PD rapid detection test compared with the laboratory-based Trinity Biotech quantitative spectrophotometric assay. Findings suggest that the CareStart test was 90% sensitive for detecting individuals with severe deficiency and 84.8% sensitive for detecting individuals with moderate and severe deficiency compared with the Trinity Biotech assay. A high negative predictive value of 98.2% indicates excellent performance in determining those patients able to take PQ safely. The CareStart G6PD test holds much value for screening malaria patients to determine eligibility for PQ therapy. PMID:24778197

Performance of the CareStart glucose-6-phosphate dehydrogenase (G6PD) rapid diagnostic test in Gressier, Haiti.

PubMed

von Fricken, Michael E; Weppelmann, Thomas A; Eaton, Will T; Masse, Roseline; Beau de Rochars, Madsen V E; Okech, Bernard A

2014-07-01

Administering primaquine (PQ) to treat malaria patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency can pose a serious risk of drug-induced hemolysis (DIH). New easy to use point-of-care rapid diagnostic tests are being developed as an alternative to labor-intensive spectrophotometric methods, but they require field testing before they can be used at scale. This study screened 456 participants in Gressier, Haiti using the Access Bio CareStart qualitative G6PD rapid detection test compared with the laboratory-based Trinity Biotech quantitative spectrophotometric assay. Findings suggest that the CareStart test was 90% sensitive for detecting individuals with severe deficiency and 84.8% sensitive for detecting individuals with moderate and severe deficiency compared with the Trinity Biotech assay. A high negative predictive value of 98.2% indicates excellent performance in determining those patients able to take PQ safely. The CareStart G6PD test holds much value for screening malaria patients to determine eligibility for PQ therapy. © The American Society of Tropical Medicine and Hygiene.

[Microbiology--laboratory examinations for bacterias].

PubMed

Hen, Renjun; Imafuku, Yuji; Yoshida, Hiroshi

2002-11-01

As it has been required to identify pathogenic microbes in shorter times, simple and rapid methods have been developed and used. Here, we summarized the present situation of rapid diagnostic testing in clinical microbiology in Japan, and also presented our results on PBP2' detection. The rapid test kits available in Japan for E. coli, Helicobacter pylori, Salmonella, Streptococcus and Staphylococcus aureus were described. Rapid examination methods are based mainly on immunologic reactions, which included slide agglutination using latex particle, immunochromatography and ELISA. Times required for the identification are 10 to 15 minutes. Moreover, rapid test kits employing PCR are also marketed. Further, we evaluated MRSA-LA "Seiken" which is a rapid detection kit for PBP2' produced by MRSA. The test was shown to be highly sensitive and specific. For the rapid identification of pathogenic microbes, simple and rapid test kits described here will be used more in clinical diagnosis.

Ultrasensitive microfluidic solid-phase ELISA using an actuatable microwell-patterned PDMS chip.

PubMed

Wang, Tanyu; Zhang, Mohan; Dreher, Dakota D; Zeng, Yong

2013-11-07

Quantitative detection of low abundance proteins is of significant interest for biological and clinical applications. Here we report an integrated microfluidic solid-phase ELISA platform for rapid and ultrasensitive detection of proteins with a wide dynamic range. Compared to the existing microfluidic devices that perform affinity capture and enzyme-based optical detection in a constant channel volume, the key novelty of our design is two-fold. First, our system integrates a microwell-patterned assay chamber that can be pneumatically actuated to significantly reduce the volume of chemifluorescent reaction, markedly improving the sensitivity and speed of ELISA. Second, monolithic integration of on-chip pumps and the actuatable assay chamber allow programmable fluid delivery and effective mixing for rapid and sensitive immunoassays. Ultrasensitive microfluidic ELISA was demonstrated for insulin-like growth factor 1 receptor (IGF-1R) across at least five orders of magnitude with an extremely low detection limit of 21.8 aM. The microwell-based solid-phase ELISA strategy provides an expandable platform for developing the next-generation microfluidic immunoassay systems that integrate and automate digital and analog measurements to further improve the sensitivity, dynamic ranges, and reproducibility of proteomic analysis.

One-step, room temperature, colorimetric melamine sensing using an in-situ formation of silver nanoparticles through modified Tollens process

NASA Astrophysics Data System (ADS)

Wang, Huiying; Chen, Dinglong; Yu, Longquan; Chang, Ming; Ci, Lijie

2015-02-01

We have developed a rapid, sensitive, one-step, and selective colorimetric detection method for melamine (MEL) in milk powder based upon an in-situ formation of silver nanoparticles (AgNPs) through modified Tollens process at room temperature. The triazine ring N atoms of MEL molecule were strategically designed to complex the Ag+ through electron donor-acceptor interaction. During the AgNPs formation procedure, the MEL molecule, which has been covalently bonded with the Ag+ ions, was adsorbed to the surface of as-prepared AgNPs, resulting in the aggregation of the adjacent AgNPs with detectable decreases of absorption signal. The concentration of MEL can be determined with the naked eye or a UV-vis spectrometer at which the yellow-to-brown color change associated with aggregate enhancement takes place. This method enables rapid (less than 30 min) and sensitive (limit of detection, LOD, 10 nM) detection, and it was also able to discriminate MEL from sixteen other milk relevant coexisting compounds. This assay does not utilize organic cosolvents, enzymatic reactions, light-sensitive dye molecules, lengthy protocols, or sophisticated instrumentation thereby overcoming some of the limitations of conventional methods.

Novel Polymerase Spiral Reaction (PSR) for rapid visual detection of Bovine Herpesvirus 1 genomic DNA from aborted bovine fetus and semen.

PubMed

Malla, Javed Ahmed; Chakravarti, Soumendu; Gupta, Vikas; Chander, Vishal; Sharma, Gaurav Kumar; Qureshi, Salauddin; Mishra, Adhiraj; Gupta, Vivek Kumar; Nandi, Sukdeb

2018-02-20

Bovine herpesvirus-1 (BHV-1) is a major viral pathogen affecting bovines leading to various clinical manifestations and causes significant economic impediment in modern livestock production system. Rapid, accurate and sensitive detection of BHV-1 infection at frozen semen stations or at dairy herds remains a priority for control of BHV-1 spread to susceptible population. Polymerase Spiral Reaction (PSR), a novel addition in the gamut of isothermal techniques, has been successfully implemented in initial optimization for detection of BHV-1 genomic DNA and further validated in clinical samples. The developed PSR assay has been validated for detection of BHV-1 from bovine semen (n=99), a major source of transmission of BHV-1 from breeding bulls to susceptible dams in artificial insemination programs. The technique has also been used for screening of BHV-1 DNA from suspected aborted fetal tissues (n=25). The developed PSR technique is 100 fold more sensitive than conventional PCR and comparable to real-time PCR. The PSR technique has been successful in detecting 13 samples positive for BHV-1 DNA in bovine semen, 4 samples more than conventional PCR. The aborted fetal tissues were negative for presence of BHV-1 DNA. The presence of BHV-1 in bovine semen samples raises a pertinent concern for extensively screening of semen from breeding bulls before been used for artificial insemination process. PSR has all the attributes for becoming a method of choice for rapid, accurate and sensitive detection of BHV-1 DNA at frozen semen stations or at dairy herds in resource constrained settings. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid and sensitive approach to simultaneous detection of genomes of hepatitis A, B, C, D and E viruses.

PubMed

Kodani, Maja; Mixson-Hayden, Tonya; Drobeniuc, Jan; Kamili, Saleem

2014-10-01

Five viruses have been etiologically associated with viral hepatitis. Nucleic acid testing (NAT) remains the gold standard for diagnosis of viremic stages of infection. NAT methodologies have been developed for all hepatitis viruses; however, a NAT-based assay that can simultaneously detect all five viruses is not available. We designed TaqMan card-based assays for detection of HAV RNA, HBV DNA, HCV RNA, HDV RNA and HEV RNA. The performances of individual assays were evaluated on TaqMan Array Cards (TAC) for detecting five viral genomes simultaneously. Sensitivity and specificity were determined by testing 329 NAT-tested clinical specimens. All NAT-positive samples for HCV (n = 32), HDV (n = 28) and HEV (n = 14) were also found positive in TAC (sensitivity, 100%). Forty-three of 46 HAV-NAT positive samples were also positive in TAC (sensitivity, 94%), while 36 of 39 HBV-NAT positive samples were positive (sensitivity, 92%). No false-positives were detected for HBV (n = 32), HCV (n = 36), HDV (n = 30), and HEV (n = 31) NAT-negative samples (specificity 100%), while 38 of 41 HAV-NAT negative samples were negative by TAC (specificity 93%). TAC assay was concordant with corresponding individual NATs for hepatitis A-E viral genomes and can be used for their detection simultaneously. The TAC assay has potential for use in hepatitis surveillance, for screening of donor specimens and in outbreak situations. Wider availability of TAC-ready assays may allow for customized assays, for improving acute jaundice surveillance and for other purposes for which there is need to identify multiple pathogens rapidly. Published by Elsevier B.V.

Analytical sensitivity of current best-in-class malaria rapid diagnostic tests.

PubMed

Jimenez, Alfons; Rees-Channer, Roxanne R; Perera, Rushini; Gamboa, Dionicia; Chiodini, Peter L; González, Iveth J; Mayor, Alfredo; Ding, Xavier C

2017-03-24

Rapid diagnostic tests (RDTs) are today the most widely used method for malaria diagnosis and are recommended, alongside microscopy, for the confirmation of suspected cases before the administration of anti-malarial treatment. The diagnostic performance of RDTs, as compared to microscopy or PCR is well described but the actual analytical sensitivity of current best-in-class tests is poorly documented. This value is however a key performance indicator and a benchmark value needed to developed new RDTs of improved sensitivity. Thirteen RDTs detecting either the Plasmodium falciparum histidine rich protein 2 (HRP2) or the plasmodial lactate dehydrogenase (pLDH) antigens were selected from the best performing RDTs according to the WHO-FIND product testing programme. The analytical sensitivity of these products was evaluated using a range of reference materials including P. falciparum and Plasmodium vivax whole parasite samples as well as recombinant proteins. The best performing HRP2-based RDTs could detect all P. falciparum cultured samples at concentrations as low as 0.8 ng/mL of HRP2. The limit of detection of the best performing pLDH-based RDT specifically detecting P. vivax was 25 ng/mL of pLDH. The analytical sensitivity of P. vivax and Pan pLDH-based RDTs appears to vary considerably from product to product, and improvement of the limit-of-detection for P. vivax detecting RDTs is needed to match the performance of HRP2 and Pf pLDH-based RDTs for P. falciparum. Different assays using different reference materials produce different values for antigen concentration in a given specimen, highlighting the need to establish universal reference assays.

Rapid detection of Bacillus anthracis spores using a super-paramagnetic lateral-flow immunological detection system.

PubMed

Wang, Dian-Bing; Tian, Bo; Zhang, Zhi-Ping; Deng, Jiao-Yu; Cui, Zong-Qiang; Yang, Rui-Fu; Wang, Xu-Ying; Wei, Hong-Ping; Zhang, Xian-En

2013-04-15

There is an urgent need for convenient, sensitive, and specific methods to detect the spores of Bacillus anthracis, the causative agent of anthrax, because of the bioterrorism threat posed by this bacterium. In this study, we firstly develop a super-paramagnetic lateral-flow immunological detection system for B. anthracis spores. This system involves the use of a portable magnetic assay reader, super-paramagnetic iron oxide particles, lateral-flow strips and two different monoclonal antibodies directed against B. anthracis spores. This detection system specifically recognises as few as 400 pure B. anthracis spores in 30 min. This system has a linear range of 4×10³-10⁶ CFU ml⁻¹ and reproducible detection limits of 200 spores mg⁻¹ milk powder and 130 spores mg⁻¹ soil for simulated samples. In addition, this approach shows no obvious cross-reaction with other related Bacillus spores, even at high concentrations, and has no significant dependence on the duration of the storage of the immunological strips. Therefore, this super-paramagnetic lateral-flow immunological detection system is a promising tool for the rapid and sensitive detection of Bacillus anthracis spores under field conditions. Copyright © 2012 Elsevier B.V. All rights reserved.

Electrochemical aptasensor for detecting tetracycline in milk

NASA Astrophysics Data System (ADS)

Hanh Le, Thi; Phuc Pham, Van; Huyen La, Thi; Binh Phan, Thi; Huan Le, Quang

2016-03-01

A rapid, simple and sensitive biosensor system for tetracycline detection is very important in food safety. In this paper we developed a label-free aptasensor for electrochemical detection of tetracycline. According to the electrochemical impendence spectroscopy (EIS) analysis, there was a linear relationship between the concentration of tetracycline and the electron transfer resistance from 10 to 3000 ng ml-1 of the tetracycline concentration. The detection limit was 10 ng ml-1 in 15 min detection duration. The prepared aptasensor showed a good reproducibility with an acceptable stability in tetracycline detection. The recoveries of tetracycline in spiked milk samples were in the range of 88.1%-94.2%. The aptasensor has sensitivity 98% and specificity of 100%.

Accuracy of an Immunochromatographic Diagnostic Test (ICT Malaria Combo Cassette Test) Compared to Microscopy among under Five-Year-Old Children when Diagnosing Malaria in Equatorial Guinea

PubMed Central

Portero, José-Luis; Rubio-Yuste, Maria; Descalzo, Miguel Angel; Raso, Jose; Lwanga, Magdalena; Obono, Jaquelina; Nseng, Gloria; Benito, Agustin; Cano, Jorge

2010-01-01

Conventional malaria diagnosis based on microscopy raises serious difficulties in weak health systems. Cost-effective and sensitive rapid diagnostic tests have been recently proposed as alternatives to microscopy. In Equatorial Guinea, a study was conducted to assess the reliability of a rapid diagnostic test compared to microscopy. The study was designed in accordance with the directives of the Standards for Reporting Diagnostic Accuracy Initiative (STARD). Peripheral thick and thin films for the microscopy diagnosis and a rapid immunochromatographic test (ICT Malaria Combo Cassette Test) were performed on under five-year-old children with malaria suspicion. The ICT test detected Plasmodium spp. infection with a sensitivity of 81.5% and a specificity of 81.9% while P. falciparum diagnosis occurred with a sensitivity of 69.7% and a specificity of 73.7%. The sensitivity of the ICT test increased with higher parasitemias. The general results showed little concordance between the ICT test and microscopy (kappa = 0.28, se: 0.04). In Equatorial Guinea, the ICT Malaria Combo Cassette Test has proven to be an acceptable test to detect high P. falciparum parasitemias. However, the decrease of sensitivity at medium and low parasitemias hampers that ICT can replace properly performed microscopy at present in the diagnosis of malaria in children. PMID:22332024

Detection of Citrus tristeza virus by using fluorescence resonance energy transfer-based biosensor

NASA Astrophysics Data System (ADS)

Shojaei, Taha Roodbar; Salleh, Mohamad Amran Mohd; Sijam, Kamaruzaman; Rahim, Raha Abdul; Mohsenifar, Afshin; Safarnejad, Reza; Tabatabaei, Meisam

2016-12-01

Due to the low titer or uneven distribution of Citrus tristeza virus (CTV) in field samples, detection of CTV by using conventional detection techniques may be difficult. Therefore, in the present work, the cadmium-telluride quantum dots (QDs) was conjugated with a specific antibody against coat protein (CP) of CTV, and the CP were immobilized on the surface of gold nanoparticles (AuNPs) to develop a specific and sensitive fluorescence resonance energy transfer (FRET)-based nanobiosensor for detecting CTV. The maximum FRET efficiency for the developed nano-biosensor was observed at 60% in AuNPs-CP/QDs-Ab ratio of 1:8.5. The designed system showed higher sensitivity and specificity over enzyme linked immunosorbent assay (ELISA) with a limit of detection of 0.13 μg mL- 1 and 93% and 94% sensitivity and specificity, respectively. As designed sensor is rapid, sensitive, specific and efficient in detecting CTV, this could be envisioned for diagnostic applications, surveillance and plant certification program.

Rapid, Sensitive, Enzyme-Immunodotting Assay for Detecting Cow Milk Adulteration in Sheep Milk: A Modern Laboratory Project

NASA Astrophysics Data System (ADS)

Inda, Luis A.; Razquín, Pedro; Lampreave, Fermín; Alava, María A.; Calvo, Miguel

1998-12-01

Specificity, sensitivity, and experimental simplicity make the immunoenzymatic assay suitable for a variety of laboratories dedicated to diverse activities such as research, quality control in food analysis, or clinical biochemistry. In these assays, the antibody that specifically recognizes the antigen is covalently attached to an enzyme. Once the antigen-antibody immunocomplex is formed, the enzymatic reaction gives a colored product that allows the detection of the initial antigen. The aim of this work was the design of a new laboratory project appropriate for use in courses of biochemistry, immunochemistry, or analytical chemistry. The assay described here detects the presence of cow milk in milk of other species. The main application is the detection of cow milk in sheep milk and cheese. Specific proteins, immunoglobulins (IgG) of the fraudulent bovine milk, are specifically recognized and retained by antibodies immobilized on a membrane. The binding of a second antibody covalently attached to horseradish peroxidase (HRP) allows the development of a visible signal. Thus, students can rapidly detect milk adulterations using a specific, sensitive, and safe experimental approach. The experiment allows students to apply their theoretical knowledge, resulting in a stimulating experience of solving a real problem during a 4-hour laboratory period.

Evaluation of a cow-side milk progesterone assay and assessment of the positive predictive value of oestrus diagnosis by dairy farmers in New South Wales.

PubMed

Ingenhoff, L; Hall, E; House, J K

2016-12-01

The three objectives of this study were to determine the positive predictive value (PPV) of oestrus diagnosis (heat detection accuracy) by dairy farmers, calculate the diagnostic sensitivity and specificity of the P4 Rapid milk progesterone assay for detecting a corpus luteum and evaluate the economics of using a cow-side milk progesterone assay designed to aid oestrus diagnosis. Milk samples were collected from 752 cows diagnosed in oestrus by farm personnel on 14 dairy farms. Samples were tested using the P4 Rapid milk progesterone assay to estimate the PPV of oestrus diagnosis at each farm and a crude pooled mean of PPV of oestrus diagnosis across all farms. A further 156 milk samples were collected from cows with luteal tissue status determined by transrectal ultrasound and tested by the P4 Rapid assay to enable calculation of the sensitivity and specificity of the P4 Rapid assay. For pooled farm samples, the PPV was 97.0%, with a range between farms of 88.9-100%. Sensitivity of the P4 Rapid milk progesterone assay for detecting a corpus luteum was 90.1% and specificity was 98.7%. Misclassification of oestrus in cows previously identified as pregnant was the most common cause of false-positive oestrus diagnoses by farm personnel. Routine testing of milk progesterone in all cows diagnosed in oestrus is not economically justified and may even slightly reduce submission rates; conversely, strategic use of cow-side milk progesterone assays can improve herd reproductive performance by facilitating decisions on whether to rebreed cows previously diagnosed as pregnant. © 2016 Australian Veterinary Association.

Rapid and sensitive sandwich enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxin B in cheese.

PubMed Central

Morissette, C; Goulet, J; Lamoureux, G

1991-01-01

A rapid and sensitive screening sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of staphylococcal enterotoxin B (SEB) in cheese by using a highly avid anti-SEB antibody (Ab) as the capture Ab (CAb) and as the biotinylated Ab conjugate. The glutaraldehyde fixation method for the immobilization of CAb on polystyrene dipsticks was superior to the adsorption fixation and the adsorption-glutaraldehyde fixation methods. The glutaraldehyde fixation method resulted in a higher surface-saturating CAb concentration as evaluated by the peroxidase saturation technique and by the ability of the CAb-coated dipstick to discriminate between positive and negative controls (index of discrimination). Of nine blocking agents used alone or in pairs, lysine-human serum albumin, bovine serum albumin, human serum albumin, and gelatin effectively saturated available sites on the CAb-coated dipsticks without causing interference with the antigen-Ab reactions. The addition of 1% polyethylene glycol to the diluent of the biotinylated anti-SEB Ab conjugate improved the detection of SEB. A concentration of 4% polyethylene glycol allowed a 5-min reaction time for the streptavidin-biotin-horseradish peroxidase conjugate. Cheddar cheese homogenate reduced the sensitivity of the SEB assay; however, the sensitivity was restored when 1.6% (wt/vol) of either a nonionic detergent (Mega-9) or two zwitterionic detergents (Zwittergent 3-10 and 3-12 detergent) was added to the diluent. By using the rapid sandwich ELISA, a minimum of 0.5 to 1.0 ng of SEB per ml was detected within 45 min. The whole procedure for the analysis of the cheddar cheese samples was completed within 1 h.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2039234

Flow cytometry as a rapid test for detection of penicillin resistance directly in bacterial cells in Enterococcus faecalis and Staphylococcus aureus.

PubMed

Jarzembowski, T; Wiśniewska, K; Józwik, A; Bryl, E; Witkowski, J

2008-08-01

We studied the usefulness of flow cytometry for detection of penicillin resistance in E. faecalis and S. aureus by direct binding of commercially available fluorescent penicillin, Bocillin FL, to cells obtained from culture. There were significantly lower percentages of fluorescent cells and median and mean fluorescence values per particle in penicillin-resistant than in penicillin-sensitive strains of both species observed. The method allows rapid detection of penicillin resistance in S. aureus and E. faecalis. The results encourage further investigations on the detection of antibiotic resistance in bacteria using flow cytometry.

Evaluation of the Clearview® Malaria pLDH Malaria Rapid Diagnostic Test in a non-endemic setting.

PubMed

Houzé, Sandrine; Hubert, Véronique; Cohen, Dorit Pessler; Rivetz, Baruch; Le Bras, Jacques

2011-09-27

Malaria Rapid Diagnostic Tests (RDTs) are widely used to diagnose malaria. The present study evaluated a new RDT, the Clearview® Malaria pLDH test targeting the pan-Plasmodium antigen lactate dehydrogenase (pLDH). The Clearview® Malaria pLDH test was evaluated on fresh samples obtained in returned international travellers using microscopy corrected by PCR as the reference method. Included samples were Plasmodium falciparum (139), Plasmodium vivax (22), Plasmodium ovale (20), Plasmodium malariae (7), and 102 negative. Overall sensitivity for the detection of Plasmodium spp was 93.2%. For P. falciparum, the sensitivity was 98.6%; for P. vivax, P. ovale and P. malariae, overall sensitivities were 90.9%, 60.0% and 85.7% respectively. For P. falciparum and for P. vivax, the sensitivities increased to 100% at parasite densities above 100/μl. The specificity was 100%. The test was easily to perform and the result was stable for at least 1 hour. The Clearview® Malaria pLDH was efficient for the diagnosis of malaria. The test was very sensitive for P. falciparum and P. vivax detection. The sensitivities for P. ovale and P. malariae were better than other RDTs.

Detection of influenza A virus subtypes using a solid-phase PCR microplate chip assay.

PubMed

Sun, Xin-Cheng; Wang, YunLong; Yang, Liping; Zhang, HuiRu

2015-01-01

A rapid and sensitive microplate chip based on solid PCR was developed to identify influenza A subtypes. A simple ultraviolet cross-linking method was used to immobilize DNA probes on pretreated microplates. Solid-phase PCR was proven to be a convenient method for influenza A screening. The sensitivity of the microplate chip was 10(-3) μg/mL for the enzymatic colorimetric method and 10(-4) μg/mL for the fluorescence method. The 10 sets of primers and probes for the microplate chip were highly specific and did not interfere with each other. These results suggest that the microplate chip based on solid PCR can be used to rapidly detect universal influenza A and its subtypes. This platform can also be used to detect other pathogenic microorganisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Design, optimisation and preliminary validation of a human specific loop-mediated amplification assay for the rapid detection of human DNA at forensic crime scenes.

PubMed

Hird, H J; Brown, M K

2017-11-01

The identification of samples at a crime scene which require forensic DNA typing has been the focus of recent research interest. We propose a simple, but sensitive analysis system which can be deployed at a crime scene to identify crime scene stains as human or non-human. The proposed system uses the isothermal amplification of DNA in a rapid assay format, which returns results in as little as 30min from sampling. The assay system runs on the Genie II device, a proven in-field detection system which could be deployed at a crime scene. The results presented here demonstrate that the system was sufficiently specific and sensitive and was able to detect the presence of human blood, semen and saliva on mock forensic samples. Copyright © 2017. Published by Elsevier B.V.

Biconically tapered fiber optic probes for rapid label-free immunoassays.

PubMed

Miller, John; Castaneda, Angelica; Lee, Kun Ho; Sanchez, Martin; Ortiz, Adrian; Almaz, Ekrem; Almaz, Zuleyha Turkoglu; Murinda, Shelton; Lin, Wei-Jen; Salik, Ertan

2015-04-01

We report use of U-shaped biconically tapered optical fibers (BTOF) as probes for label-free immunoassays. The tapered regions of the sensors were functionalized by immobilization of immunoglobulin-G (Ig-G) and tested for detection of anti-IgG at concentrations of 50 ng/mL to 50 µg/mL. Antibody-antigen reaction creates a biological nanolayer modifying the waveguide structure leading to a change in the sensor signal, which allows real-time monitoring. The kinetics of the antibody (mouse Ig-G)-antigen (rabbit anti-mouse IgG) reactions was studied. Hydrofluoric acid treatment makes the sensitive region thinner to enhance sensitivity, which we confirmed by experiments and simulations. The limit of detection for the sensor was estimated to be less than 50 ng/mL. Utilization of the rate of the sensor peak shift within the first few minutes of the antibody-antigen reaction is proposed as a rapid protein detection method.

Comparative performance of aldolase and lactate dehydrogenase rapid diagnostic tests in Plasmodium vivax detection

PubMed Central

2014-01-01

Background Misdiagnosis of malaria by commercial rapid diagnostic tests (RDTs) is a major cause of concern in the diagnosis of malaria. This retrospective study was aimed at assessing the relative performance of four RDTs with emphasis on the detection of two Plasmodium vivax antigens: aldolase and lactate dehydrogenase (LDH). Methods Three commercially available Plasmodium LDH or aldolase antigen detection kits (One Step Malaria P.f/P.v, ParaHit Total ver. 1.0, SD Bioline Malaria) and an anti-P. vivax aldolase-specific monoclonal antibody (mAb) pair 1C3-12 F10 were evaluated with P. vivax positive as well as non-P. vivax samples and healthy samples using blood smear examination as standard. Each test was read according to the manufacturer’s instructions. Results MAb 1C3-12 F10 pair targeting P. vivax-specific aldolase exhibited very good specificity and sensitivity of 100 and 97.4%, respectively. Positive predictive value (PPV) and negative predictive value (NPV) of 100 and 99.5%, respectively, were also observed. The anti-P. vivax LDH in the One-Step Malaria P.f/P.v test showed sensitivity, specificity, PPV and NPV of 93.5, 98.0, 88.9 and 98.8%, respectively. ParaHit Total ver. 1.0 targeting the pan-aldolase antigen showed sensitivity, specificity of 97.4 and 99.6%, respectively. PPV and NPV were both 99.5%. SD Bioline had sensitivity, specificity, PPV and NPV of 93.5, 100, 100 and 98.8%, respectively. The overall sensitivity and specificity of all four RDTs were acceptable, especially for the aldolase detection tests. Five (6.5%) of the P. vivax-positive samples (n = 77) that were confirmed by microscopic examination as well as the two aldolase detection RDTs (mAb 1C3-12 F10 and ParaHit Total ver.1.0) were undetected by the two LDH detection RDTs (One Step Malaria P.f/P.v and SD Bioline). Similarly, two positive samples (2.6%) that were positively confirmed by the LDH detection RDTs were also undetected by the aldolase detection test kits. Conclusion Aldolase and LDH antigens perform differently in different P. vivax samples; hence there is a high risk of misdiagnosis when monoclonal antibodies are used against only one particular antigen in the test. A combination of both aldolase and LDH in RDTs for the rapid diagnosis of P. vivax will enhance the sensitivity of the assay and reduce misdiagnosis. PMID:25015737

Amperometric biosensor for Salmonella typhimurium detection in milk

USDA-ARS?s Scientific Manuscript database

This paper reports an amperometric biosensor for rapid and sensitive Salmonella Typhimurium detection in milk. The biosensor was assembled from the self-assembled monolayers technique on a gold surface. In this device, polyclonal antibodies were oriented by protein A. The biosensor structure was cha...

The development of methods for the detection of Salmonella in chickens by a combination of immunomagnetic separation and PCRs.

PubMed

Dai, Fengying; Zhang, Miao; Xu, Dixin; Yang, Yin; Wang, Jiaxiao; Li, Mingzhen; Du, Meihong

2017-11-01

Micro- and nanoimmunomagnetic beads (MIMBs and NIMBs) used for immunomagnetic separation (IMS) with PCR were studied for the rapid detection of Salmonella. The capture efficiency of the two different IMBs was evaluated by a conventional plate counting method, and the binding pattern was studied using scanning electron microscopy. The specificity of the IMBs was tested with Salmonella, Shigella flexneri, enterohemorrhagic Escherichia coli O157:H7, and Listeria monocytogenes. By comparing the pre-enrichment IMS and the IMS enrichment steps with a 5.5-H enrichment time, this study developed a rapid and sensitive method for the detection of Salmonella in chicken. The method was implemented by IMS enrichment and PCR with MIMBs and NIMBs, with a total analysis time of 8 H. We showed that the method was sensitive based on NIMBs with a detection limit of 10° CFU for Salmonella in 25 g of chicken. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Rapid multiplex detection of 10 foodborne pathogens with an up-converting phosphor technology-based 10-channel lateral flow assay

PubMed Central

Zhao, Yong; Wang, Haoran; Zhang, Pingping; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Yang, Ruifu; Wang, Chengbin; Zhou, Lei

2016-01-01

The rapid high-throughput detection of foodborne pathogens is essential in controlling food safety. In this study, a 10-channel up-converting phosphor technology-based lateral flow (TC-UPT-LF) assay was established for the rapid and simultaneous detection of 10 epidemic foodborne pathogens. Ten different single-target UPT-LF strips were developed and integrated into one TC-UPT-LF disc with optimization. Without enrichment the TC-UPT-LF assay had a detection sensitivity of 104 CFU mL−1 or 105 CFU mL−1 for each pathogen, and after sample enrichment it was 10 CFU/0.6 mg. The assay also showed good linearity, allowing quantitative detection, with a linear fitting coefficient of determination (R2) of 0.916–0.998. The 10 detection channels did not cross-react, so multiple targets could be specifically detected. When 279 real food samples were tested, the assay was highly consistent (100%) with culture-based methods. The results for 110 food samples artificially contaminated with single or multiple targets showed a high detection rate (≥80%) for most target bacteria. Overall, the TC-UPT-LF assay allows the rapid, quantitative, and simultaneous detection of 10 kinds of foodborne pathogens within 20 min, and is especially suitable for the rapid detection and surveillance of foodborne pathogens in food and water. PMID:26884128

Rapid multiplex detection of 10 foodborne pathogens with an up-converting phosphor technology-based 10-channel lateral flow assay.

PubMed

Zhao, Yong; Wang, Haoran; Zhang, Pingping; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Yang, Ruifu; Wang, Chengbin; Zhou, Lei

2016-02-17

The rapid high-throughput detection of foodborne pathogens is essential in controlling food safety. In this study, a 10-channel up-converting phosphor technology-based lateral flow (TC-UPT-LF) assay was established for the rapid and simultaneous detection of 10 epidemic foodborne pathogens. Ten different single-target UPT-LF strips were developed and integrated into one TC-UPT-LF disc with optimization. Without enrichment the TC-UPT-LF assay had a detection sensitivity of 10(4) CFU mL(-1) or 10(5) CFU mL(-1) for each pathogen, and after sample enrichment it was 10 CFU/0.6 mg. The assay also showed good linearity, allowing quantitative detection, with a linear fitting coefficient of determination (R(2)) of 0.916-0.998. The 10 detection channels did not cross-react, so multiple targets could be specifically detected. When 279 real food samples were tested, the assay was highly consistent (100%) with culture-based methods. The results for 110 food samples artificially contaminated with single or multiple targets showed a high detection rate (≥ 80%) for most target bacteria. Overall, the TC-UPT-LF assay allows the rapid, quantitative, and simultaneous detection of 10 kinds of foodborne pathogens within 20 min, and is especially suitable for the rapid detection and surveillance of foodborne pathogens in food and water.

A comparative study of blood smear, QBC and antigen detection for diagnosis of malaria.

PubMed

Parija, S C; Dhodapkar, Rahul; Elangovan, Subashini; Chaya, D R

2009-01-01

Rapid diagnosis is prerequisite for effective treatment and reducing mortality and morbidity of malaria. This study was taken up to compare the efficacy of various methods available, i.e., thick and thin smear, quantitative buffy coat (QBC), plasmodium lactate dehydrogenase and aldolase in blood of patient. A total of 411 samples were collected from patients presenting with classic symptoms of malaria. For traditional microscopy; thick and thin smears were prepared and stained with Leishman's stain, taking thick smear as gold standard, thin smear had a sensitivity and specificity of 54.8% and 100%, respectively. QBC and antigen detection was done using commercially available kits; out of 411 samples, QBC and Malariagen were positive in 66 and 62 cases, with a sensitivity of 78% and 75%, respectively. Leishman's thick smear, although cost effective, is difficult to interpret for inexperienced microscopists; so if facilities are available, QBC should be used for routine diagnosis. In places where facilities are not available, rapid, simple and easy to interpret antigen detection test can be used despite low sensitivity.

Comparison of two antigen assays for rapid intrapartum detection of vaginal group B streptococcal colonization.

PubMed

Green, M; Dashefsky, B; Wald, E R; Laifer, S; Harger, J; Guthrie, R

1993-01-01

As part of a clinical investigation evaluating the efficacy of intrapartum antigen detection for screening for heavy vaginal colonization with group B streptococci (GBS), we compared the performance of modified Bactigen and Directigen GBS latex particle agglutination (LPA) kits. Paired vaginal swabs obtained from women in labor were rapidly transported to the laboratory and used for culturing (both swabs) and LPA testing (one swab by each method). GBS growth was estimated semiquantitatively and further designated as light or heavy growth. Performance specifications for each method were determined by comparing LPA and culture results from the same swab. A total of 4,251 paired swabs were evaluated during the study period. The performance specifications for detecting GBS growth of any degree for Bactigen and Directigen, respectively, were as follows: sensitivity, 20 and 24%; specificity, 99 and 99%. The performance specifications for heavy colonization for Bactigen and Directigen, respectively, were as follows: sensitivity, 57 and 62%; specificity, 99 and 99%. Neither LPA kit was a sensitive indicator of vaginal colonization with GBS or neonatal infection.

Loop-mediated isothermal amplification (LAMP) method for detection of genetically modified maize T25.

PubMed

Xu, Junyi; Zheng, Qiuyue; Yu, Ling; Liu, Ran; Zhao, Xin; Wang, Gang; Wang, Qinghua; Cao, Jijuan

2013-11-01

The loop-mediated isothermal amplification (LAMP) assay indicates a potential and valuable means for genetically modified organism (GMO) detection especially for its rapidity, simplicity, and low cost. We developed and evaluated the specificity and sensitivity of the LAMP method for rapid detection of the genetically modified (GM) maize T25. A set of six specific primers was successfully designed to recognize six distinct sequences on the target gene, including a pair of inner primers, a pair of outer primers, and a pair of loop primers. The optimum reaction temperature and time were verified to be 65°C and 45 min, respectively. The detection limit of this LAMP assay was 5 g kg(-1) GMO component. Comparative experiments showed that the LAMP assay was a simple, rapid, accurate, and specific method for detecting the GM maize T25.

Rapid detection and differentiation of avian infectious bronchitis virus: an application of Mass genotype by melting temperature analysis in RT-qPCR using SYBR Green I

PubMed Central

OKINO, Cintia Hiromi; MONTASSIER, Maria de Fátima Silva; de OLIVEIRA, Andressa Peres; MONTASSIER, Helio José

2018-01-01

A method based on Melting Temperature analysis of Hypervariable regions (HVR) of S1 gene within a RT-qPCR was developed to detect different genotypes of avian infectious bronchitis virus (IBV) and identify the Mass genotype. The method was able to rapidly identify the Mass genotype among IBV field isolates, vaccine attenuated strains and reference M41 strain in allantoic liquid and also directly in tissues. The RT-qPCR developed detected the virus in both tracheal and pulmonary samples from M41-infected or H120-infected birds, in a larger post-infection period compared to detection by standard method of virus isolation. RT-qPCR method tested provided a sensitivity and rapid approach for screening on IBV detection and Mass genotyping from IBV isolates. PMID:29491226

LAMP assay for rapid diagnosis of cow DNA in goat milk and meat samples.

PubMed

Deb, R; Sengar, G S; Singh, U; Kumar, S; Raja, T V; Alex, R; Alyethodi, R R; Prakash, B

2017-01-01

Animal species detection is one of the crucial steps for consumer's food analysis. In the present study we developed an in-house built loop-mediated isothermal amplification (LAMP) assay for rapid detection of adulterated cow DNA in goat milk/meat samples. The cow milk/tissue DNA in goat milk/meat samples were identified in the developed LAMP assay by either naked eye visualizing with SYBR Green I dyes or by detecting the typical ladder pattern on gel electrophoresis. This test can detect up to minimum 5% level of cow components admixed in goat milk/meat samples and can be completed within 1 h 40 min starting from DNA extraction from milk/meat samples and can be performed in a water bath. Developed LAMP methodology is simple; rapid and sensitive techniques that can detect adulterant like cow components in goat milk/meat are more accurate than other existing DNA based technologies.

Loop-mediated isothermal amplification (LAMP) method for detection of genetically modified maize T25

PubMed Central

Xu, Junyi; Zheng, Qiuyue; Yu, Ling; Liu, Ran; Zhao, Xin; Wang, Gang; Wang, Qinghua; Cao, Jijuan

2013-01-01

The loop-mediated isothermal amplification (LAMP) assay indicates a potential and valuable means for genetically modified organism (GMO) detection especially for its rapidity, simplicity, and low cost. We developed and evaluated the specificity and sensitivity of the LAMP method for rapid detection of the genetically modified (GM) maize T25. A set of six specific primers was successfully designed to recognize six distinct sequences on the target gene, including a pair of inner primers, a pair of outer primers, and a pair of loop primers. The optimum reaction temperature and time were verified to be 65°C and 45 min, respectively. The detection limit of this LAMP assay was 5 g kg−1 GMO component. Comparative experiments showed that the LAMP assay was a simple, rapid, accurate, and specific method for detecting the GM maize T25. PMID:24804053

LAMP assay for rapid diagnosis of cow DNA in goat milk and meat samples

PubMed Central

Deb, R.; Sengar, G. S.; Singh, U.; Kumar, S.; Raja, T. V.; Alex, R.; Alyethodi, R. R.; Prakash, B.

2017-01-01

Animal species detection is one of the crucial steps for consumer’s food analysis. In the present study we developed an in-house built loop-mediated isothermal amplification (LAMP) assay for rapid detection of adulterated cow DNA in goat milk/meat samples. The cow milk/tissue DNA in goat milk/meat samples were identified in the developed LAMP assay by either naked eye visualizing with SYBR Green I dyes or by detecting the typical ladder pattern on gel electrophoresis. This test can detect up to minimum 5% level of cow components admixed in goat milk/meat samples and can be completed within 1 h 40 min starting from DNA extraction from milk/meat samples and can be performed in a water bath. Developed LAMP methodology is simple; rapid and sensitive techniques that can detect adulterant like cow components in goat milk/meat are more accurate than other existing DNA based technologies. PMID:28775755

Recombinase polymerase amplification applied to plant virus detection and potential implications.

PubMed

Babu, Binoy; Ochoa-Corona, Francisco M; Paret, Mathews L

2018-04-01

Several isothermal techniques for the detection of plant pathogens have been developed with the advent of molecular techniques. Among them, Recombinase Polymerase Amplification (RPA) is becoming an important technique for the rapid, sensitive and cost-effective detection of plant viruses. The RPA technology has the advantage to be implemented in field-based scenarios because the method requires a minimal sample preparation, and is performed at constant low temperature (37-42 °C). The RPA technique is rapidly becoming a promising tool for use in rapid detection and further diagnostics in plant clinics and monitoring quarantine services. This paper presents a review of studies conducted using RPA for detection/diagnosis of plant viruses with either DNA genomes (Banana bunchy top virus, Bean golden yellow mosaic virus, Tomato mottle virus, Tomato yellow leaf curl virus) or RNA genomes (Little Cherry virus 2, Plum pox virus and Rose rosette virus). Copyright © 2018 Elsevier Inc. All rights reserved.

Development of a simple and rapid reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for sensitive detection of Citrus tristeza virus.

PubMed

Warghane, Ashish; Misra, Pragati; Bhose, Sumit; Biswas, Kajal Kumar; Sharma, Ashwani Kumar; Reddy, M Krishna; Ghosh, Dilip Kumar

2017-12-01

Tristeza is a devastating disease of citrus and reported to be present in almost all countries where it is cultivated as a commercial crop. The etiological agent of this disease is Citrus tristeza virus (CTV), a member of the genus Closterovirus with in the family Closteroviridae. The pathogen is restricted to the phloem tissue of the infected citrus plant and has a monopartite ss (+) RNA genome of ∼20kb size. Till date, there is no effective control measure available for this virus. Management of tristeza depends on destruction of CTV infected field plants, production of virus-free planting material for new orchard establishment and controlling viruliferous aphid vectors responsible for field spread of the pathogen. Availability of rapid diagnostic assay is essential for rapid and efficient detection of the pathogen. In the present investigation, RT-LAMP (reverse transcription-loop mediated isothermal amplification), a highly sensitive, robust and low cost assay has been developed for rapid detection of CTV in infected citrus plant samples. Based on conserved nucleotide sequences available in GenBank and specific to p25 gene (major coat protein gene) of predominant CTV isolates of India, four primer sets (CTV-F3, CTV-B3, CTV-FIP and CTV-BIP) ware designed and custom synthesized. The amplified LAMP products obtained after maintaining isothermal condition of 65°C for 60min duration could be visible easily with necked eyes in presence of SYBR Green I (100X). Subsequently, LAMP products were verified by electrophoresis run in 1.5% agarose gel. The RT-LAMP results obtained with known CTV isolates maintained in screen house of CCRI, Nagpur were validated using field samples and thereafter it was further confirmed by conventional RT-PCR (reveres transcription-polymerase chain reaction) assay. The sensitivity of CTV-RT-LAMP protocol standardized in the present study was 100 times more than conventional one step RT-PCR assay. It also has maximum detection limit up to 0.0001ng RNA in individual reaction mixture. CTV-RT-LAMP assay is a simple, sensitive, rapid and less costly detection technique. This assay could be used for CTV diagnosis in pathology laboratories having limited facility and resources and even by citrus nurseries situated in remote locations. As per our knowledge and available literature, the present study reports first time about the usefulness of RT-LAMP assay for detection of CTV from India. Copyright © 2017 Elsevier B.V. All rights reserved.

AMPEROMETRIC THICK-FILM STRIP ELECTRODES FOR MONITORING ORGANOPHOSPHATE NERVE AGENTS BASED ON IMMOBILIZED ORGANOPHOSPHORUS HYDROLASE. (R823663)

EPA Science Inventory

An amperometric biosensor based on the immobilization of organophosphorus hydrolase
(OPH) onto screen-printed carbon electrodes is shown useful for the rapid, sensitive, and low-cost
detection of organophosphate (OP) nerve agents. The sensor relies upon the sensitive and ra...

Irradiation influence on the detection of genetic-modified soybeans

NASA Astrophysics Data System (ADS)

Villavicencio, A. L. C. H.; Araújo, M. M.; Baldasso, J. G.; Aquino, S.; Konietzny, U.; Greiner, R.

2004-09-01

Three soybean varieties were analyzed to evaluate the irradiation influence on the detection of genetic modification. Samples were treated in a 60Co facility at dose levels of 0, 500, 800, and 1000Gy. The seeds were at first analyzed by Comet Assay as a rapid screening irradiation detection method. Secondly, germination test was performed to detect the viability of irradiated soybeans. Finally, because of its high sensitivity, its specificity and rapidity the polimerase chain reaction was the method applied for genetic modified organism detection. The analysis of DNA by the single technique of microgel electrophoresis of single cells (DNA Comet Assay) showed that DNA damage increased with increasing radiation doses. No negative influence of irradiation on the genetic modification detection was found.

Colorimetry and SERS dual-mode detection of telomerase activity: combining rapid screening with high sensitivity.

PubMed

Zong, Shenfei; Wang, Zhuyuan; Chen, Hui; Hu, Guohua; Liu, Min; Chen, Peng; Cui, Yiping

2014-01-01

As an important biomarker and therapeutic target, telomerase has attracted considerable attention concerning its detection and monitoring. Here, we present a colorimetry and surface enhanced Raman scattering (SERS) dual-mode telomerase activity detection method, which has several distinctive advantages. First, colorimetric functionality allows rapid preliminary discrimination of telomerase activity by the naked eye. Second, the employment of SERS technique results in greatly improved detection sensitivity. Third, the combination of colorimetry and SERS into one detection system can ensure highly efficacious and sensitive screening of numerous samples. Besides, the avoidance of polymerase chain reaction (PCR) procedures further guarantees fine reliability and simplicity. Generally, the presented method is realized by an "elongate and capture" procedure. To be specific, gold nanoparticles modified with Raman molecules and telomeric repeat complementary oligonucleotide are employed as the colorimetric-SERS bifunctional reporting nanotag, while magnetic nanoparticles functionalized with telomerase substrate oligonucleotide are used as the capturing substrate. Telomerase can synthesize and elongate telomeric repeats onto the capturing substrate. The elongated telomeric repeats subsequently facilitate capturing of the reporting nanotag via hybridization between telomeric repeat and its complementary strand. The captured nanotags can cause a significant difference in the color and SERS intensity of the magnetically separated sediments. Thus both the color and SERS can be used as indicators of the telomerase activity. With fast screening ability and outstanding sensitivity, we anticipate that this method would greatly promote practical application of telomerase-based early-stage cancer diagnosis.

Detection of gonococcal infection : pros and cons of a rapid test.

PubMed

Vickerman, Peter; Peeling, Rosanna W; Watts, Charlotte; Mabey, David

2005-01-01

WHO estimates that 62 million cases of gonorrhea occur annually worldwide. Untreated infection can cause serious long-term complications, especially in women. In addition, Neisseria gonorrheae infection can facilitate HIV transmission, and babies born to infected mothers are at risk of ocular infection, which can lead to blindness. Where diagnostic facilities are lacking, gonorrhea can be treated syndromically. However, this inevitably leads to over-treatment, especially in women in whom the syndrome of vaginal discharge may be due not to N. gonorrheae infection but to several other more prevalent conditions. Over-treatment is a major concern because of widespread N. gonorrheae antibiotic resistance. Moreover, a high proportion of gonorrhea cases are asymptomatic and so do not present for syndromic management. Such cases will only be detected by screening tests. The gold standard test for the detection of N. gonorrheae is culture, which has high sensitivity and specificity. However, it requires well trained staff and its performance is affected by specimen transport conditions. Other options include microscopy and tests that detect gonococcal antigen or nucleic acid. Nucleic acid amplification tests (NAATs) have higher sensitivity and can be used on non-invasive samples (urine). However, they can cross-react with other Neisseria species and are expensive, requiring highly trained staff and sophisticated equipment. In settings where patients are asked to return for laboratory results, some infected patients never receive treatment as they fail to return for their test results. This reduction in treatment, and the possible onward transmission of N. gonorrheae during any delay in treatment, means that a rapid test of lower sensitivity may be more effective if it results in patients being treated at the initial visit. Indeed, even with the low sensitivity of currently available rapid tests (50-70%), modeling shows that they can outperform gold standard tests in populations with high sexual activity and/or low return rates. Unfortunately, however, most of the rapid tests currently available are immunoassays that are quite expensive and involve many steps, which limit their current usefulness. In summary, the pros and cons of using a rapid test are dependent on the setting. Culture or NAATs remain the best choice in an ideal setting. However, in settings where laboratory facilities are not available, or in high-risk populations where return rates are low, rapid tests may be the most effective way of diagnosing gonorrhea. Their optimal use in these settings requires the development of simpler and cheaper rapid tests.

A multiplex immunochromatographic test using gold nanoparticles for the rapid and simultaneous detection of four nitrofuran metabolites in fish samples.

PubMed

Wang, Quan; Liu, Yingchun; Wang, Mingyan; Chen, Yongjun; Jiang, Wei

2018-01-01

There is an urgent need for the rapid and simultaneous detection of multiple analytes present in a sample matrix. Here, a multiplex immunochromatographic test (multi-ICT) was developed that successfully allowed for the rapid and simultaneous detection of four major nitrofuran metabolites, i.e., 3-amino-2-oxazolidinone (AOZ), semicarbazide (SEM), 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), and 1-aminohydantoin (AHD), in fish samples. Four different antigens were separately immobilized in four test lines on a nitrocellulose membrane. Goat anti-mouse immunoglobulin (IgG) was used as a control. Sensitive and specific monoclonal antibodies (mAbs) that recognize the corresponding antigens were selected for the assay, and no cross-reactivity between the antibodies in the detection assay was observed. The free analytes in samples or standards were pre-incubated with freeze-dried mAb-gold conjugates to improve the sensitivity of the detection assay. The multi-ICT detection was accomplished in less than 15 min by the naked eye. The cutoff values for the strip test were 0.5 ng/mL for AOZ and 0.75 ng/mL for AHD, SEM, and AMOZ, which were all below the maximum residue levels set by the European Union and China. A high degree of consistency was observed between the multi-ICT method and commercially available enzyme-linked immunosorbent assay (ELISA) kits using spiked, incurred, and "blind" fish samples, indicating the accuracy, reproducibility, and reliability of the novel test strip. This newly developed multi-ICT strip assay is suitable for the rapid and high-throughput screening of four nitrofuran metabolites in fish samples on-site, with no treatment or devices required. Graphical abstract A multiplex immunochromatographic test (multi-ICT) was developed that successfully allowed for the rapid and simultaneous detection of four major nitrofuran metabolites (AOZ, SEM, AMOZ, and AHD) in fish samples.

Evaluation of two line probe assays for rapid detection of Mycobacterium tuberculosis, tuberculosis (TB) drug resistance, and non-TB Mycobacteria in HIV-infected individuals with suspected TB.

PubMed

Luetkemeyer, Anne F; Kendall, Michelle A; Wu, Xingye; Lourenço, Maria Cristina; Jentsch, Ute; Swindells, Susan; Qasba, Sarojini S; Sanchez, Jorge; Havlir, Diane V; Grinsztejn, Beatriz; Sanne, Ian M; Firnhaber, Cynthia

2014-04-01

Limited performance data from line probe assays (LPAs), nucleic acid tests used for the rapid diagnosis of tuberculosis (TB), nontuberculosis mycobacteria (NTM), and Mycobacterium tuberculosis drug resistance are available for HIV-infected individuals, in whom paucibacillary TB is common. In this study, the strategy of testing sputum with GenoType MTBDRplus (MTBDR-Plus) and GenoType Direct LPA (Direct LPA) was compared to a gold standard of one mycobacterial growth indicator tube (MGIT) liquid culture. HIV-positive (HIV(+)) individuals with suspected TB from southern Africa and South America with <7 days of TB treatment had 1 sputum specimen tested with Direct LPA, MTBDR-Plus LPA, smear microscopy, MGIT, biochemical identification of mycobacterial species, and culture-based drug-susceptibility testing (DST). Of 639 participants, 59.3% were MGIT M. tuberculosis culture positive, of which 276 (72.8%) were acid-fast bacillus (AFB) smear positive. MTBDR-Plus had a sensitivity of 81.0% and a specificity of 100%, with sensitivities of 44.1% in AFB smear-negative versus 94.6% in AFB smear-positive specimens. For specimens that were positive for M. tuberculosis by MTBDR-Plus, the sensitivity and specificity for rifampin resistance were 91.7% and 96.6%, respectively, and for isoniazid (INH) they were 70.6% and 99.1%. The Direct LPA had a sensitivity of 88.4% and a specificity of 94.6% for M. tuberculosis detection, with a sensitivity of 72.5% in smear-negative specimens. Ten of 639 MGIT cultures grew Mycobacterium avium complex or Mycobacterium kansasii, half of which were detected by Direct LPA. Both LPA assays performed well in specimens from HIV-infected individuals, including in AFB smear-negative specimens, with 72.5% sensitivity for M. tuberculosis identification with the Direct LPA and 44.1% sensitivity with MTBDR-Plus. LPAs have a continued role for use in settings where rapid identification of INH resistance and clinically relevant NTM are priorities.

Highly sensitive oligothiophene-phenylamine-based dual-functional fluorescence "turn-on" sensor for rapid and simultaneous detection of Al3+ and Fe3+ in environment and food samples.

PubMed

Guo, Zongrang; Niu, Qingfen; Li, Tianduo

2018-07-05

Developing low-cost and efficient sensors for rapid, selective and sensitive detection of the transition metal ions in environmental and food science is very important. In this study, a novel dual-functional fluorescent "turn-on" sensor 3TP based on oligothiophene-phenylamine Schiff base has been synthesized for discrimination and simultaneous detection of both Al 3+ and Fe 3+ ions with high selectivity and anti-interference over other metal ions. Sensor 3TP displayed a very fast fluorescence-enhanced response towards Al 3+ and Fe 3+ ions with low detection limits (0.177μM for Al 3+ and 0.172μM for Fe 3+ ) and wide pH response range (4.0-12.0). The Al 3+ /Fe 3+ sensing mechanisms were investigated by fluorescence experiments, 1 H NMR titrations, FT-IR and ESI-MS spectra. Importantly, sensor 3TP was served as an efficient solid material for the highly sensitive and selective detection of Fe 3+ on TLC plates. Moreover, the sensor 3TP has been successfully used to detect trace Al 3+ and Fe 3+ in environment and food samples with satisfactory results and good recoveries, revealing a convenient, reliable and accurate method for Al 3+ and Fe 3+ analysis in real samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Flexible, transparent and highly sensitive SERS substrates with cross-nanoporous structures for fast on-site detection.

PubMed

Wang, Yingcheng; Jin, Yuanhao; Xiao, Xiaoyang; Zhang, Tianfu; Yang, Haitao; Zhao, Yudan; Wang, Jiaping; Jiang, Kaili; Fan, Shoushan; Li, Qunqing

2018-05-30

A flexible and transparent film assembled from the cross-nanoporous structures of Au on PET (CNS of Au@PET) is developed as a versatile and effective SERS substrate for rapid, on-site trace analysis with high sensitivity. The fabrication of the CNS of Au can be achieved on a large scale at low cost by employing an etching process with super-aligned carbon nanotubes as a mask, followed by metal deposition. A strongly enhanced Raman signal with good uniformity can be obtained, which is attributed to the excitation of "hot spots" around the metal nanogaps and sharp edges. Using the CNS of Au@PET film as a SERS platform, real-time and on-site SERS detection of the food contaminant crystal violet (CV) is achieved, with a detection limit of CV solution on a tomato skin of 10-7 M. Owing to its ability to efficiently extract trace analytes, the resulting substrate also achieves detection of 4-ATP contaminants and thiram pesticides by swabbing the skin of an apple. A SERS detection signal for 4-ATP has a relative standard deviation of less than 10%, revealing the excellent reproducibility of the substrate. The flexible, transparent and highly sensitive substrates fabricated using this simple and cost-effective strategy are promising for practical application in rapid, on-site SERS-based detection.

Gold nanoparticle-based enzyme-linked antibody-aptamer sandwich assay for detection of Salmonella Typhimurium.

PubMed

Wu, Wenhe; Li, Jun; Pan, Dun; Li, Jiang; Song, Shiping; Rong, Mingge; Li, Zixi; Gao, Jimin; Lu, Jianxin

2014-10-08

Enzyme-linked immunosorbent assay (ELISA) provides a convenient means for the detection of Salmonella enterica serovar Typhimurium (STM), which is important for rapid diagnosis of foodborne pathogens. However, conventional ELISA is limited by antibody-antigen immunoreactions and suffers from poor sensitivity and tedious sample pretreatment. Therefore, development of novel ELISA remains challenging. Herein, we designed a comprehensive strategy for rapid, sensitive, and quantitative detection of STM with high specificity by gold nanoparticle-based enzyme-linked antibody-aptamer sandwich (nano-ELAAS) method. STM was captured and preconcentrated from samples with aptamer-modified magnetic particles, followed by binding with detector antibodies. Then nanoprobes carrying a large amount of reporter antibodies and horseradish peroxidase molecules were used for colorimetric signal amplification. Under the optimized reaction conditions, the nano-ELAAS assay had a quantitative detection range from 1 × 10(3) to 1 × 10(8) CFU mL(-1), a limit of detection of 1 × 10(3) CFU mL(-1), and a selectivity of >10-fold for STM in samples containing other bacteria at higher concentration with an assay time less than 3 h. In addition, the developed nanoprobes were improved in terms of detection range and/or sensitivity when compared with two commercial enzyme-labeled antibody signal reporters. Finally, the nano-ELAAS method was demonstrated to work well in milk samples, a common source of STM contamination.

Highly sensitive oligothiophene-phenylamine-based dual-functional fluorescence "turn-on" sensor for rapid and simultaneous detection of Al3+ and Fe3+ in environment and food samples

NASA Astrophysics Data System (ADS)

Guo, Zongrang; Niu, Qingfen; Li, Tianduo

2018-07-01

Developing low-cost and efficient sensors for rapid, selective and sensitive detection of the transition metal ions in environmental and food science is very important. In this study, a novel dual-functional fluorescent "turn-on" sensor 3TP based on oligothiophene-phenylamine Schiff base has been synthesized for discrimination and simultaneous detection of both Al3+ and Fe3+ ions with high selectivity and anti-interference over other metal ions. Sensor 3TP displayed a very fast fluorescence-enhanced response towards Al3+ and Fe3+ ions with low detection limits (0.177 μM for Al3+ and 0.172 μM for Fe3+) and wide pH response range (4.0-12.0). The Al3+/Fe3+ sensing mechanisms were investigated by fluorescence experiments, 1H NMR titrations, FT-IR and ESI-MS spectra. Importantly, sensor 3TP was served as an efficient solid material for the highly sensitive and selective detection of Fe3+ on TLC plates. Moreover, the sensor 3TP has been successfully used to detect trace Al3+ and Fe3+ in environment and food samples with satisfactory results and good recoveries, revealing a convenient, reliable and accurate method for Al3+ and Fe3+ analysis in real samples.

Detecting and Genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Call, Douglas R.; Brockman, Fred J.; Chandler, Darrell P.

2000-12-01

Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification.more » The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.« less

Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Call, Douglas R.; Brockman, Fred J.; Chandler, Darrell P.

2001-07-05

Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification.more » The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFUs ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.« less

Rapid detection of Cu(2+) by a paper-based microfluidic device coated with bovine serum albumin (BSA)-Au nanoclusters.

PubMed

Fang, Xueen; Zhao, Qianqian; Cao, Hongmei; Liu, Juan; Guan, Ming; Kong, Jilie

2015-11-21

In this work, bovine serum albumin (BSA)-Au nanoclusters were used to coat a paper-based microfluidic device. This device acted as a Cu(2+) biosensor that showed fluorescence quenching on detection of copper ions. The detection limit of this sensor could be adjusted by altering the water absorbing capacity of the device. Qualitative and semi-quantitative results could be obtained visually without the aid of any advanced instruments. This sensor could test Cu(2+) rapidly with high specificity and sensitivity, which would be useful for point-of-care testing (POCT).

Highly Sensitive, Label-Free Detection of 2,4-Dichlorophenoxyacetic Acid Using an Optofluidic Chip.

PubMed

Feng, Xueling; Zhang, Gong; Chin, Lip Ket; Liu, Ai Qun; Liedberg, Bo

2017-07-28

A highly sensitive approach for rapid and label-free detection of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) using an optofluidic chip is demonstrated. The optofluidic chip is prepared by covalent immobilization of 2,4-D-bovine serum albumin (2,4-D-BSA) conjugate to an integrated microring resonator. Subsequent detection of 2,4-D carried out in a competitive immunoreaction format enables selective detection of 2,4-D in different types of water samples, including bottled, tap, and lake water, at a limit of detection (LOD) of 4.5 pg/mL and in a quantitative range of 15-10 5 pg/mL. The microring resonator-based optofluidic chip is reusable with ultrahigh sensitivity that offers real-time and on-site detection of low-molecular-weight targets for potential applications in food safety and environmental monitoring.

Rapid quantification of viable Legionella in nuclear cooling tower waters using filter cultivation, fluorescent in situ hybridization and solid-phase cytometry.

PubMed

Baudart, J; Guillaume, C; Mercier, A; Lebaron, P; Binet, M

2015-05-01

To develop a rapid and sensitive method to quantify viable Legionella spp. in cooling tower water samples. A rapid, culture-based method capable of quantifying as few as 600 Legionella microcolonies per litre within 2 days in industrial waters was developed. The method combines a short cultivation step of microcolonies on GVPC agar plate, specific detection of Legionella cells by a fluorescent in situ hybridization (FISH) approach, and a sensitive enumeration using a solid-phase cytometer. Following optimization of the cultivation conditions, the qualitative and quantitative performance of the method was assessed and the method was applied to 262 nuclear power plant cooling water samples. The performance of this method was in accordance with the culture method (NF-T 90-431) for Legionella enumeration. The rapid detection of viable Legionella in water is a major concern to the effective monitoring of this pathogenic bacterium in the main water sources involved in the transmission of legionellosis infection (Legionnaires' disease). The new method proposed here appears to be a robust, efficient and innovative means for rapidly quantifying cultivable Legionella in cooling tower water samples within 48 h. © 2015 The Society for Applied Microbiology.

Comparison of Xpert Flu rapid nucleic acid testing with rapid antigen testing for the diagnosis of influenza A and B.

PubMed

DiMaio, Michael A; Sahoo, Malaya K; Waggoner, Jesse; Pinsky, Benjamin A

2012-12-01

Influenza infections are associated with thousands of hospital admissions and deaths each year. Rapid detection of influenza is important for prompt initiation of antiviral therapy and appropriate patient triage. In this study the Cepheid Xpert Flu assay was compared with two rapid antigen tests, BinaxNOW Influenza A & B and BD Directigen EZ Flu A+B, as well as direct fluorescent antibody testing for the rapid detection of influenza A and B. Using real-time, hydrolysis probe-based, reverse transcriptase PCR as the reference method, influenza A sensitivity was 97.3% for Xpert Flu, 95.9% for direct fluorescent antibody testing, 62.2% for BinaxNOW, and 71.6% for BD Directigen. Influenza B sensitivity was 100% for Xpert Flu and direct fluorescent antibody testing, 54.5% for BinaxNOW, and 48.5% for BD Directigen. Specificity for influenza A was 100% for Xpert Flu, BinaxNOW, and BD Directigen, and 99.2% for direct fluorescent antibody testing. All methods demonstrated 100% specificity for influenza B. These findings support the use of the Xpert Flu assay in settings requiring urgent diagnosis of influenza A and B. Copyright © 2012 Elsevier B.V. All rights reserved.

Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the malachite green decolourisation assay

PubMed Central

Coban, Ahmet Yilmaz; Uzun, Meltem

2013-01-01

Early detection of drug resistance in Mycobacterium tuberculosis isolates allows for earlier and more effective treatment of patients. The aim of this study was to investigate the performance of the malachite green decolourisation assay (MGDA) in detecting isoniazid (INH) and rifampicin (RIF) resistance in M. tuberculosis clinical isolates. Fifty M. tuberculosis isolates, including 19 multidrug-resistant, eight INH-resistant and 23 INH and RIF-susceptible samples, were tested. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and agreement of the assay for INH were 92.5%, 91.3%, 92.5%, 91.3% and 92%, respectively. Similarly, the sensitivity, specificity, PPV, NPV and agreement of the assay for RIF were 94.7%, 100%, 100%, 96.8% and 98%, respectively. There was a major discrepancy in the tests of two isolates, as they were sensitive to INH by the MGDA test, but resistant by the reference method. There was a minor discrepancy in the tests of two additional isolates, as they were sensitive to INH by the reference method, but resistant by the MGDA test. The drug susceptibility test results were obtained within eight-nine days. In conclusion, the MGDA test is a reliable and accurate method for the rapid detection of INH and RIF resistance compared with the reference method and the MGDA test additionally requires less time to obtain results. PMID:24402143

Rapid and ratiometric detection of hypochlorite with real application in tap water: molecules to low cost devices (TLC sticks).

PubMed

Goswami, Shyamaprosad; Manna, Abhishek; Paul, Sima; Quah, Ching Kheng; Fun, Hoong-Kun

2013-12-25

We have designed a chemodosimeter DPNO (weak fluorescence) which can be oxidized to HPNO (strong blue fluorescence) by OCl(-) with high selectivity and sensitivity in a ratiometric approach with a noticeably lower detection limit. The sensor could be useful for the detection of hypochlorites in tap water.

Bacteriophage-based rapid and sensitive detection of Escherichia coli O157:H7 isolates from ground beef

USDA-ARS?s Scientific Manuscript database

We investigated efficacy of bacteriophage-based detection technology to detect Escherichia coli O157:H7 from ground beef. The assay involved 8 h enrichment of cold stressed beef samples in presence of antimicrobials followed by capture of the pathogen on O157:H7-specific immunomagnetic beads and sp...

Privileged Detection of Conspecifics: Evidence from Inversion Effects during Continuous Flash Suppression

ERIC Educational Resources Information Center

Stein, Timo; Sterzer, Philipp; Peelen, Marius V.

2012-01-01

The rapid visual detection of other people in our environment is an important first step in social cognition. Here we provide evidence for selective sensitivity of the human visual system to upright depictions of conspecifics. In a series of seven experiments, we assessed the impact of stimulus inversion on the detection of person silhouettes,…

Field-Deployable Reverse Transcription-Insulated Isothermal PCR (RT-iiPCR) Assay for Rapid and Sensitive Detection of Foot-and-Mouth Disease Virus.

PubMed

Ambagala, A; Fisher, M; Goolia, M; Nfon, C; Furukawa-Stoffer, T; Ortega Polo, R; Lung, O

2017-10-01

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals, which can decimate the livestock industry and economy of countries previously free of this disease. Rapid detection of foot-and-mouth disease virus (FMDV) is critical to containing an FMD outbreak. Availability of a rapid, highly sensitive and specific, yet simple and field-deployable assay would support local decision-making during an FMDV outbreak. Here we report validation of a novel reverse transcription-insulated isothermal PCR (RT-iiPCR) assay that can be performed on a commercially available, compact and portable POCKIT ™ analyser that automatically analyses data and displays '+' or '-' results. The FMDV RT-iiPCR assay targets the 3D region of the FMDV genome and was capable of detecting 9 copies of in vitro-transcribed RNA standard with 95% confidence. It accurately identified 63 FMDV strains belonging to all seven serotypes and showed no cross-reactivity with viruses causing similar clinical diseases in cloven-hoofed animals. The assay was able to identify FMDV RNA in multiple sample types including oral, nasal and lesion swabs, epithelial tissue suspensions, vesicular and oral fluid samples, even before the appearance of clinical signs. Clinical sensitivity of the assay was comparable or slightly higher than the laboratory-based real-time RT-PCR assay in use. The assay was able to detect FMDV RNA in vesicular fluid samples without nucleic acid extraction. For RNA extraction from more complex sample types, a commercially available taco ™ mini transportable magnetic bead-based, automated extraction system was used. This assay provides a potentially useful field-deployable diagnostic tool for rapid detection of FMDV in an outbreak in FMD-free countries or for routine diagnostics in endemic countries with less structured laboratory systems. © 2016 Her Majesty the Queen in Right of Canada.

A real-time reverse transcription loop-mediated isothermal amplification assay for the rapid detection of yellow fever virus.

PubMed

Kwallah, Allan ole; Inoue, Shingo; Muigai, Anne W T; Kubo, Toru; Sang, Rosemary; Morita, Kouichi; Mwau, Matilu

2013-10-01

Yellow fever, a mosquito-borne disease, is an important viral hemorrhagic fever in Africa and South America where it is endemic. Detection of yellow fever virus (YFV) in Africa remains a challenge due to a lack of highly specific tests. The aim of this study was to develop and optimize a rapid detection reverse transcription loop-mediated isothermal amplification (RT-LAMP) for YFV. The RT-LAMP was done isothermally at 62 °C using a real-time turbidimeter that allowed detection within 1h. Specificity of the RT-LAMP was determined using RNA from flaviviruses and other related viruses where only YFV RNA was detected: West Nile virus, dengue viruses, Japanese encephalitis virus, Rift Valley fever virus, and chikungunya virus. In addition, equal sensitivity was also observed when the RT-LAMP and the real-time RT-PCR were compared using YFV-spiked human serum samples with a detection limit of 0.29 PFU/ml. Two Kenyan YFV wild strains showed an equal detection limit as the vaccine strain 17D in this study. The RT-LAMP reduced the time of reaction from 3h to 1h and increased sensitivity tenfold compared to RT-PCR. Therefore, this test offers a simple, rapid and reliable diagnostic tool for yellow fever when there are outbreaks of acute hemorrhagic fever in Kenya and other African countries. Copyright © 2013 Elsevier B.V. All rights reserved.

Colorimetric Detection of Small Molecules in Complex Matrixes via Target-Mediated Growth of Aptamer-Functionalized Gold Nanoparticles.

PubMed

Soh, Jun Hui; Lin, Yiyang; Rana, Subinoy; Ying, Jackie Y; Stevens, Molly M

2015-08-04

A versatile and sensitive colorimetric assay that allows the rapid detection of small-molecule targets using the naked eye is demonstrated. The working principle of the assay integrates aptamer-target recognition and the aptamer-controlled growth of gold nanoparticles (Au NPs). Aptamer-target interactions modulate the amount of aptamer strands adsorbed on the surface of aptamer-functionalized Au NPs via desorption of the aptamer strands when target molecules bind with the aptamer. Depending on the resulting aptamer coverage, Au NPs grow into morphologically varied nanostructures, which give rise to different colored solutions. Au NPs with low aptamer coverage grow into spherical NPs, which produce red-colored solutions, whereas Au NPs with high aptamer coverage grow into branched NPs, which produce blue-colored solutions. We achieved visible colorimetric response and nanomolar detection limits for the detection of ochratoxin A (1 nM) in red wine samples, as well as cocaine (1 nM) and 17β-estradiol (0.2 nM) in spiked synthetic urine and saliva, respectively. The detection limits were well within clinically and physiologically relevant ranges, and below the maximum food safety limits. The assay is highly sensitive, specific, and able to detect an array of analytes rapidly without requiring sophisticated equipment, making it relevant for many applications, such as high-throughput drug and clinical screening, food sampling, and diagnostics. Furthermore, the assay is easily adapted as a chip-based platform for rapid and portable target detection.

Multifunctional Nanotechnology-Enabled Sensors for Rapid Capture and Detection of Pathogens.

PubMed

Mustafa, Fatima; Hassan, Rabeay Y A; Andreescu, Silvana

2017-09-15

Nanomaterial-based sensing approaches that incorporate different types of nanoparticles (NPs) and nanostructures in conjunction with natural or synthetic receptors as molecular recognition elements provide opportunities for the design of sensitive and selective assays for rapid detection of contaminants. This review summarizes recent advancements over the past ten years in the development of nanotechnology-enabled sensors and systems for capture and detection of pathogens. The most common types of nanostructures and NPs, their modification with receptor molecules and integration to produce viable sensing systems with biorecognition, amplification and signal readout are discussed. Examples of all-in-one systems that combine multifunctional properties for capture, separation, inactivation and detection are also provided. Current trends in the development of low-cost instrumentation for rapid assessment of food contamination are discussed as well as challenges for practical implementation and directions for future research.

Rapid and sensitive detection of Curvularia lunata associated with maize leaf spot based on its Clg2p gene using semi-nested PCR.

PubMed

Hou, J M; Ma, B C; Zuo, Y H; Guo, L L; Gao, S G; Wang, Y Y; Liu, T

2013-04-01

Curvularia lunata (Wakker) Boed, the causative agent of Curvularia leaf spot in maize, was determined according to conidiophore and conidium morphology in a previous study. In the current study, a sensitive polymerase chain reaction assay was developed for the detection of C. lunata. Two specific forward (ClgD1/ClgD2) and one reverse primers (ClgD3) were designed based on a Ras-related (Clg2p) gene. Eight C. lunata isolates that represent different virulent strains in maize, six other Curvularia spp., and 22 fungal plant pathogens were used to test the specificity of the primers. PCR amplification using ClgD1/ClgD3 as the first-round primers resulted in an 870-bp band from the C. lunata isolates. The detection sensitivity using ClgD1/ClgD3 was 100 pg of genomic DNA. In the second round of PCR, a 1 : 50 dilution of the first-round PCR products was used as a template with the ClgD2/ClgD3 primer pair, which increased the detection sensitivity to 1 fg. This semi-nested PCR procedure could also be used to detect C. lunata from infected maize leaves. The proposed PCR-based assay may be used for diagnosing and monitoring maize Curvularia leaf spot. The semi-nested PCR assay may provide researchers and laboratory technologists a tool to rapidly detect C. lunata, which causes maize Curvularia leaf spot, compared with histological examination. © 2012 The Society for Applied Microbiology.

Rapid and Sensitive Detection of sFAT-1 Transgenic Pigs by Visual Loop-Mediated Isothermal Amplification.

PubMed

Tao, Chenyu; Yang, Yalan; Li, Xunbi; Zheng, Xinmin; Ren, Hongyan; Li, Kui; Zhou, Rong

2016-07-01

Genetically modified (GM) livestock have the potential to contribute to improving the environment and human health, with consumption of fewer resources and reduced waste production. However, the transgene process also poses risks. The safety assessment and control of transgenic animal products have drawn wide attention, and the relevant regulations and technology are being developed. Quick testing technology plays a significant role in on-site and customs sampling. Nowadays, loop-mediated isothermal amplification (LAMP) was widely applied in nucleic acid analysis because of its simplicity, rapidity, high efficiency and specificity. In this study, a specific, sensitive detection system for detecting sFAT-1 transgenic pigs was designed. A set of six primers including two loop primers was designed for the target sequence. The DNA samples were amplified in less than 1 h at the optimized temperature and detecting by both Nephelometer LA-320c and unaided eyes directly adding calcein. The detection limit of sFAT-1 LAMP was as low as 1.26 ng/μL. Furthermore, blind tests of transgenic and non-transgenic DNA samples were all correctly detected. Hence, the results in this study demonstrated that LAMP is a very useful tool for transgenic detection.

Surface grafted antibodies: controlled architecture permits enhanced antigen detection.

PubMed

Sebra, Robert P; Masters, Kristyn S; Bowman, Christopher N; Anseth, Kristi S

2005-11-22

The attachment of antibodies to substrate surfaces is useful for achieving specific detection of antigens and toxins associated with clinical and field diagnostics. Here, acrylated whole antibodies were produced through conjugation chemistry, with the goal of covalently photografting these proteins from surfaces in a controlled fashion, to facilitate rapid and sensitive antigenic detection. A living radical photopolymerization chemistry was used to graft the acrylated whole antibodies on polymer surfaces at controlled densities and spatial locations by controlling the exposure time and area, respectively. Copolymer grafts containing these antibodies were synthesized to demonstrate two principles. First, PEG functionalities were introduced to prevent nonspecific protein interactions and improve the reaction kinetics by increasing solvation and mobility of the antibody-containing chains. Both of these properties lead to sensitive (pM) and rapid (<20 min) detection of antigens with this surface modification technique. Second, graft composition was tailored to include multiple antibodies on the same grafted chains, establishing a means for simultaneously detecting multiple antigens on one grafted surface area. Finally, the addition of PEG spacers between the acrylate functionality and the pendant detection antibodies was tuned to enhance the detection of a short-half-life molecule, glucagon, in a complex biological environment, plasma.

Magnetic particles for in vitro molecular diagnosis: From sample preparation to integration into microsystems.

PubMed

Tangchaikeeree, Tienrat; Polpanich, Duangporn; Elaissari, Abdelhamid; Jangpatarapongsa, Kulachart

2017-10-01

Colloidal magnetic particles (MPs) have been developed in association with molecular diagnosis for several decades. MPs have the great advantage of easy manipulation using a magnet. In nucleic acid detection, these particles can act as a capture support for rapid and simple biomolecule separation. The surfaces of MPs can be modified by coating with various polymer materials to provide functionalization for different applications. The use of MPs enhances the sensitivity and specificity of detection due to the specific activity on the surface of the particles. Practical applications of MPs demonstrate greater efficiency than conventional methods. Beyond traditional detection, MPs have been successfully adopted as a smart carrier in microfluidic and lab-on-a-chip biosensors. The versatility of MPs has enabled their integration into small single detection units. MPs-based biosensors can facilitate rapid and highly sensitive detection of very small amounts of a sample. In this review, the application of MPs to the detection of nucleic acids, from sample preparation to analytical readout systems, is described. State-of-the-art integrated microsystems containing microfluidic and lab-on-a-chip biosensors for the nucleic acid detection are also addressed. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of Rapid Detection and Genetic Characterization of Salmonella in Poultry Breeder Feeds

PubMed Central

Jarquin, Robin; Hanning, Irene; Ahn, Soohyoun; Ricke, Steven C.

2009-01-01

Salmonella is a leading cause of foodborne illness in the United States, with poultry and poultry products being a primary source of infection to humans. Poultry may carry some Salmonella serovars without any signs or symptoms of disease and without causing any adverse effects to the health of the bird. Salmonella may be introduced to a flock by multiple environmental sources, but poultry feed is suspected to be a leading source. Detecting Salmonella in feed can be challenging because low levels of the bacteria may not be recovered using traditional culturing techniques. Numerous detection methodologies have been examined over the years for quantifying Salmonella in feeds and many have proven to be effective for Salmonella isolation and detection in a variety of feeds. However, given the potential need for increased detection sensitivity, molecular detection technologies may the best candidate for developing rapid sensitive methods for identifying small numbers of Salmonella in the background of large volumes of feed. Several studies have been done using polymerase chain reaction (PCR) assays and commercial kits to detect Salmonella spp. in a wide variety of feed sources. In addition, DNA array technology has recently been utilized to track the dissemination of a specific Salmonella serotype in feed mills. This review will discuss the processing of feeds and potential points in the process that may introduce Salmonella contamination to the feed. Detection methods currently used and the need for advances in these methods also will be discussed. Finally, implementation of rapid detection for optimizing control methods to prevent and remove any Salmonella contamination of feeds will be considered. PMID:22346699

RAPID DETECTION OF ALGAL TOXINS - PHASE I

EPA Science Inventory

The proposed program will demonstrate the ability of a detector based on measuring the conductance of nicotinic acetylcholine receptor (nAChR) ion channels to detect and quantify anatoxin-a contamination in drinking water systems.  The nAChR is extremely sensitive to the p...

Rapid Detection of Nivalenol and Deoxynivalenol in Wheat Using Surface Plasmon Resonance Immunoassay

USDA-ARS?s Scientific Manuscript database

Surface plasmon resonance immunoassay using a monoclonal antibody was developed to measure nivalenol (NIV) and deoxynivalenol (DON) contamination in wheat. A DON-immobilized sensor chip having high sensitivity and stability was prepared, and an SPR detection procedure was developed. The competitiv...

Comparison of Directigen Group A Strep Test with a traditional culture technique for detection of group A beta-hemolytic streptococci.

PubMed Central

McCusker, J J; McCoy, E L; Young, C L; Alamares, R; Hirsch, L S

1984-01-01

The Directigen Group A Strep Test (DGAST), a new rapid method of detecting group A beta-hemolytic streptococci directly from throat swabs, was compared with a traditional culture technique for the detection of group A beta-hemolytic streptococci. Five hundred oropharyngeal swabs from pediatric and adult patients were cultured and then processed by using the DGAST. Of the 144 specimens positive by culture, 131 were DGAST positive (sensitivity, 90.9%). Of the 356 specimens negative by culture, 353 were DGAST negative (specificity, 99.2%). Twelve of the 13 false-negative DGAST results were from pediatric patients. One hundred isolates of non-group A beta-hemolytic streptococci were recovered, primarily groups C, F, and G. The DGAST is easy to perform, rapid, sensitive, and very specific for detection of group A beta-hemolytic streptococci directly from swabs. Supplementing the DGAST with a culture on a 5% sheep blood agar plate would enhance detection of group A beta-hemolytic streptococci, especially in pediatric patients. PMID:6386884

Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification.

PubMed

Hammond, Rosemarie W; Zhang, Shulu

2016-10-01

A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39°C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in infected leaf and seed tissues. The performance of the AmplifyRP(®) Acceler8™ RT-RPA diagnostic assay, utilizing a lateral flow strip contained within an amplicon detection chamber, was evaluated and the results were compared with a standard RT-PCR assay. The AmplifyRP(®) Acceler8™ assay was specific for TCDVd in leaf and seed tissues, its sensitivity was comparable to conventional RT-PCR in leaf tissues, and it does not require extensive sample purification, specialized equipment, or technical expertise. This is the first report utilizing an RT-RPA assay to detect viroids and the assay can be used both in the laboratory and in the field for TCDVd detection. Published by Elsevier B.V.

Nano-particle enhanced impedimetric biosensor for detedtion of foodborne pathogens

NASA Astrophysics Data System (ADS)

Kim, G.; Om, A. S.; Mun, J. H.

2007-03-01

Recent outbreaks of foodborne illness have been increased the need for rapid and sensitive methods for detection of these pathogens. Conventional methods for pathogens detection and identification involve prolonged multiple enrichment steps. Even though some immunological rapid assays are available, these assays still need enrichment steps result in delayed detection. Biosensors have shown great potential for rapid detection of foodborne pathogens. They are capable of direct monitoring the antigen-antibody reactions in real time. Among the biosensors, impedimetric biosensors have been widely adapted as an analysis tool for the study of various biological binding reactions because of their high sensitivity and reagentless operation. In this study a nanoparticle-enhanced impedimetric biosensor for Salmonella enteritidis detection was developed which detected impedance changes caused by the attachment of the cells to the anti-Salmonella antibodies immobilized on interdigitated gold electrodes. Successive immobilization of neutravidin followed by anti-Salmonella antibodies was performed to the sensing area to create a biological detection surface. To enhance the impedance responses generated by antigen-antibody reactions, anti-Salmonella antibody conjugated nanoparticles were introduced on the sensing area. Using a portable impedance analyzer, the impedance across the interdigital electrodes was measured after the series of antigen-antibody bindings. Bacteria cells present in solution attached to capture antibodies and became tethered to the sensor surface. Attached bacteria cells changed the dielectric constant of the media between the electrodes thereby causing a change in measured impedance. Optimum input frequency was determined by analyzing frequency characteristics of the biosensor over ranges of applied frequencies from 10 Hz to 400 Hz. At 100 Hz of input frequency, the biosensor was most sensitive to the changes of the bacteria concentration and this frequency was used for the detection experiments. The biosensor was able to detect 106 CFU/mL in phosphate buffered saline (PBS) with a detection time of 3 minutes. Additional use of nanoparticles significantly enhanced the detection performance. By using the nanoparticles the biosensor could detect 104 CFU/mL of Salmonella enteritidis in PBS and 105 CFU/mL of cells in milk.

Microscopy outperformed in a comparison of five methods for detecting Trichomonas vaginalis in symptomatic women.

PubMed

Nathan, B; Appiah, J; Saunders, P; Heron, D; Nichols, T; Brum, R; Alexander, S; Baraitser, P; Ison, C

2015-03-01

In the UK, despite its low sensitivity, wet mount microscopy is often the only method of detecting Trichomonas vaginalis infection. A study was conducted in symptomatic women to compare the performance of five methods for detecting T. vaginalis: an in-house polymerase chain reaction (PCR); Aptima T. vaginalis kit; OSOM ®Trichomonas Rapid Test; culture and microscopy. Symptomatic women underwent routine testing; microscopy and further swabs were taken for molecular testing, OSOM and culture. A true positive was defined as a sample that was positive for T. vaginalis by two or more different methods. Two hundred and forty-six women were recruited: 24 patients were positive for T. vaginalis by two or more different methods. Of these 24 patients, 21 patients were detected by real-time PCR (sensitivity 88%); 22 patients were detected by the Aptima T. vaginalis kit (sensitivity 92%); 22 patients were detected by OSOM (sensitivity 92%); nine were detected by wet mount microscopy (sensitivity 38%); and 21 were detected by culture (sensitivity 88%). Two patients were positive by just one method and were not considered true positives. All the other detection methods had a sensitivity to detect T. vaginalis that was significantly greater than wet mount microscopy, highlighting the number of cases that are routinely missed even in symptomatic women if microscopy is the only diagnostic method available. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Rapid and sensitive detection of redspotted grouper nervous necrosis virus (RGNNV) infection by aptamer-coat protein-aptamer sandwich enzyme-linked apta-sorbent assay (ELASA).

PubMed

Zhou, L; Li, P; Ni, S; Yu, Y; Yang, M; Wei, S; Qin, Q

2017-12-01

Redspotted grouper nervous necrosis virus (RGNNV) is one of the most devastating pathogens in the aquaculture of the grouper, Epinephlus sp., worldwide. The early and rapid diagnosis of RGNNV is important for the prevention of RGNNV infection. In this study, an aptamer (A10)-based sandwich enzyme-linked apta-sorbent assay (ELASA) was developed for RGNNV diagnosis. This sandwich ELASA showed high specificity for the RGNNV coat protein (CP) and virions in virus-infected cells and tissues. At the optimized working concentration of 200 nM of aptamer, the ELASA could detect RGNNV in the lysates of as few as 4 × 10 3 RGNNV-infected GB cells. Incubation for 10 min was sufficient to produce accurate results. The sandwich ELASA was most stable at incubation temperatures of 4-25°C, but could still distinguish RGNNV-infected samples from the controls at 37°C. It could detect RGNNV infection in brain lysates diluted 1/10, with results consistent with those of reverse transcription PCR, although with 10% less sensitivity. The main equipment required includes dissection tools, a water bath, Pierce™ Streptavidin Coated Plates and a microplate reader. The sandwich ELASA has great potential utility for the rapid and sensitive diagnosis of RGNNV in its early stages by fish farmers. © 2017 John Wiley & Sons Ltd.

A vertically aligned carbon nanotube-based impedance sensing biosensor for rapid and high sensitive detection of cancer cells.

PubMed

Abdolahad, Mohammad; Taghinejad, Mohammad; Taghinejad, Hossein; Janmaleki, Mohsen; Mohajerzadeh, Shams

2012-03-21

A novel vertically aligned carbon nanotube based electrical cell impedance sensing biosensor (CNT-ECIS) was demonstrated for the first time as a more rapid, sensitive and specific device for the detection of cancer cells. This biosensor is based on the fast entrapment of cancer cells on vertically aligned carbon nanotube arrays and leads to mechanical and electrical interactions between CNT tips and entrapped cell membranes, changing the impedance of the biosensor. CNT-ECIS was fabricated through a photolithography process on Ni/SiO(2)/Si layers. Carbon nanotube arrays have been grown on 9 nm thick patterned Ni microelectrodes by DC-PECVD. SW48 colon cancer cells were passed over the surface of CNT covered electrodes to be specifically entrapped on elastic nanotube beams. CNT arrays act as both adhesive and conductive agents and impedance changes occurred as fast as 30 s (for whole entrapment and signaling processes). CNT-ECIS detected the cancer cells with the concentration as low as 4000 cells cm(-2) on its surface and a sensitivity of 1.7 × 10(-3)Ω cm(2). Time and cell efficiency factor (TEF and CEF) parameters were defined which describe the sensor's rapidness and resolution, respectively. TEF and CEF of CNT-ECIS were much higher than other cell based electrical biosensors which are compared in this paper.

Thermo-optical Characterization of Photothermal Optical Phase Shift Detection in Extended-Nano Channels and UV Detection of Biomolecules.

PubMed

Shimizu, Hisashi; Miyawaki, Naoya; Asano, Yoshihiro; Mawatari, Kazuma; Kitamori, Takehiko

2017-06-06

The expansion of microfluidics research to nanofluidics requires absolutely sensitive and universal detection methods. Photothermal detection, which utilizes optical absorption and nonradiative relaxation, is promising for the sensitive detection of nonlabeled biomolecules in nanofluidic channels. We have previously developed a photothermal optical phase shift (POPS) detection method to detect nonfluorescent molecules sensitively, while a rapid decrease of the sensitivity in nanochannels and the introduction of an ultraviolet (UV) excitation system were issues to be addressed. In the present study, our primary aim is to characterize the POPS signal in terms of the thermo-optical properties and quantitatively evaluate the causes for the decrease in sensitivity. The UV excitation system is then introduced into the POPS detector to realize the sensitive detection of nonlabeled biomolecules. The UV-POPS detection system is designed and constructed from scratch based on a symmetric microscope. The results of simulations and experiments reveal that the sensitivity decreases due to a reduction of the detection volume, dissipation of the heat, and cancellation of the changes in the refractive indices. Finally, determination of the concentration of a nonlabeled protein (bovine serum albumin) is performed in a very thin 900 nm deep nanochannel. As a result, the limit of detection (LOD) is 2.3 μM (600 molecules in the 440 attoliter detection volume), which is as low as that previously obtained for our visible POPS detector. UV-POPS detection is thus expected be a powerful technique for the study of biomolecules, including DNAs and proteins confined in nanofluidic channels.

Microwave-Accelerated Method for Ultra-Rapid Extraction of Neisseria gonorrhoeae DNA for Downstream Detection

PubMed Central

Melendez, Johan H.; Santaus, Tonya M.; Brinsley, Gregory; Kiang, Daniel; Mali, Buddha; Hardick, Justin; Gaydos, Charlotte A.; Geddes, Chris D.

2016-01-01

Nucleic acid-based detection of gonorrhea infections typically require a two-step process involving isolation of the nucleic acid, followed by the detection of the genomic target often involving PCR-based approaches. In an effort to improve on current detection approaches, we have developed a unique two-step microwave-accelerated approach for rapid extraction and detection of Neisseria gonorrhoeae (GC) DNA. Our approach is based on the use of highly-focused microwave radiation to rapidly lyse bacterial cells, release, and subsequently fragment microbial DNA. The DNA target is then detected by a process known as microwave-accelerated metal-enhanced fluorescence (MAMEF), an ultra-sensitive direct DNA detection analytical technique. In the present study, we show that highly focused microwaves at 2.45 GHz, using 12.3 mm gold film equilateral triangles, are able to rapidly lyse both bacteria cells and fragment DNA in a time- and microwave power-dependent manner. Detection of the extracted DNA can be performed by MAMEF, without the need for DNA amplification in less than 10 minutes total time or by other PCR-based approaches. Collectively, the use of a microwave-accelerated method for the release and detection of DNA represents a significant step forward towards the development of a point-of-care (POC) platform for detection of gonorrhea infections. PMID:27325503

Rapid and Sensitive Detection of Vibrio parahaemolyticus and Vibrio vulnificus by Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification Technique.

PubMed

Wang, Yi; Li, Dongxun; Wang, Yan; Li, Kewei; Ye, Changyun

2016-01-19

Vibrio parahaemolyticus and Vibrio vulnificus are two marine seafood-borne pathogens causing severe illnesses in humans and aquatic animals. In this study, a recently developed novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully developed and evaluated for simultaneous detection of V. parahaemolyticus and V. vulnificus strains in only a single reaction. Two MERT-LAMP primer sets were designed to specifically target toxR gene of V. parahaemolyticus and rpoS gene of V. vulnificus. The MERT-LAMP reactions were conducted at 62 °C, and the positive results were produced in as short as 19 min with the genomic DNA templates extracted from the V. parahaemolyticus and V. vulnificus strains. The two target pathogens present in the same sample could be simultaneously detected and correctly differentiated based on distinct fluorescence curves in a real-time format. The sensitivity of MERT-LAMP assay was 250 fg and 125 fg DNA per reaction with genomic templates of V. parahaemolyticus and V. vulnificus strains, which was in conformity with conventional LAMP detection. Compared with PCR-based techniques, the MERT-LAMP technology was 100- and 10-fold more sensitive than that of PCR and qPCR methods. Moreover, the limit of detection of MERT-LAMP approach for V. parahaemolyticus isolates and V. vulnificus isolates detection in artificially-contaminated oyster samples was 92 CFU and 83 CFU per reaction. In conclusion, the MERT-LAMP assay presented here was a rapid, specific, and sensitive tool for the detection of V. parahaemolyticus and V. vulnificus, and could be adopted for simultaneous screening of V. parahaemolyticus and V. vulnificus in a wide variety of samples.

CE with chemiluminescence detection for the determination of thyroxine in human serum.

PubMed

Mu, Xiaomei; Li, Shuting; Lu, Xin; Zhao, Shulin

2014-04-01

A sensitive and rapid approach to perform thyroxine (T4) assay by CE with chemiluminescence (CL) detection was developed. The sensitive detection was based on the enhancement effect of T4 on the CL reaction between luminol and potassium permanganate (KMnO4 ) in alkaline solution. A laboratory-built reaction flow cell and a photon counter were deployed for the CL detection. Experimental conditions for CL detection were studied in detail to achieve maximum assay sensitivity. Optimal conditions were found to be 5.0 × 10(-4) M luminol added to the CE running buffer and 9.2 × 10(-5) M KMnO4 in 0.072 M NaOH solution introduced postcolumn. In the optimized experimental conditions, the linear range for T4 detection was 6.0 × 10(-8) -6.0 × 10(-6) M, with the detection limit of 2.0 × 10(-8) M (S/N = 3). Six human serum samples from healthy subjects, hyperthyroid patients and hypothyroid patients were analyzed by the presented method. The serum level of T4 in healthy subjects was found be 9.0 × 10(-8) M, whereas the T4 level was found to be 15.6 × 10(-8) M in hyperthyroid patients and 1.3 × 10(-8) M in hypothyroid patients. The results suggested a potential application of the proposed assay in rapid primary diagnosis of diseases such as hyperthyroid and hypothyroid. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Highly selective and sensitive fluorescent paper sensor for nitroaromatic explosive detection.

PubMed

Ma, Yingxin; Li, Hao; Peng, Shan; Wang, Leyu

2012-10-02

Rapid, sensitive, and selective detection of explosives such as 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenol (TNP), especially using a facile paper sensor, is in high demand for homeland security and public safety. Although many strategies have been successfully developed for the detection of TNT, it is not easy to differentiate the influence from TNP. Also, few methods were demonstrated for the selective detection of TNP. In this work, via a facile and versatile method, 8-hydroxyquinoline aluminum (Alq(3))-based bluish green fluorescent composite nanospheres were successfully synthesized through self-assembly under vigorous stirring and ultrasonic treatment. These polymer-coated nanocomposites are not only water-stable but also highly luminescent. Based on the dramatic and selective fluorescence quenching of the nanocomposites via adding TNP into the aqueous solution, a sensitive and robust platform was developed for visual detection of TNP in the mixture of nitroaromatics including TNT, 2,4-dinitrotoluene (DNT), and nitrobenzene (NB). Meanwhile, the fluorescence intensity is proportional to the concentration of TNP in the range of 0.05-7.0 μg/mL with the 3σ limit of detection of 32.3 ng/mL. By handwriting or finger printing with TNP solution as ink on the filter paper soaked with the fluorescent nanocomposites, the bluish green fluorescence was instantly and dramatically quenched and the dark patterns were left on the paper. Therefore, a convenient and rapid paper sensor for TNP-selective detection was fabricated.

Detection of Papaya leaf distortion mosaic virus by reverse-transcription loop-mediated isothermal amplification.

PubMed

Shen, Wentao; Tuo, Decai; Yan, Pu; Li, Xiaoying; Zhou, Peng

2014-01-01

Papaya leaf distortion mosaic virus (PLDMV) can infect transgenic papaya resistant to a related pathogen, Papaya ringspot virus (PRSV), posing a substantial threat to papaya production in China. Current detection methods, however, are unable to be used for rapid detection in the field. Here, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of PLDMV, using a set of four RT-LAMP primers designed based on the conserved sequence of PLDMV CP. The RT-LAMP method detected specifically PLDMV and was highly sensitive, with a detection limit of 1.32×10(-6) μg of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR, while also requiring significantly less time and equipment. The effectiveness of RT-LAMP and one-step RT-PCR in detecting the virus were compared using 90 field samples of non-transgenic papaya and 90 field samples of commercialized PRSV-resistant transgenic papaya from Hainan Island. None of the non-transgenic papaya tested positive for PLDMV using either method. In contrast, 19 of the commercialized PRSV-resistant transgenic papaya samples tested positive by RT-LAMP assay, and 6 of those tested negative by RT-PCR. Therefore, the PLDMV-specific RT-LAMP is a simple, rapid, sensitive, and cost-effective tool in the field diagnosis and control of PLDMV. Copyright © 2013 Elsevier B.V. All rights reserved.

Rapid detection of Shigella and enteroinvasive Escherichia coli in produce enrichments by a conventional multiplex PCR assay.

PubMed

Binet, Rachel; Deer, Deanne M; Uhlfelder, Samantha J

2014-06-01

Faster detection of contaminated foods can prevent adulterated foods from being consumed and minimize the risk of an outbreak of foodborne illness. A sensitive molecular detection method is especially important for Shigella because ingestion of as few as 10 of these bacterial pathogens can cause disease. The objectives of this study were to compare the ability of four DNA extraction methods to detect Shigella in six types of produce, post-enrichment, and to evaluate a new and rapid conventional multiplex assay that targets the Shigella ipaH, virB and mxiC virulence genes. This assay can detect less than two Shigella cells in pure culture, even when the pathogen is mixed with background microflora, and it can also differentiate natural Shigella strains from a control strain and eliminate false positive results due to accidental laboratory contamination. The four DNA extraction methods (boiling, PrepMan Ultra [Applied Biosystems], InstaGene Matrix [Bio-Rad], DNeasy Tissue kit [Qiagen]) detected 1.6 × 10(3)Shigella CFU/ml post-enrichment, requiring ∼18 doublings to one cell in 25 g of produce pre-enrichment. Lower sensitivity was obtained, depending on produce type and extraction method. The InstaGene Matrix was the most consistent and sensitive and the multiplex assay accurately detected Shigella in less than 90 min, outperforming, to the best of our knowledge, molecular assays currently in place for this pathogen. Published by Elsevier Ltd.

A thermal-sensitive device fabricated with diamond film and a planar microelectrode

DOE Office of Scientific and Technical Information (OSTI.GOV)

Changzhi Gu; Zengsun Jin; Xianyi Lu

1995-12-31

Polycrystalline diamond film were deposited by means of the hot filament CVD technique (HFCVD) onto a planar interdigital Ti microelectrode arrays, and forming a thermal-sensitive device, The resistor changes of diamond film caused by temperature are shown to be sensitive, reproducible, rapid and stable thermal-sensitive device. The characteristics of thermal-sensitive for this device was study. Functionalized diamond film deposited onto planar microelectrode arrays can easily detect temperature from 20{degrees}C to 700{degrees}C.

Development of an in situ magnetic beads based RT-PCR method for electrochemiluminescent detection of rotavirus

NASA Astrophysics Data System (ADS)

Zhan, Fangfang; Zhou, Xiaoming

2012-12-01

Rotaviruses are double-stranded RNA viruses belonging to the family of enteric pathogens. It is a major cause of diarrhoeal disease in infants and young children worldwide. Consequently, rapid and accurate detection of rotaviruses is of great importance in controlling and preventing food- and waterborne diseases and outbreaks. Reverse transcription-polymerase chain reaction (RT-PCR) is a reliable method that possesses high specificity and sensitivity. It has been widely used to detection of viruses. Electrochemiluminescence (ECL) can be considered as an important and powerful tool in analytical and clinical application with high sensitivity, excellent specificity, and low cost. Here we have developed a method for the detection of rotavirus by combining in situ magnetic beads (MBs) based RT-PCR with ECL. RT of rotavirus RNA was carried out in a traditional way and the resulting cDNA was directly amplified on MBs. Forward primers were covalently bounded to MBs and reverse primers were labeled with tris-(2, 2'-bipyridyl) ruthenium (TBR). During the PCR cycling, the TBR labeled products were directly loaded and enriched on the surface of MBs. Then the MBs-TBR complexes could be analyzed by a magnetic ECL platform without any post-modification or post-incubation which avoid some laborious manual operations and achieve rapid yet sensitive detection. In this study, rotavirus from fecal specimens was successfully detected within 2 h, and the limit of detection was estimated to be 104copies/μL. This novel in situ MBs based RT-PCR with ECL detection method can be used for pathogen detection in food safety field and clinical diagnosis.

Laboratory Evaluation of a Smartphone-Based Electronic Reader of Rapid Dual Point-of-Care Tests for Antibodies to Human Immunodeficiency Virus and Treponema pallidum Infections.

PubMed

Herbst de Cortina, Sasha; Bristow, Claire C; Humphries, Romney; Vargas, Silver Keith; Konda, Kelika A; Caceres, Carlos F; Klausner, Jeffrey D

2017-07-01

Dual point-of-care tests for antibodies to human immunodeficiency virus (HIV) and Treponema pallidum allow for same-day testing and treatment and have been demonstrated to be cost-effective in preventing the adverse outcomes of HIV infection and syphilis. By recording and transmitting data as they are collected, electronic readers address challenges related to the decentralization of point-of-care testing. We evaluated a smartphone-based electronic reader using 201 sera tested with 2 dual rapid tests for detection of antibodies to HIV and T. pallidum in Los Angeles, USA, and Lima, Peru. Tests were read both visually and with the electronic reader. Enzyme immunoassay followed by Western blot and T. pallidum particle agglutination were the reference tests for HIV and T. pallidum, respectively. The sensitivities of the 2 rapid tests for detection of HIV were 94.1% and 97.0% for electronic readings. Both tests had a specificity of 100% for detection of HIV by electronic reading. The sensitivities of the 2 rapid tests for detection of T. pallidum were 86.5% and 92.4% for electronic readings. The specificities for detection of T. pallidum were 99.1% and 99.0% by electronic reading. There were no significant differences between the accuracies of visual and electronic readings, and the performance did not differ between the 2 study sites. Our results show the electronic reader to be a promising option for increasing the use of point-of-care testing programs.

Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Method for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus

PubMed Central

Thai, Hong Thi Cam; Le, Mai Quynh; Vuong, Cuong Duc; Parida, Manmohan; Minekawa, Harumi; Notomi, Tsugunori; Hasebe, Futoshi; Morita, Kouichi

2004-01-01

The development and evaluation of a one-step single-tube accelerated real-time quantitative reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay is reported for rapid detection of the severe acute respiratory syndrome coronavirus (SARS-CoV) replicase gene. A total of 49 samples (15 throat washes, 13 throat swabs, and 21 combined throat and nasal swabs) collected from patients admitted to the Hanoi-French and Ninhbinh hospitals in Vietnam during the SARS epidemic were evaluated and compared to conventional RT-PCR. The RT-LAMP assay demonstrated 100-fold-greater sensitivity, with a detection limit of 0.01 PFU. The sensitivity and specificity of RT-LAMP assay for detecting viral RNA in clinical specimens with regard to RT-PCR were 100 and 87%, respectively. The specificity of the RT-LAMP assay was further validated by restriction analysis as well as nucleotide sequencing of the amplified product. The concentration of virus in most of the clinical samples was 0.1 PFU (0.1 to 102 PFU), as determined from the standard curve of SARS RT-LAMP and based on the time of positivity. The assay procedure is quite simple, wherein the amplification is carried out in a single tube under isothermal conditions at 63°C, and the result can be obtained in less than 1 h (as early as 11 min). Thus, the RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection and will be useful for rapid and reliable clinical diagnosis of SARS-CoV in developing countries. PMID:15131154

Usefulness of an inexpensive, Paracheck test in detecting asymptomatic infectious reservoir of plasmodium falciparum during dry season in an inaccessible terrain in central India.

PubMed

Singh, N; Saxena, A; Sharma, V P

2002-10-01

The performance of a new indigenous rapid diagnostic test, Paracheck Pf was evaluated in detection of Plasmodium falciparum in asymptomatic children in remote forest villages of Mandla district, central India to determine the lower limits of sensitivity and specificity of rapid test. A finger prick blood sample was collected to prepare blood smear and for testing with the Paracheck test. The blood smears were read by an experienced technician blinded to the Paracheck results. The figures for specificity, sensitivity, accuracy and predictive values were calculated using microscopy as gold standard. The new diagnostic test had a sensitivity of 94% and a specificity of 89%. The positive and negative predictive values were 71% and 98%, respectively. The J -index was 0.83%. The rapid test was found to be very easy to perform and the result could be read reliably by field workers. The field evaluation with this new inexpensive test, ($0.65/test) indicates that it could be used as an epidemiological tool in the management of malaria particularly in areas where microscopy is not operationally feasible to attain the goal of the roll back malaria initiative.

Comparison of two commercial rapid in-clinic serological tests for detection of antibodies against Leishmania spp. in dogs.

PubMed

Athanasiou, Labrini V; Petanides, Theodoros A; Chatzis, Manolis K; Kasabalis, Dimitrios; Apostolidis, Kosmas N; Saridomichelakis, Manolis N

2014-03-01

Antibodies against Leishmania spp. are detected in most dogs with clinical signs of leishmaniasis due to Leishmania infantum. Accurate, rapid in-clinic serological tests may permit immediate confirmation of the diagnosis and implementation of therapeutic measures. The aim of the current study was to evaluate the diagnostic accuracy of 2 commercial, rapid in-clinic serological tests for the detection of anti-Leishmania antibodies in sera of dogs, the Snap Canine Leishmania Antibody Test kit (IDEXX Laboratories Inc., Westbrook, Maine) and the ImmunoRun Antibody Detection kit (Biogal Galed Labs, Kibbutz Galed, Israel), using indirect fluorescent antibody test (IFAT) as the reference method. A total of 109 sera collected from 65 seropositive and 44 seronegative dogs were used. The sensitivities of the Snap and ImmunoRun kits were 89.23% (95% confidence interval: 79.05-95.54%) and 86.15% (95% confidence interval: 75.33-93.45%), respectively, and the specificity of both tests was 100%. A good agreement between each of the rapid in-clinic serological tests and IFAT and between the 2 rapid in-clinic serological tests was witnessed. Both rapid in-clinic serological tests showed an adequate diagnostic accuracy and can be used for the fast detection of antibodies against L. infantum in dogs.

A biosensor platform for rapid detection of E. coli in drinking water.

PubMed

Hesari, Nikou; Alum, Absar; Elzein, Mohamad; Abbaszadegan, Morteza

2016-02-01

There remains a need for rapid, specific and sensitive assays for the detection of bacterial indicators for water quality monitoring. In this study, a strategy for rapid detection of Escherichia coli in drinking water has been developed. This strategy is based on the use of the substrate 4-methylumbelliferyl-β-d-glucuronide (MUG), which is hydrolyzed rapidly by the action of E. coli β-d-glucuronidase (GUD) enzyme to yield a fluorogenic 4-methylumbelliferone (4-MU) product that can be quantified and related to the number of E. coli cells present in water samples. In this study, the detection time required for the biosensor response ranged between 20 and 120 min, depending on the number of bacteria in the sample. This approach does not need extensive sample processing with a rapid detection capability. The specificity of the MUG substrate was examined in both, pure cultures of non-target bacterial genera such as Klebsiella, Salmonella, Enterobacter and Bacillus. Non-target substrates that included 4-methylumbelliferyl-β-d-galactopyranoside (MUGal) and l-leucine β-naphthylamide aminopeptidase (LLβ-N) were also investigated to identify nonspecific patterns of enzymatic activities in E. coli. GUD activity was found to be specific for E. coli and no further enzymatic activity was detected by other species. In addition, fluorescence assays were performed for the detection of E. coli to generate standard curves; and the sensitivity of the GUD enzymatic response was measured and repeatedly determined to be less than 10 E. coli cells in a reaction vial. The applicability of the method was tested by performing multiple fluorescence assays under pure and mixed bacterial flora in environmental samples. The results of this study showed that the fluorescence signals generated in samples using specific substrate molecules can be utilized to develop a bio-sensing platform for the detection of E. coli in drinking water. Furthermore, this system can be applied independently or in conjunction with other methods as a part of an array of biochemical assays in order to reliably detect E. coli in water. Copyright © 2015 Elsevier Inc. All rights reserved.

Reverse Transcription Recombinase Polymerase Amplification Assay for the Detection of Middle East Respiratory Syndrome Coronavirus

PubMed Central

Abd El Wahed, Ahmed; Patel, Pranav; Heidenreich, Doris; Hufert, Frank T.; Weidmann, Manfred

2013-01-01

The emergence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in the eastern Mediterranean and imported cases to Europe has alerted public health authorities. Currently, detection of MERS-CoV in patient samples is done by real-time RT-PCR. Samples collected from suspected cases are sent to highly-equipped centralized laboratories for screening. A rapid point-of-care test is needed to allow more widespread mobile detection of the virus directly from patient material. In this study, we describe the development of a reverse transcription isothermal Recombinase Polymerase Amplification (RT-RPA) assay for the identification of MERS-CoV. A partial nucleocapsid gene RNA molecular standard of MERS-coronavirus was used to determine the assay sensitivity. The isothermal (42°C) MERS-CoV RT-RPA was as sensitive as real-time RT-PCR (10 RNA molecules), rapid (3-7 minutes) and mobile (using tubescanner weighing 1kg). The MERS-CoV RT-RPA showed cross-detection neither of any of the RNAs of several coronaviruses and respiratory viruses affecting humans nor of the human genome. The developed isothermal real-time RT-RPA is ideal for rapid mobile molecular MERS-CoV monitoring in acute patients and may also facilitate the search for the animal reservoir of MERS-CoV. PMID:24459611

Development of 2, 7-Diamino-1, 8-Naphthyridine (DANP) Anchored Hairpin Primers for RT-PCR Detection of Chikungunya Virus Infection

PubMed Central

Chen, Huixin; Parimelalagan, Mariya; Takei, Fumie; Hapuarachchi, Hapuarachchige Chanditha; Koay, Evelyn Siew-Chuan; Ng, Lee Ching; Ho, Phui San; Nakatani, Kazuhiko; Chu, Justin Jang Hann

2016-01-01

A molecular diagnostic platform with DANP-anchored hairpin primer was developed and evaluated for the rapid and cost-effective detection of Chikungunya virus (CHIKV) with high sensitivity and specificity. The molecule 2, 7-diamino-1, 8-naphthyridine (DANP) binds to a cytosine-bulge and emits fluorescence at 450 nm when it is excited by 400 nm light. Thus, by measuring the decline in fluorescence emitted from DANP—primer complexes after PCR reaction, we could monitor the PCR progress. By adapting this property of DANP, we have previously developed the first generation DANP-coupled hairpin RT-PCR assay. In the current study, we improved the assay performance by conjugating the DANP molecule covalently onto the hairpin primer to fix the DANP/primer ratio at 1:1; and adjusting the excitation emission wavelength to 365/430 nm to minimize the background signal and a ‘turn-on’ system is achieved. After optimizing the PCR cycle number to 30, we not only shortened the total assay turnaround time to 60 minutes, but also further reduced the background fluorescence. The detection limit of our assay was 0.001 PFU per reaction. The DANP-anchored hairpin primer, targeting nsP2 gene of CHIKV genome, is highly specific to CHIKV, having no cross-reactivity to a panel of other RNA viruses tested. In conclusion, we report here a molecular diagnostic assay that is sensitive, specific, rapid and cost effective for CHIKV detection and can be performed where no real time PCR instrumentation is required. Our results from patient samples indicated 93.62% sensitivity and 100% specificity of this method, ensuring that it can be a useful tool for rapid detection of CHIKV for outbreaks in many parts of the world. PMID:27571201

Development of 2, 7-Diamino-1, 8-Naphthyridine (DANP) Anchored Hairpin Primers for RT-PCR Detection of Chikungunya Virus Infection.

PubMed

Chen, Huixin; Parimelalagan, Mariya; Takei, Fumie; Hapuarachchi, Hapuarachchige Chanditha; Koay, Evelyn Siew-Chuan; Ng, Lee Ching; Ho, Phui San; Nakatani, Kazuhiko; Chu, Justin Jang Hann

2016-08-01

A molecular diagnostic platform with DANP-anchored hairpin primer was developed and evaluated for the rapid and cost-effective detection of Chikungunya virus (CHIKV) with high sensitivity and specificity. The molecule 2, 7-diamino-1, 8-naphthyridine (DANP) binds to a cytosine-bulge and emits fluorescence at 450 nm when it is excited by 400 nm light. Thus, by measuring the decline in fluorescence emitted from DANP-primer complexes after PCR reaction, we could monitor the PCR progress. By adapting this property of DANP, we have previously developed the first generation DANP-coupled hairpin RT-PCR assay. In the current study, we improved the assay performance by conjugating the DANP molecule covalently onto the hairpin primer to fix the DANP/primer ratio at 1:1; and adjusting the excitation emission wavelength to 365/430 nm to minimize the background signal and a 'turn-on' system is achieved. After optimizing the PCR cycle number to 30, we not only shortened the total assay turnaround time to 60 minutes, but also further reduced the background fluorescence. The detection limit of our assay was 0.001 PFU per reaction. The DANP-anchored hairpin primer, targeting nsP2 gene of CHIKV genome, is highly specific to CHIKV, having no cross-reactivity to a panel of other RNA viruses tested. In conclusion, we report here a molecular diagnostic assay that is sensitive, specific, rapid and cost effective for CHIKV detection and can be performed where no real time PCR instrumentation is required. Our results from patient samples indicated 93.62% sensitivity and 100% specificity of this method, ensuring that it can be a useful tool for rapid detection of CHIKV for outbreaks in many parts of the world.

Establishment of the cross-clade antigen detection system for H5 subtype influenza viruses using peptide monoclonal antibodies specific for influenza virus H5 hemagglutinin.

PubMed

Takahashi, Hitoshi; Nagata, Shiho; Odagiri, Takato; Kageyama, Tsutomu

2018-04-15

The H5 subtype of highly pathogenic avian influenza (H5 HPAI) viruses is a threat to both animal and human public health and has the potential to cause a serious future pandemic in humans. Thus, specific and rapid detection of H5 HPAI viruses is required for infection control in humans. To develop a simple and rapid diagnostic system to detect H5 HPAI viruses with high specificity and sensitivity, we attempted to prepare monoclonal antibodies (mAbs) that specifically recognize linear epitopes in hemagglutinin (HA) of H5 subtype viruses. Nine mAb clones were obtained from mice immunized with a synthetic partial peptide of H5 HA molecules conserved among various H5 HPAI viruses. The antigen-capture enzyme-linked immunosorbent assay using the most suitable combination of these mAbs, which bound specifically to lysed H5 HA under an optimized detergent condition, was specific for H5 viruses and could broadly detect H5 viruses in multiple different clades. Taken together, these peptide mAbs, which recognize linear epitopes in a highly conserved region of H5 HA, may be useful for specific and highly sensitive detection of H5 HPAI viruses and can help in the rapid diagnosis of human, avian, and animal H5 virus infections. Copyright © 2018 Elsevier Inc. All rights reserved.

Loop-Mediated Isothermal Amplification of the sefA Gene for Rapid Detection of Salmonella Enteritidis and Salmonella Gallinarum in Chickens.

PubMed

Gong, Jiansen; Zhuang, Linlin; Zhu, Chunhong; Shi, Shourong; Zhang, Di; Zhang, Linji; Yu, Yan; Dou, Xinhong; Xu, Bu; Wang, Chengming

2016-04-01

Salmonella spp. pose a threat to both human and animal health, with more than 2600 serovars having been reported to date. Salmonella serovars are usually identified by slide agglutination tests, which are labor intensive and time consuming. In an attempt to develop a more rapid screening method for the major poultry Salmonella serovars, we developed a loop-mediated isothermal amplification (LAMP) assay, which directly detected the sefA gene, a fimbrial operon gene existing in several specific serovars of Salmonella enterica including the major poultry serovars, namely Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) and Salmonella enterica serovar Gallinarum (Salmonella Gallinarum). With the 177 bacterial strains we tested, positive reactions were only observed with 85 strains of serovar Salmonella Enteritidis and Salmonella Gallinarum. The detection limit of the LAMP assay was 4 CFU/reaction with genomic DNAs of Salmonella Enteritidis (ATCC 13076) from pure culture and 400 CFU/ reaction with DNA extracted from spiked chicken feces. The LAMP assay was more sensitive than conventional culture, especially without enrichment, in detecting Salmonella Enteritidis (CMCC 50041) in the spiked fecal samples. The results show the sefA LAMP method is a rapid, sensitive, specific, and practical method for directly detection of Salmonella Enteritidis and Salmonella Gallinarum in chickens. The sefA LAMP assay can potentially serve as new on-site diagnostics in the poultry industry.

Development of real-time and lateral flow dipstick recombinase polymerase amplification assays for rapid detection of goatpox virus and sheeppox virus.

PubMed

Yang, Yang; Qin, Xiaodong; Zhang, Xiangle; Zhao, Zhixun; Zhang, Wei; Zhu, Xueliang; Cong, Guozheng; Li, Yanmin; Zhang, Zhidong

2017-07-17

Goatpox virus (GTPV) and sheeppox virus (SPPV), which belong to the Capripoxvirus (CaPV), are economically important pathogens of small ruminants. Therefore, a sensitive, specific and rapid diagnostic assay for detection of GTPV and SPPV is necessary to accurately and promptly control these diseases. Recombinase polymerase amplification (RPA) assays combined with a real-time fluorescent detection (real-time RPA assay) and lateral flow dipstick (RPA LFD assay) were developed targeting the CaPV G-protein-coupled chemokine receptor (GPCR) gene, respectively. The sensitivity of both CaPV real-time RPA assay and CaPV RPA LFD assay were 3 × 10 2 copies per reaction within 20 min at 38 °C. Both assays were highly specific for CaPV, with no cross-reactions with peste des petits ruminants virus, foot-and-mouth disease virus and Orf virus. The evaluation of the performance of these two assays with clinical sample (n = 107) showed that the CaPV real-time RPA assay and CaPV RPA LFD assay were able to specially detect SPPV or GTPV present in samples of ovine in liver, lung, kidney, spleen, skin and blood. This study provided a highly time-efficient and simple alternative for rapid detection of GTPV and SPPV.

Rapid and broad detection of H5 hemagglutinin by an immunochromatographic kit using novel monoclonal antibody against highly pathogenic avian influenza virus belonging to the genetic clade 2.3.4.4.

PubMed

Nguyen, Lam Thanh; Nakaishi, Kazunari; Motojima, Keiko; Ohkawara, Ayako; Minato, Erina; Maruyama, Junki; Hiono, Takahiro; Matsuno, Keita; Okamatsu, Masatoshi; Kimura, Takashi; Takada, Ayato; Kida, Hiroshi; Sakoda, Yoshihiro

2017-01-01

Highly pathogenic avian influenza viruses (HPAIVs) of H5 subtype have persistently caused outbreaks in domestic poultry and wild birds worldwide and sporadically infected humans. Rapid and accurate diagnosis is one of the key strategies for the control of H5 HPAIVs. However, the sensitivity of the diagnosis of H5 HPAIVs has gradually reduced due to extensive antigenic variation during their evolution. Particularly, the previously developed immunochromatographic diagnosis kit for H5 viruses, Linjudge Flu A/H5, exhibits reduced detection of H5 HPAIVs isolated in recent years. In the present study, we established a new advanced H5 rapid immunochromatographic detection kit (New Linjudge Flu A/H5) by a combination of two anti-H5 hemagglutinin monoclonal antibodies, A64/1 previously applied in the Linjudge Flu A/H5 and A32/2, a novel monoclonal antibody generated from a clade 2.3.4.4 H5 HPAIV. The new kit broadly detected all classical and recent H5 influenza viruses and showed a higher specificity and sensitivity than the original Linjudge Flu A/H5 with recently circulating H5 HPAIVs. Furthermore, the applicability of the New Linjudge Flu A/H5 was demonstrated by detecting antigens from the swabs and tissue homogenates of naturally infected birds and experimentally infected chickens with H5N6 HPAIVs belonging to the genetic clade 2.3.4.4. Our study, therefore, can provide an effective point-of-care rapid antigen detection kit for the surveillance of H5 avian influenza viruses and as a prompt countermeasure against the current widespread of the clade 2.3.4.4 H5 HPAIVs in domestic and wild birds.

SPECIES-SPECIFIC DETECTION OF THREE HUMAN-PATHOGENIC MICROSPORIDIAL SPECIES FROM THE GENUS ENCEPHALITOZOON VIA FLUOROGENIC 5' NUCLEASE PCR ASSAYS

EPA Science Inventory

This describes fluorogenic 5' nuclease PCR assays suitable for rapid, sensitive, quantitative, high-throughput detection of the human-pathogenic microsporidial species Encephalitozoon hellem, E. cunicli and E. intestinalis. The assays utilize species-specific primer sets and a g...

Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe

PubMed Central

Xue, Yong; Wilkes, Jon G.; Moskal, Ted J.; Williams, Anna J.; Cooper, Willie M.; Nayak, Rajesh; Rafii, Fatemeh; Buzatu, Dan A.

2016-01-01

Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts. PMID:26913737

Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe.

PubMed

Xue, Yong; Wilkes, Jon G; Moskal, Ted J; Williams, Anna J; Cooper, Willie M; Nayak, Rajesh; Rafii, Fatemeh; Buzatu, Dan A

2016-01-01

Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts.

Comparison of Detection Limits of 4th Generation Combination HIV Antigen/Antibody, p24 Antigen and Viral Load Assays on Diverse HIV Isolates.

PubMed

Stone, Mars; Bainbridge, John; Sanchez, Ana M; Keating, Sheila M; Pappas, Andrea; Rountree, Wes; Todd, Chris; Bakkour, Sonia; Manak, Mark; Peel, Sheila A; Coombs, Robert W; Ramos, Eric M; Shriver, M Kathleen; Contestable, Paul; Nair, Sangeetha Vijaysri; Wilson, David H; Stengelin, Martin; Murphy, Gary; Hewlett, Indira; Denny, Thomas N; Busch, Michael P

2018-05-23

Detection of acute HIV infection is critical for HIV public health and diagnostics. Clinical 4 th generation antigen-antibody (Ag/Ab) combination (combo) and p24 Ag immunoassays have enhanced detection of acute infection compared to Ab alone assays, but require ongoing evaluation with currently circulating diverse subtypes. Genetically and geographically diverse HIV clinical isolates were used to assess clinical HIV diagnostic, blood screening and next generation assays. Blinded 300 member panels of 20 serially diluted well-characterized antibody negative HIV isolates were distributed to manufacturers and end-user labs to assess relative analytic sensitivity of currently approved and pre-approved clinical HIV 4 th generation Ag/Ab combo or p24 Ag alone immunoassays across diverse subtypes. The limits of virus detection (LODs) were estimated for different subtypes relative to confirmed viral loads. Analysis of immunoassay sensitivity was benchmarked against confirmed viral load measurements on the blinded panel. Based on the proportion of positive results on 300 observations all Ag/Ab combo and standard sensitivity p24 Ag assays performed similarly and within half log LODs, illustrating similar breadth of reactivity and diagnostic utility. Ultrasensitive p24 Ag assays achieved dramatically increased sensitivities, while the rapid combo-assays performed poorly. Similar performance of the different commercially available 4 th gen assays on diverse subtypes supports their use in broad geographic settings with locally circulating HIV clades and recombinant strains. Next generation pre-clinical ultrasensitive p24 Ag assays achieved dramatically improved sensitivity, while p24 Ag detection by rapid 4 th gen assays performed poorly. Copyright © 2018 American Society for Microbiology.

Use of bacteriophage cell wall-binding proteins for rapid diagnostics of Listeria.

PubMed

Schmelcher, Mathias; Loessner, Martin J

2014-01-01

Diagnostic protocols for food-borne bacterial pathogens such as Listeria need to be sensitive, specific, rapid, and inexpensive. Conventional culture methods are hampered by lengthy enrichment and incubation steps. Bacteriophage-derived high-affinity binding molecules (cell wall-binding domains, CBDs) specific for Listeria cells have recently been introduced as tools for detection and differentiation of this pathogen in foods. When coupled with magnetic separation, these proteins offer advantages in sensitivity and speed compared to the standard diagnostic methods. Furthermore, fusion of CBDs to differently colored fluorescent reporter proteins enables differentiation of Listeria strains in mixed cultures. This chapter provides protocols for detection of Listeria in food by CBD-based magnetic separation and subsequent multiplexed identification of strains of different serotypes with reporter-CBD fusion proteins.

Electrochemical Nanoparticle-Enzyme Sensors for Screening Bacterial Contamination in Drinking Water

PubMed Central

Chen, Juhong; Jiang, Ziwen; Ackerman, Jonathan D.; Yazdani, Mahdieh; Hou, Singyuk

2015-01-01

Traditional plating and culturing methods used to quantify bacteria commonly require hours to days from sampling to results. We present here a simple, sensitive and rapid electrochemical method for bacteria detection in drinking water based on gold nanoparticle-enzyme complexes. The gold nanoparticles were functionalized with positively charged quaternary amine headgroups that could bind to enzymes through electrostatic interactions, resulting in inhibition of enzymatic activity. In the presence of bacteria, the nanoparticles released from the enzymes and preferentially bound to the bacteria, resulting in an increase in enzyme activity, releasing a redox-active phenol from the substrate. We employed this strategy for the electrochemical sensing of Escherichia coli and Staphylococcus aureus, resulting in a rapid detection (<1h) with high sensitivity (102 CFU·mL−1). PMID:26042607

Rapid screening for human-pathogenic Mucorales using rolling circle amplification.

PubMed

Dolatabadi, S; Najafzadeh, M J; de Hoog, G S

2014-12-01

Mucormycosis has emerged as a relatively common severe mycosis in patients with haematological and allogeneic stem cell transplantation. Source of transmission is from unidentified sources in the environment. Early diagnosis of infection and its source of contamination are paramount for rapid and appropriate therapy. In this study, rolling circle amplification (RCA) is introduced as a sensitive, specific and reproducible isothermal DNA amplification technique for rapid molecular identification of six of the most virulent species (Rhizopus microsporus, R. arrhizus var. arrhizus, R. arrhizus var. delemar, Mucor irregularis, Mucor circinelloides, Lichtheimia ramosa, Lichtheimia corymbifera). DNAs of target species were successfully amplified, with no cross reactivity between species. RCA can be considered as a rapid detection method with high specificity and sensitivity, suitable for large screening. © 2014 Blackwell Verlag GmbH.

Electrolyte-free Amperometric Immunosensor using a Dendritic Nanotip†

PubMed Central

Kim, Jong-Hoon; Hiraiwa, Morgan; Lee, Hyun-Boo; Lee, Kyong-Hoon; Cangelosi, Gerard A.; Chung, Jae-Hyun

2013-01-01

Electric detection using a nanocomponent may lead to platforms for rapid and simple biosensing. Sensors composed of nanotips or nanodots have been described for highly sensitive amperometry enabled by confined geometry. However, both fabrication and use of nanostructured sensors remain challenging. This paper describes a dendritic nanotip used as an amperometric biosensor for highly sensitive detection of target bacteria. A dendritic nanotip is structured by Si nanowires coated with single-walled carbon nanotubes (SWCNTs) for generation of a high electric field. For reliable measurement using the dendritic structure, Si nanowires were uniformly fabricated by ultraviolet (UV) lithography and etching. The dendritic structure effectively increased the electric current density near the terminal end of the nanotip according to numerical computation. The electrical characteristics of a dendritic nanotip with additional protein layers was studied by cyclic voltammetry and I–V measurement in deionized (DI) water. When the target bacteria dielectrophoretically captured onto a nanotip were bound with fluorescence antibodies, the electric current through DI water decreased. Measurement results were consistent with fluorescence- and electron microscopy. The sensitivity of the amperometry was 10 cfu/sample volume (103 cfu/mL), which was equivalent to the more laborious fluorescence measurement method. The simple configuration of a dendritic nanotip can potentially offer an electrolyte-free detection platform for sensitive and rapid biosensors. PMID:23585927

Electrolyte-free Amperometric Immunosensor using a Dendritic Nanotip.

PubMed

Kim, Jong-Hoon; Hiraiwa, Morgan; Lee, Hyun-Boo; Lee, Kyong-Hoon; Cangelosi, Gerard A; Chung, Jae-Hyun

2013-01-01

Electric detection using a nanocomponent may lead to platforms for rapid and simple biosensing. Sensors composed of nanotips or nanodots have been described for highly sensitive amperometry enabled by confined geometry. However, both fabrication and use of nanostructured sensors remain challenging. This paper describes a dendritic nanotip used as an amperometric biosensor for highly sensitive detection of target bacteria. A dendritic nanotip is structured by Si nanowires coated with single-walled carbon nanotubes (SWCNTs) for generation of a high electric field. For reliable measurement using the dendritic structure, Si nanowires were uniformly fabricated by ultraviolet (UV) lithography and etching. The dendritic structure effectively increased the electric current density near the terminal end of the nanotip according to numerical computation. The electrical characteristics of a dendritic nanotip with additional protein layers was studied by cyclic voltammetry and I-V measurement in deionized (DI) water. When the target bacteria dielectrophoretically captured onto a nanotip were bound with fluorescence antibodies, the electric current through DI water decreased. Measurement results were consistent with fluorescence- and electron microscopy. The sensitivity of the amperometry was 10 cfu/sample volume (10 3 cfu/mL), which was equivalent to the more laborious fluorescence measurement method. The simple configuration of a dendritic nanotip can potentially offer an electrolyte-free detection platform for sensitive and rapid biosensors.

Evaluation of a rapid immunodiagnostic test kit for rabies virus.

PubMed

Kang, BoKyu; Oh, JinSik; Lee, ChulSeung; Park, Bong-Kyun; Park, YoungNam; Hong, KyungSoo; Lee, KyungGi; Cho, ByungKi; Song, DaeSub

2007-10-01

A rapid immunodiagnostic test kit for rabies virus detection was evaluated using 51 clinical samples and 4 isolates of rabies virus. The quick detection of rabies virus under field conditions may be helpful in determining if post-exposure prophylaxis is needed, thereby avoiding unnecessary treatments, as well as undue economic burden. There are several widely used diagnostic methods for rabies, including fluorescent antibody tests, reverse transcription polymerase chain reaction, and electron microscopy; however, these methods include time-consuming, intricate, and costly procedures. The rapid immunodiagnostic test was able to detect rabies virus in clinical samples, including brain tissue and saliva, in addition to 10(3.2) 50% lethal dose (LD(50))/mL cell-adapted rabies virus. The assay was not cross-reactive with non-rabies virus microbes. When the performance of the rapid immunodiagnostic test was compared to a fluorescent antibody test, the rapid immunodiagnostic test had a sensitivity of 91.7% and specificity of 100% (95.8% CI).

Utility of a stressed-SNP real-time PCR assay for the rapid identification of measles vaccine strain in patient samples.

PubMed

Tran, Thomas; Kostecki, Renata; Catton, Michael; Druce, Julian

2018-05-09

Rapid differentiation of wild-type measles virus from measles vaccine strains is crucial during a measles outbreak and in a measles elimination setting. A real-time RT-PCR for the rapid detection of measles vaccine strains was developed with high specificity and greater sensitivity than when compared to traditional measles genotyping methods. The "stressed" minor grove binder TaqMan probe design approach achieves specificity to vaccine strains only, without compromising sensitivity. This assay has proven to be extremely useful in outbreak settings, without requiring sequence genotyping, for over 4 years at the Regional Measles Reference Laboratory for the Western Pacific Region. Copyright © 2018 Tran et al.

Rapid Detection of Staphylococcus aureus and Methicillin-Resistant S. aureus (MRSA) in Wound Specimens and Blood Cultures: Multicenter Preclinical Evaluation of the Cepheid Xpert MRSA/SA Skin and Soft Tissue and Blood Culture Assays▿

PubMed Central

Wolk, D. M.; Struelens, M. J.; Pancholi, P.; Davis, T.; Della-Latta, P.; Fuller, D.; Picton, E.; Dickenson, R.; Denis, O.; Johnson, D.; Chapin, K.

2009-01-01

A multicenter preclinical evaluation was conducted to evaluate the performance of two Cepheid Xpert assays for detection of methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus. Sensitivity was 97.1% and 98.3% for MRSA in wound and blood culture specimens, respectively. Sensitivity was 100% for S. aureus from both specimen types. PMID:19144803

Amperometric biosensor based on a single antibody of dual function for rapid detection of Streptococcus agalactiae.

PubMed

Vásquez, Gersson; Rey, Alba; Rivera, Camilo; Iregui, Carlos; Orozco, Jahir

2017-01-15

Pathogenic bacteria are responsible for several diseases in humans and in a variety of hosts. Detection of pathogenic bacteria is imperative to avoid and/or fight their potential harmful effects. This work reports on the first amperometric biosensor for the rapid detection of Streptococcus agalactiae (S. agalactiae). The biosensor relies on a single biotinylated antibody that immobilizes the bacteria on a screen-printed carbon electrode while is further linked to a streptavidin-conjugated HRP reporter. The biotinylated antibody provides selectivity to the biosensor whereas serves as an anchoring point to the reporter for further amplification of the electrochemical signal. The resultant immunosensor is simple, responds rapidly, and allows for the selective and highly sensitive quantification of S. agalactiae cells in a concentration range of 10 1 -10 7 CFUml -1 , with a detection limit of 10CFUml -1 . The approach not only enables a rapid detection and quantification of S. agalactiae in environmental samples but also opens up new opportunities for the simple fabrication of electrochemical immunosensors for different target pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

Rapid detection of urinary polyomavirus BK by heterodyne-based surface plasmon resonance biosensor

NASA Astrophysics Data System (ADS)

Su, Li-Chen; Tian, Ya-Chung; Chang, Ying-Feng; Chou, Chien; Lai, Chao-Sung

2014-01-01

In renal transplant patients, immunosuppressive therapy may result in the reactivation of polyomavirus BK (BKV), leading to polyomavirus-associated nephropathy (PVAN), which inevitably causes allograft failure. Since the treatment outcomes of PVAN remain unsatisfactory, early identification and continuous monitoring of BKV reactivation and reduction of immunosuppressants are essential to prevent PVAN development. The present study demonstrated that the developed dual-channel heterodyne-based surface plasmon resonance (SPR) biosensor is applicable for the rapid detection of urinary BKV. The use of a symmetrical reference channel integrated with the poly(ethylene glycol)-based low-fouling self-assembled monolayer to reduce the environmental variations and the nonspecific noise was proven to enhance the sensitivity in urinary BKV detection. Experimentally, the detection limit of the biosensor for BKV detection was estimated to be around 8500 copies/mL. In addition, urine samples from five renal transplant patients were tested to rapidly distinguish PVAN-positive and PVAN-negative renal transplant patients. By virtue of its simplicity, rapidity, and applicability, the SPR biosensor is a remarkable potential to be used for continuous clinical monitoring of BKV reactivation.

A rapid and ultrasensitive SERRS assay for histidine and tyrosine based on azo coupling.

PubMed

Sui, Huimin; Wang, Yue; Yu, Zhi; Cong, Qian; Han, Xiao Xia; Zhao, Bing

2016-10-01

A simple and highly sensitive surface-enhanced resonance Raman scattering (SERRS)-based approach coupled with azo coupling reaction has been put forward for quantitative analysis of histidine and tyrosine. The SERRS-based assay is simple and rapid by mixing the azo reaction products with silver nanoparticles (AgNPs) for measurements within 2min. The limits of detection (LODs) of the method are as low as 4.33×10(-11) and 8.80×10(-11)M for histidine and tyrosine, respectively. Moreover, the SERRS fingerprint information specific to corresponding amino acids guarantees the selective detection for the target histidine and tyrosine. The results from serum indicated the potential application of the proposed approach into biological samples. Compared with the methods ever reported, the main advantages of this methodology are simpleness, rapidity without time-consuming separation or pretreatment steps, high sensitivity, selectivity and the potential for determination of other molecules containing imidazole or phenol groups. Copyright © 2016 Elsevier B.V. All rights reserved.

Convenient Detection of the Citrus Greening (Huanglongbing) Bacterium ‘Candidatus Liberibacter asiaticus’ by Direct PCR from the Midrib Extract

PubMed Central

Fujikawa, Takashi; Miyata, Shin-Ichi; Iwanami, Toru

2013-01-01

A phloem-limited bacterium, ‘Candidatus Liberibacter asiaticus’ (Las) is a major pathogen of citrus greening (huanglongbing), one of the most destructive citrus diseases worldwide. The rapid identification and culling of infected trees and budwoods in quarantine are the most important control measures. DNA amplification including conventional polymerase chain reaction (PCR) has commonly been used for rapid detection and identification. However, long and laborious procedures for DNA extraction have greatly reduced the applicability of this method. In this study, we found that the Las bacterial cells in the midribs of infected leaves were extracted rapidly and easily by pulverization and centrifugation with mini homogenization tubes. We also found that the Las bacterial cells in the midrib extract were suitable for highly sensitive direct PCR. The performance of direct PCR using this extraction method was not inferior to that of conventional PCR. Thus, the direct PCR method described herein is characterized by its simplicity, sensitivity, and robustness, and is applicable to quarantine testing. PMID:23437295

Integrated Magneto-Chemical Sensor For On-Site Food Allergen Detection.

PubMed

Lin, Hsing-Ying; Huang, Chen-Han; Park, Jongmin; Pathania, Divya; Castro, Cesar M; Fasano, Alessio; Weissleder, Ralph; Lee, Hakho

2017-10-24

Adverse food reactions, including food allergies, food sensitivities, and autoimmune reaction (e.g., celiac disease) affect 5-15% of the population and remain a considerable public health problem requiring stringent food avoidance and epinephrine availability for emergency events. Avoiding problematic foods is practically difficult, given current reliance on prepared foods and out-of-home meals. In response, we developed a portable, point-of-use detection technology, termed integrated exogenous antigen testing (iEAT). The system consists of a disposable antigen extraction device coupled with an electronic keychain reader for rapid sensing and communication. We optimized the prototype iEAT system to detect five major food antigens in peanuts, hazelnuts, wheat, milk, and eggs. Antigen extraction and detection with iEAT requires <10 min and achieves high-detection sensitivities (e.g., 0.1 mg/kg for gluten, lower than regulatory limits of 20 mg/kg). When testing under restaurant conditions, we were able to detect hidden food antigens such as gluten within "gluten-free" food items. The small size and rapid, simple testing of the iEAT system should help not only consumers but also other key stakeholders such as clinicians, food industries, and regulators to enhance food safety.

A method of multiplex PCR for detection of field released Beauveria bassiana, a fungal entomopathogen applied for pest management in jute (Corchorus olitorius).

PubMed

Biswas, Chinmay; Dey, Piyali; Gotyal, B S; Satpathy, Subrata

2015-04-01

The fungal entomopathogen Beauveria bassiana is a promising biocontrol agent for many pests. Some B. bassiana strains have been found effective against jute pests. To monitor the survival of field released B. bassiana a rapid and efficient detection technique is essential. Conventional methods such as plating method or direct culture method which are based on cultivation on selective media followed by microscopy are time consuming and not so sensitive. PCR based methods are rapid, sensitive and reliable. A single primer PCR may fail to amplify some of the strains. However, multiplex PCR increases the possibility of detection as it uses multiple primers. Therefore, in the present investigation a multiplex PCR protocol was developed by multiplexing three primers SCA 14, SCA 15 and SCB 9 to detect field released B. bassiana strains from soil as well as foliage of jute field. Using our multiplex PCR protocol all the five B. bassiana strains could be detected from soil and three strains viz., ITCC 6063, ITCC 4563 and ITCC 4796 could be detected even from the crop foliage after 45 days of spray.

Study on validity of a rapid diagnostic test kit versus light microscopy for malaria diagnosis in Ahmedabad city, India.

PubMed

Vyas, S; Puwar, B; Patel, V; Bhatt, G; Kulkarni, S; Fancy, M

2014-05-01

Light microscopy of blood smears for diagnosis of malaria in the field has several limitations, notably delays in diagnosis. This study in Ahmedabad in Gujarat State, India, evaluated the diagnostic performance of a rapid diagnostic test for malaria (SD Bioline Malaria Ag P.f/Pan) versus blood smear examination as the gold standard. All fever cases presenting at 13 urban health centres were subjected to rapid diagnostic testing and thick and thin blood smears. A total of 677 cases with fever were examined; 135 (20.0%) tested positive by rapid diagnostic test and 86 (12.7%) by blood smear. The sensitivity of the rapid diagnostic test for malaria was 98.8%, specificity was 91.5%, positive predictive value 63.0% and negative predictive value 99.8%. For detection of Plasmodium falciparum the sensitivity of rapid diagnostic test was 100% and specificity was 97.3%. The results show the acceptability of the rapid test as an alternative to light microscopy in the field setting.

Microfluidic biosensor for β-Hydroxybutyrate (βHBA) determination of subclinical ketosis diagnosis.

PubMed

Weng, Xuan; Zhao, Wenting; Neethirajan, Suresh; Duffield, Todd

2015-02-12

Determination of β-hydroxybutyrate (βHBA) is a gold standard for diagnosis of Subclinical Ketosis (SCK), a common disease in dairy cows that causes significant economic loss. Early detection of SCK can help reduce the risk of the disease progressing into clinical stage, thus minimizing economic losses on dairy cattle. Conventional laboratory methods are time consuming and labor-intensive, requiring expensive and bulky equipment. Development of portable and robust devices for rapid on-site SCK diagnosis is an effective way to prevent and control ketosis and can significantly aid in the management of dairy animal health. Microfluidic technology provides a rapid, cost-effective way to develop handheld devices for on-farm detection of sub-clinical ketosis. In this study, a highly sensitive microfluidics-based biosensor for on-site SCK diagnosis has been developed. A rapid, low-cost microfluidic biosensor with high sensitivity and specificity was developed for SCK diagnosis. Determination of βHBA was employed as the indicator in the diagnosis of SCK. On-chip detection using miniaturized and cost-effective optical sensor can be finished in 1 minute with a detection limit of 0.05 mM concentration. Developed microfluidic biosensor was successfully tested with the serum samples from dairy cows affected by SCK. The results of the developed biosensor agreed well with two other laboratory methods. The biosensor was characterized by high sensitivity and specificity towards βHBA with a detection limit of 0.05 mM. The developed microfluidic biosensor provides a promising prototype for a cost-effective handheld meter for on-site SCK diagnosis. By using microfluidic method, the detection time is significantly decreased compared to other laboratory methods. Here, we demonstrate a field-deployable device to precisely identify and measure subclinical ketosis by specific labeling and quantification of β-hydroxybutyate in cow blood samples. A real-time on-site detection system will maximize convenience for the farmers.

Diagnostic Sensitivity and Specificity of Dark Adaptometry for Detection of Age-Related Macular Degeneration

PubMed Central

Jackson, Gregory R.; Scott, Ingrid U.; Kim, Ivana K.; Quillen, David A.; Iannaccone, Alessandro; Edwards, John G.

2014-01-01

Purpose. Difficulty with night vision is a common complaint of patients with age-related macular degeneration (AMD). Consistent with this complaint, dark adaptation (DA) is substantially impaired in these patients. Because of the severity of the deficit, measurement of DA has been suggested as a means for the diagnosis of AMD. Previous methods for measurement of DA were time intensive (>30 minutes), which made them unsuitable for clinical use. This study evaluated a rapid DA test (≤6.5 minutes) for the detection of AMD. Methods. Dark adaptation was measured by using the AdaptDx dark adaptometer in two groups: subjects with normal retinal health and subjects with AMD. Subjects were assigned to their group by clinical examination and grading of fundus photographs. Subjects were classified as having DA consistent with normal retinal health (rod intercept ≤ 6.5 minutes) or having dark adaptation consistent with AMD (rod intercept > 6.5 minutes). Results. The eligible sample for analysis included 21 normal adults and 127 AMD patients. The rapid test was found to have a diagnostic sensitivity of 90.6% (P < 0.001) and specificity of 90.5% (P < 0.027). Thus, abnormal DA was detected in 115 of 127 AMD patients, and normal DA was found in 19 of 21 normal adults. Conclusions. The high diagnostic sensitivity and specificity compared favorably to long-duration research methods for the measurement of DA, and slit lamp biomicroscopy performed by a retina specialist. These results suggest that a rapid DA test is useful for the detection of AMD. PMID:24550363

Rapid detection of acetamiprid in foods using surface-enhanced Raman spectroscopy (SERS).

PubMed

Wijaya, Wisiani; Pang, Shintaro; Labuza, Theodore P; He, Lili

2014-04-01

Acetamiprid is a neonicotinoid pesticide that is commonly used in modern farming. Acetamiprid residue in food commodities can be a potential harm to human and has been implicated in the honey bee hive die off crisis. In this study, we developed rapid, simple, and sensitive methods to detect acetamiprid in apple juice and on apple surfaces using surface-enhanced Raman spectroscopy (SERS). No pretreatment of apple juice sample was performed. A simple surface swab method was used to recover acetamiprid from the apple surface. Samples were incubated with silver dendrites for several minutes and SERS spectra were taken directly from the silver surface. Detection of a set of 5 apple juice samples can be done within 10 min. The swab-SERS method took 15 min for a set of 5 samples. Resulting spectral data were analyzed using principal component analysis. The highest acetamiprid peak at 634 cm(-1) was used to detect and quantify the amount of acetamiprid spiked in 1:1 water-methanol solvent, apple juice, and on apple surface. The SERS method was able to successfully detect acetamiprid at 0.5 μg/mL (0.5 ppm) in solvent, 3 μg/mL (3 ppm) in apple juice, and 0.125 μg/cm(2) on apple surfaces. The SERS methods provide simple, rapid, and sensitive ways to detect acetamiprid in beverages and on the surfaces of thick skinned fruits and vegetables. © 2014 Institute of Food Technologists®

Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus.

PubMed

Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

2016-10-01

Eucalyptus dieback disease, caused by Cylindrocladium scoparium , has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium . The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus

PubMed Central

Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

2016-01-01

Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products. PMID:27721691

Comparative Evaluation of Four Phenotypic Tests for Detection of Carbapenemase-Producing Gram-Negative Bacteria

PubMed Central

Noël, Audrey; Berhin, Catherine; Hoebeke, Martin; Bouchahrouf, Warda; Yunus, Sami; Bogaerts, Pierre; Glupczynski, Youri

2016-01-01

ABSTRACT Four screening assays aimed for rapid detection of carbapenemase production from Gram-negative bacterial isolates, i.e., the Neo-Rapid Carb kit (Rosco Diagnostica A/S), the Rapidec Carba NP test (bioMérieux SA), the β Carba test (Bio-Rad Laboratories N.V.), and a homemade electrochemical assay (BYG Carba test) were evaluated against a panel comprising 328 clinical isolates (Enterobacteriaceae [n = 198] and nonfermentative Gram-negative bacilli [n = 130]) with previously characterized resistance mechanisms to carbapenems. Among Enterobacteriaceae isolates, the BYG Carba test and the β Carba test showed excellent sensitivities (respectively, 100% and 97.3%) and specificities (respectively, 98.9% and 97.7%). The two other assays yielded poorer performances with sensitivity and specificity of 91.9% and 83.9% for the Rapidec Carba NP test and of 89.2% and 89.7% for the Neo-Rapid Carb kit, respectively. Among Pseudomonas spp., sensitivities and specificities ranged, respectively, from 87.3% to 92.7% and from 88.2% to 94.1%. Finally, all tests performed poorly against Acinetobacter spp., with sensitivities and specificities, respectively, ranging from 27.3% to 75.8% and from 75 to 100%. Among commercially available assays, the β Carba test appeared to be the most convenient for routine use and showed the best overall performances, especially against OXA-48-like producers. The excellent performance of the BYG Carba test against Enterobacteriaceae was confirmed (100% sensitivity and 98.9% specificity). PMID:27927915

Rapid and PCR-free DNA detection by nanoaggregation-enhanced chemiluminescence

Treesearch

Renu Singh; Alexandra Feltmeyer; Olga Saiapina; Jennifer Juzwik; Brett Arenz; Abdennour Abbas

2017-01-01

The aggregation of gold nanoparticles (AuNPs) is known to induce an enhancement of localized surface plasmon resonance due to the coupling of plasmonic fields of adjacent nanoparticles. Here we show that AuNPs aggregation also causes a significant enhancement of chemiluminescence in the presence of luminophores. The phenomenon is used to introduce a rapid and sensitive...

APTEC: aptamer-tethered enzyme capture as a novel rapid diagnostic test for malaria.

PubMed

Dirkzwager, Roderick M; Kinghorn, Andrew B; Richards, Jack S; Tanner, Julian A

2015-03-18

We report the rapid diagnosis of malaria by aptamer-tethered enzyme capture (APTEC) whereby an aptamer captures biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) then activity is measured colorimetrically. The robust test was sensitive (limit of detection = 4.9 ng mL(-1)) and could reliably diagnose malaria in clinical blood samples.

Molecular Detection and Species-Specific Identification of Medically Important Aspergillus Species by Real-Time PCR in Experimental Invasive Pulmonary Aspergillosis ▿

PubMed Central

Walsh, Thomas J.; Wissel, Mark C.; Grantham, Kevin J.; Petraitiene, Ruta; Petraitis, Vidmantas; Kasai, Miki; Francesconi, Andrea; Cotton, Margaret P.; Hughes, Johanna E.; Greene, Lora; Bacher, John D.; Manna, Pradip; Salomoni, Martin; Kleiboeker, Steven B.; Reddy, Sushruth K.

2011-01-01

Diagnosis of invasive pulmonary aspergillosis (IPA) remains a major challenge to clinical microbiology laboratories. We developed rapid and sensitive quantitative PCR (qPCR) assays for genus- and species-specific identification of Aspergillus infections by use of TaqMan technology. In order to validate these assays and understand their potential diagnostic utility, we then performed a blinded study of bronchoalveolar lavage (BAL) fluid specimens from well-characterized models of IPA with the four medically important species. A set of real-time qPCR primers and probes was developed by utilizing unique ITS1 regions for genus- and species-specific detection of the four most common medically important Aspergillus species (Aspergillus fumigatus, A. flavus, A. niger, and A. terreus). Pan-Aspergillus and species-specific qPCRs with BAL fluid were more sensitive than culture for detection of IPA caused by A. fumigatus in untreated (P < 0.0007) and treated (P ≤ 0.008) animals, respectively. For infections caused by A. terreus and A. niger, culture and PCR amplification from BAL fluid yielded similar sensitivities for untreated and treated animals. Pan-Aspergillus PCR was more sensitive than culture for detection of A. flavus in treated animals (P = 0.002). BAL fluid pan-Aspergillus and species-specific PCRs were comparable in sensitivity to BAL fluid galactomannan (GM) assay. The copy numbers from the qPCR assays correlated with quantitative cultures to determine the pulmonary residual fungal burdens in lung tissue. Pan-Aspergillus and species-specific qPCR assays may improve the rapid and accurate identification of IPA in immunocompromised patients. PMID:21976757

Rapid screening for inflammatory neuropathies by standardized clinical criteria

PubMed Central

Tramontozzi, Louis A.

2016-01-01

Abstract Background: Delay in recognition and treatment of inflammatory neuropathies increases morbidity and mortality. We have developed and standardized 3 clinical screening criteria that rapidly detect inflammatory neuropathies. Methods: We reviewed all patients with definite large fiber neuropathy in 2 different patient populations: 1 from a private neurology clinic and the other from a tertiary care center. Patients were divided into 2 groups: those with an inflammatory neuropathy and those with a noninflammatory neuropathy. We specifically noted the 3 key neuropathy characteristics: onset, distribution, and associated systemic features (ODS). We studied the sensitivity and specificity of ODS in differentiating between inflammatory and noninflammatory neuropathies. Results: A total of 206 patients were included: 51 from the private clinic and 155 from the tertiary care center. The sensitivity of using ODS in detecting an inflammatory neuropathy was 96% and the specificity was 85%. The positive predictive value of ODS was 0.8 and negative predictive value was 0.97. Conclusions: Rapid screening for inflammatory neuropathies by ODS clinical criteria is highly sensitive and has a high negative predictive value for noninflammatory neuropathies. ODS uses simple clinical criteria to rapidly screen for patients with a potentially treatable form of neuropathy and accelerate their diagnostic evaluation. Classification of evidence: This study provides Class IV evidence that 3 neuropathy characteristics—onset, distribution, and associated systemic features—accurately identify patients with inflammatory neuropathies. PMID:29443273

Specific detection of Angiostrongylus cantonensis in the snail Achatina fulica using a loop-mediated isothermal amplification (LAMP) assay.

PubMed

Liu, Chun-Yan; Song, Hui-Qun; Zhang, Ren-Li; Chen, Mu-Xin; Xu, Min-Jun; Ai, Lin; Chen, Xiao-Guang; Zhan, Xi-Mei; Liang, Shao-Hui; Yuan, Zi-Guo; Lin, Rui-Qing; Zhu, Xing-Quan

2011-08-01

Angiostrongylus cantonensis, a rat lungworm, can cause eosinophilic meningitis and angiostrongyliasis in humans following ingestion of contaminated foods or intermediate/paratenic hosts with infective larvae. The snail Achatina fulica is one of the important intermediate hosts of A. cantonensis and is commonly eaten by humans in some countries. In the present study, we developed a loop-mediated isothermal amplification (LAMP) method for the specific detection of A. cantonensis in Ac. fulica. Primers for LAMP were designed based on the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA) of A. cantonensis. Specificity tests showed that only the products of A. cantonensis were detected when DNA samples of A. cantonensis and the heterologous control samples Anisakis simplex s.s, Trichuris trichiura, Toxocara canis, Trichinella spiralis and Ascaris lumbricoides were amplified by LAMP. Sensitivity evaluation indicated that the LAMP assay is 10 times more sensitive than the conventional polymerase chain reaction (PCR) assay. The established LAMP assay is rapid, inexpensive and easy to be performed. It can be used in clinical applications for rapid and sensitive detection of A. cantonensis in snails, which has implications for the effective control of angiostrongyliasis. Copyright © 2011 Elsevier Ltd. All rights reserved.

Grapefruit photonic crystal fiber long period gratings sensor for DNT sensing application

NASA Astrophysics Data System (ADS)

Tao, Chuanyi; Li, Jingke; Zhu, Tenglong

2016-10-01

The detection of explosives and their residues is of great importance in public health, antiterrorism and homeland security applications. The vapor pressures of most explosive compounds are extremely low and attenuation of the available vapor is often great due to diffusion in the environment, making direct vapor detection difficult. In reality bomb dogs are still the most efficient way to quickly detect explosives on the spot. Many formulations of TNT-based explosives contain DNT residues. The use of long period gratings (LPGs) formed in grapefruit photonic crystal fiber (PCF) with thin-film overlay coated on the inner surface of air holes for gas sensing is demonstrated. A gas analyteinduced index variation of the thin-film immobilized on the inner surface of the holey region of the fiber can be observed by a shift of the resonance wavelength. We demonstrate a 2,4-dinitrotoluene (DNT) sensor using grapefruit PCF-LPGs. Coating with gas-sensitive thin-film on the inner surface of the air holes of the grapefruit PCF-LPG could provide a promising platform for rapid highly sensitive gas sensing. A rapid and highly sensitive detection of DNT has been demonstrated using the grapefruit PCF-LPG sensor to show the feasibility of the proposed approach.

Multiplex polymerase chain reaction assay developed to diagnose adult bacterial meningitis in Taiwan.

PubMed

Lee, Chi-Tsung; Hsiao, Kuang-Ming; Chen, Jin-Cherng; Su, Cheng-Chuan

2015-11-01

Acute bacterial meningitis causes high morbidity and mortality; the associated clinical symptoms often are insensitive or non-specific; and the pathogenic bacteria are geographically diverse. Clinical diagnosis requires a rapid and accurate methodology. This study aimed to develop a new multiplex polymerase chain reaction (mPCR) assay to detect simultaneously six major bacteria that cause adult bacterial meningitis in Taiwan: Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, and Acinetobacter baumannii. Species-specific primers for the six bacteria were developed using reference strains. The specificities of the mPCRs for these bacteria were validated, and the sensitivities were evaluated via serial dilutions. The mPCR assay specifically detected all of the six pathogens, particularly with sensitivities of 12 colony forming units (CFU)/mL, 90 CFU/mL, and 390 CFU/mL for E. coli, S. pneumoniae, and K. pneumoniae, respectively. This mPCR assay is a rapid and specific tool to detect the six major bacterial pathogens that cause acute adult meningitis in Taiwan, particularly sensitive for detecting E. coli, S. pneumoniae, and K. pneumoniae. The assay may facilitate early diagnosis and guidance for antimicrobial therapy for adult patients with this deadly disease in Taiwan. © 2015 APMIS. Published by John Wiley & Sons Ltd.

Development of real-time and lateral flow strip reverse transcription recombinase polymerase Amplification assays for rapid detection of peste des petits ruminants virus.

PubMed

Yang, Yang; Qin, Xiaodong; Song, Yiming; Zhang, Wei; Hu, Gaowei; Dou, Yongxi; Li, Yanmin; Zhang, Zhidong

2017-02-07

Peste des petits ruminants (PPR) is an economically important, Office International des Epizooties (OIE) notifiable, transboundary viral disease of small ruminants such as sheep and goat. PPR virus (PPRV), a negative-sense single-stranded RNA virus, is the causal agent of PPR. Therefore, sensitive, specific and rapid diagnostic assay for the detection of PPRV are necessary to accurately and promptly diagnose suspected case of PPR. In this study, reverse transcription recombinase polymerase amplification assays using real-time fluorescent detection (real-time RT-RPA assay) and lateral flow strip detection (LFS RT-RPA assay) were developed targeting the N gene of PPRV. The sensitivity of the developed real-time RT-RPA assay was as low as 100 copies per reaction within 7 min at 40 °C with 95% reliability; while the sensitivity of the developed LFS RT-RPA assay was as low as 150 copies per reaction at 39 °C in less than 25 min. In both assays, there were no cross-reactions with sheep and goat pox viruses, foot-and-mouth disease virus and Orf virus. These features make RPA assay promising candidates either in field use or as a point of care diagnostic technique.

Highly sensitive and specific colorimetric detection of cancer cells via dual-aptamer target binding strategy.

PubMed

Wang, Kun; Fan, Daoqing; Liu, Yaqing; Wang, Erkang

2015-11-15

Simple, rapid, sensitive and specific detection of cancer cells is of great importance for early and accurate cancer diagnostics and therapy. By coupling nanotechnology and dual-aptamer target binding strategies, we developed a colorimetric assay for visually detecting cancer cells with high sensitivity and specificity. The nanotechnology including high catalytic activity of PtAuNP and magnetic separation & concentration plays a vital role on the signal amplification and improvement of detection sensitivity. The color change caused by small amount of target cancer cells (10 cells/mL) can be clearly distinguished by naked eyes. The dual-aptamer target binding strategy guarantees the detection specificity that large amount of non-cancer cells and different cancer cells (10(4) cells/mL) cannot cause obvious color change. A detection limit as low as 10 cells/mL with detection linear range from 10 to 10(5) cells/mL was reached according to the experimental detections in phosphate buffer solution as well as serum sample. The developed enzyme-free and cost effective colorimetric assay is simple and no need of instrument while still provides excellent sensitivity, specificity and repeatability, having potential application on point-of-care cancer diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Multifunctional Nanotechnology-Enabled Sensors for Rapid Capture and Detection of Pathogens

PubMed Central

Mustafa, Fatima; Andreescu, Silvana

2017-01-01

Nanomaterial-based sensing approaches that incorporate different types of nanoparticles (NPs) and nanostructures in conjunction with natural or synthetic receptors as molecular recognition elements provide opportunities for the design of sensitive and selective assays for rapid detection of contaminants. This review summarizes recent advancements over the past ten years in the development of nanotechnology-enabled sensors and systems for capture and detection of pathogens. The most common types of nanostructures and NPs, their modification with receptor molecules and integration to produce viable sensing systems with biorecognition, amplification and signal readout are discussed. Examples of all-in-one systems that combine multifunctional properties for capture, separation, inactivation and detection are also provided. Current trends in the development of low-cost instrumentation for rapid assessment of food contamination are discussed as well as challenges for practical implementation and directions for future research. PMID:28914769

A new strategy for fast radiofrequency CW EPR imaging: Direct detection with rapid scan and rotating gradients

PubMed Central

Subramanian, Sankaran; Koscielniak, Janusz W.; Devasahayam, Nallathamby; Pursley, Randall H.; Pohida, Thomas J.; Krishna, Murali C.

2007-01-01

Rapid field scan on the order of T/s using high frequency sinusoidal or triangular sweep fields superimposed on the main Zeeman field, was used for direct detection of signals without low-frequency field modulation. Simultaneous application of space-encoding rotating field gradients have been employed to perform fast CW EPR imaging using direct detection that could, in principle, approach the speed of pulsed FT EPR imaging. The method takes advantage of the well-known rapid-scan strategy in CW NMR and EPR that allows arbitrarily fast field sweep and the simultaneous application of spinning gradients that allows fast spatial encoding. This leads to fast functional EPR imaging and, depending on the spin concentration, spectrometer sensitivity and detection band width, can provide improved temporal resolution that is important to interrogate dynamics of spin perfusion, pharmacokinetics, spectral spatial imaging, dynamic oxymetry, etc. PMID:17350865

Detection of Botulinum Neurotoxin Serotype A, B, and F Proteolytic Activity in Complex Matrices with Picomolar to Femtomolar Sensitivity

PubMed Central

Dunning, F. Mark; Ruge, Daniel R.; Piazza, Timothy M.; Stanker, Larry H.; Zeytin, Füsûn N.

2012-01-01

Rapid, high-throughput assays that detect and quantify botulinum neurotoxin (BoNT) activity in diverse matrices are required for environmental, clinical, pharmaceutical, and food testing. The current standard, the mouse bioassay, is sensitive but is low in throughput and precision. In this study, we present three biochemical assays for the detection and quantification of BoNT serotype A, B, and F proteolytic activities in complex matrices that offer picomolar to femtomolar sensitivity with small assay volumes and total assay times of less than 24 h. These assays consist of magnetic beads conjugated with BoNT serotype-specific antibodies that are used to purify BoNT from complex matrices before the quantification of bound BoNT proteolytic activity using the previously described BoTest reporter substrates. The matrices tested include human serum, whole milk, carrot juice, and baby food, as well as buffers containing common pharmaceutical excipients. The limits of detection were below 1 pM for BoNT/A and BoNT/F and below 10 pM for BoNT/B in most tested matrices using 200-μl samples and as low as 10 fM for BoNT/A with an increased sample volume. Together, these data describe rapid, robust, and high-throughput assays for BoNT detection that are compatible with a wide range of matrices. PMID:22923410

Digital sensing and sizing of vesicular stomatitis virus pseudotypes in complex media: a model for Ebola and Marburg detection.

PubMed

Daaboul, George G; Lopez, Carlos A; Chinnala, Jyothsna; Goldberg, Bennett B; Connor, John H; Ünlü, M Selim

2014-06-24

Rapid, sensitive, and direct label-free capture and characterization of nanoparticles from complex media such as blood or serum will broadly impact medicine and the life sciences. We demonstrate identification of virus particles in complex samples for replication-competent wild-type vesicular stomatitis virus (VSV), defective VSV, and Ebola- and Marburg-pseudotyped VSV with high sensitivity and specificity. Size discrimination of the imaged nanoparticles (virions) allows differentiation between modified viruses having different genome lengths and facilitates a reduction in the counting of nonspecifically bound particles to achieve a limit-of-detection (LOD) of 5 × 10(3) pfu/mL for the Ebola and Marburg VSV pseudotypes. We demonstrate the simultaneous detection of multiple viruses in a single sample (composed of serum or whole blood) for screening applications and uncompromised detection capabilities in samples contaminated with high levels of bacteria. By employing affinity-based capture, size discrimination, and a "digital" detection scheme to count single virus particles, we show that a robust and sensitive virus/nanoparticle sensing assay can be established for targets in complex samples. The nanoparticle microscopy system is termed the Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and is capable of high-throughput and rapid sizing of large numbers of biological nanoparticles on an antibody microarray for research and diagnostic applications.

Gold nanoparticles based colorimetric nanodiagnostics for cancer and infectious diseases

NASA Astrophysics Data System (ADS)

Valentini, Paola; Persano, Stefano; Cecere, Paola; Sabella, Stefania; Pompa, Pier Paolo

2014-03-01

Traditional in vitro diagnostics requires specialized laboratories and costly instrumentation, both for the amplification of nucleic acid targets (usually achieved by PCR) and for the assay readout, often based on fluorescence. We are developing hybrid nanomaterials-based sensors for the rapid and low-cost diagnosis of various disease biomarkers, for applications in portable platforms for diagnostics at the point-of-care. To this aim, we exploited the size and distancedependent optical properties of gold nanoparticles (AuNPs) to achieve colorimetric detection. Moreover, in order to avoid the complexity of thermal cycles associated to traditional PCR, the design of our systems includes signal amplification schemes, achieved by the use of enzymes (nucleases, helicase) or DNAzymes. Focused on instrument-free and sensitive detection, we carefully combined the intrinsic sensitivity by multivalency of functionalized AuNPs with isothermal and non-stringent enzyme-aided reaction conditions, controlled AuNPs aggregates, universal reporters and magnetic microparticles, the latter used both as a substrate and as a means for the colorimetric detection. We obtained simple and robust assays for the sensitive (pM range or better) naked-eye detection of cancer or infectious diseases (HPV, HCV) biomarkers, requiring no instrumentation except for a simple heating plate. Finally, we are also developing non-medical applications of these bio-nanosensors, such as in the development of on-field rapid tests for the detection of pollutants and other food and water contaminants.

Carbon Nanomaterials Based Electrochemical Sensors/Biosensors for the Sensitive Detection of Pharmaceutical and Biological Compounds

PubMed Central

Adhikari, Bal-Ram; Govindhan, Maduraiveeran; Chen, Aicheng

2015-01-01

Electrochemical sensors and biosensors have attracted considerable attention for the sensitive detection of a variety of biological and pharmaceutical compounds. Since the discovery of carbon-based nanomaterials, including carbon nanotubes, C60 and graphene, they have garnered tremendous interest for their potential in the design of high-performance electrochemical sensor platforms due to their exceptional thermal, mechanical, electronic, and catalytic properties. Carbon nanomaterial-based electrochemical sensors have been employed for the detection of various analytes with rapid electron transfer kinetics. This feature article focuses on the recent design and use of carbon nanomaterials, primarily single-walled carbon nanotubes (SWCNTs), reduced graphene oxide (rGO), SWCNTs-rGO, Au nanoparticle-rGO nanocomposites, and buckypaper as sensing materials for the electrochemical detection of some representative biological and pharmaceutical compounds such as methylglyoxal, acetaminophen, valacyclovir, β-nicotinamide adenine dinucleotide hydrate (NADH), and glucose. Furthermore, the electrochemical performance of SWCNTs, rGO, and SWCNT-rGO for the detection of acetaminophen and valacyclovir was comparatively studied, revealing that SWCNT-rGO nanocomposites possess excellent electrocatalytic activity in comparison to individual SWCNT and rGO platforms. The sensitive, reliable and rapid analysis of critical disease biomarkers and globally emerging pharmaceutical compounds at carbon nanomaterials based electrochemical sensor platforms may enable an extensive range of applications in preemptive medical diagnostics. PMID:26404304

Fast real-time polymerase chain reaction for quantitative detection of Lactobacillus delbrueckii bacteriophages in milk.

PubMed

Martín, Maria Cruz; del Rio, Beatriz; Martínez, Noelia; Magadán, Alfonso H; Alvarez, Miguel A

2008-12-01

One of the main microbiological problems of the dairy industry is the susceptibility of starter bacteria to virus infections. Lactobacillus delbrueckii, a component of thermophilic starter cultures used in the manufacture of several fermented dairy products, including yogurt, is also sensitive to bacteriophage attacks. To avoid the problems associated with these viruses, quick and sensitive detection methods are necessary. In the present study, a fast real-time quantitative polymerase chain reaction assay for the direct detection and quantification of L. delbrueckii phages in milk was developed. A set of primers and a TaqMan MGB probe was designed, based on the lysin gene sequence of different L. delbrueckii phages. The results show the proposed method to be a rapid (total processing time 30 min), specific and highly sensitive technique for detecting L. delbrueckii phages in milk.

Rapid Prototyping of a High Sensitivity Graphene Based Glucose Sensor Strip.

PubMed

Tehrani, Farshad; Reiner, Lisa; Bavarian, Behzad

2015-01-01

A rapid prototyping of an inexpensive, disposable graphene and copper nanocomposite sensor strip using polymeric flexible substrate for highly sensitive and selective nonenzymatic glucose detection has been developed and tested for direct oxidization of glucose. The CuNPs were electrochemically deposited on to the graphene sheets to improve electron transfer rates and to enhance electrocatalytic activity toward glucose. The graphene based electrode with CuNPs demonstrated a high degree of sensitivity (1101.3 ± 56 μA/mM.cm2), excellent selectivity (without an interference with Ascorbic Acid, Uric Acid, Dopamine, and Acetaminophen), good stability with a linear response to glucose ranging from 0.1 mM to 0.6 mM concentration, and detection limits of 0.025 mM to 0.9 mM. Characterization of the electrodes was performed by scanning electron microscopy (FESEM and SEM). The electrochemical properties of the modified graphene electrodes were inspected by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and amperometry.

Rapid and sensitive method for determination of withaferin-A in human plasma by HPLC.

PubMed

Patial, Pankaj; Gota, Vikram

2011-02-01

To develop and validate a rapid and sensitive high-performance liquid chromatographic method for determination of withaferin-A in human plasma. Withaferin-A, the active molecule of a traditional Indian herb, has demonstrated several biological activities in preclinical models. A validated bioassay is not available for its pharmacokinetic evaluation. The chromatographic system used a reverse-phase C18 column with UV-visible detection at 225 nm. The mobile phase consisted of water and acetonitrile applied in a gradient flow. Withaferin-A was extracted by simple protein-precipitation technique. The calibration curve was linear in the concentration range of 0.05-1.6 µg/ml. The method has the desired sensitivity to detect the plasma concentration range of withaferin-A that is likely to show biological activity based on in vitro data. This is the first HPLC method ever described for the estimation of withaferin-A in human plasma which could be applied for pharmacokinetic studies.

Rapid Prototyping of a High Sensitivity Graphene Based Glucose Sensor Strip

PubMed Central

Tehrani, Farshad; Reiner, Lisa; Bavarian, Behzad

2015-01-01

A rapid prototyping of an inexpensive, disposable graphene and copper nanocomposite sensor strip using polymeric flexible substrate for highly sensitive and selective nonenzymatic glucose detection has been developed and tested for direct oxidization of glucose. The CuNPs were electrochemically deposited on to the graphene sheets to improve electron transfer rates and to enhance electrocatalytic activity toward glucose. The graphene based electrode with CuNPs demonstrated a high degree of sensitivity (1101.3±56 μA/mM.cm2), excellent selectivity (without an interference with Ascorbic Acid, Uric Acid, Dopamine, and Acetaminophen), good stability with a linear response to glucose ranging from 0.1 mM to 0.6 mM concentration, and detection limits of 0.025 mM to 0.9 mM. Characterization of the electrodes was performed by scanning electron microscopy (FESEM and SEM). The electrochemical properties of the modified graphene electrodes were inspected by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and amperometry. PMID:26678700

Effectiveness of rapid prescreening and 10% rescreening in liquid-based Papanicolaou testing.

PubMed

Currens, Heather S; Nejkauf, Katharine; Wagner, Lynn; Raab, Stephen S

2012-01-01

Although rapid prescreening (RPS) has been shown to be an effective quality control procedure for detecting false-negative conventional Papanicolaou (Pap) tests, RPS has not been widely implemented in the United States. In our laboratory, cytotechnologists performed RPS in 3,567 liquid-based Pap tests: 1,911 SurePath (BD Diagnostics-TriPath, Burlington, NC) preparations that were manually screened and 1,656 ThinPrep Pap tests (Hologic, Bedford, MA) that were imaged using the ThinPrep Imaging System (Hologic). We compared the sensitivity of RPS, 10% rescreening (R-10%), and routine screening (RS). In contrast with previously published findings, we found that RS + RPS did not improve screening sensitivity compared with RS + R-10%. These results support the following hypotheses: (1) Higher baseline RS sensitivity as a result of Pap test diagnoses standardization implemented for quality improvement purposes decreases the performance impact of RPS. (2) R-10% and RPS quality assurance methods detect diagnostic failures caused by different types of cognitive errors.

Evaluation of the Clearview® malaria pLDH malaria rapid diagnostic test in a non-endemic setting

PubMed Central

2011-01-01

Background Malaria Rapid Diagnostic Tests (RDTs) are widely used to diagnose malaria. The present study evaluated a new RDT, the Clearview® Malaria pLDH test targeting the pan-Plasmodium antigen lactate dehydrogenase (pLDH). Methods The Clearview® Malaria pLDH test was evaluated on fresh samples obtained in returned international travellers using microscopy corrected by PCR as the reference method. Included samples were Plasmodium falciparum (139), Plasmodium vivax (22), Plasmodium ovale (20), Plasmodium malariae (7), and 102 negative. Results Overall sensitivity for the detection of Plasmodium spp was 93.2%. For P. falciparum, the sensitivity was 98.6%; for P. vivax, P. ovale and P. malariae, overall sensitivities were 90.9%, 60.0% and 85.7% respectively. For P. falciparum and for P. vivax, the sensitivities increased to 100% at parasite densities above 100/μl. The specificity was 100%. The test was easily to perform and the result was stable for at least 1 hour. Conclusion The Clearview® Malaria pLDH was efficient for the diagnosis of malaria. The test was very sensitive for P. falciparum and P. vivax detection. The sensitivities for P. ovale and P. malariae were better than other RDTs PMID:21951996

Development and Evaluation of a Rapid and Sensitive EBOV-RPA Test for Rapid Diagnosis of Ebola Virus Disease.

PubMed

Yang, Mingjuan; Ke, Yuehua; Wang, Xuesong; Ren, Hang; Liu, Wei; Lu, Huijun; Zhang, Wenyi; Liu, Shiwei; Chang, Guohui; Tian, Shuguang; Wang, Lihua; Huang, Liuyu; Liu, Chao; Yang, Ruifu; Chen, Zeliang

2016-06-01

Confirming Ebola virus disease (EVD), a deadly infectious disease, requires real-time RT-PCR, which takes up to a few hours to yield results. Therefore, a rapid diagnostic assay is imperative for EVD diagnosis. A rapid nucleic acid test based on recombinase polymerase amplification (EBOV-RPA) was developed to specifically detect the 2014 outbreak strains. The EBOV-RPA assay was evaluated by testing samples from suspected EVD patients in parallel with RT-PCR. An EBOV-RPA, which could be completed in 20 min, was successfully developed. Of 271 patients who tested positive for Ebola virus by RT-PCR, 264 (sensitivity: 97%, 95% CI: 95.5-99.3%) were positive by EBOV-RPA; 101 of 104 patients (specificity: 97%, 95% CI: 93.9-100%) who tested negative by RT-PCR were also negative by EBOV-RPA. The sensitivity values for samples with a Ct value of <34, which accounted for 95.59% of the samples, was 100%. Discordant samples positive by RT-PCR but negative by EBOV-RPA had significantly high Ct values. Results of external quality assessment samples with EBOV-RPA were 100%, consistent with those of RT-PCR. The EBOV-RPA assay showed 97% sensitivity and 97% specificity for all EVD samples tested, making it a rapid and sensitive test for EVD diagnosis.

Noncontact optical detection of explosive particles via photodissociation followed by laser-induced fluorescence.

PubMed

Wynn, C M; Palmacci, S; Kunz, R R; Aernecke, M

2011-09-12

High-sensitivity (ng/cm²) optical detection of the explosive 2,4,6-trinitrotoluene (TNT) is demonstrated using photodissociation followed by laser-induced fluorescence (PD-LIF). Detection occurs rapidly, within 6 laser pulses (~7 ns each) at a range of 15 cm. Dropcasting is used to create calibrated samples covering a wide range of TNT concentrations; and a correspondence between fractional area covered by TNT and PD-LIF signal strength is observed. Dropcast data are compared to that of an actual fingerprint. These results demonstrate that PD-LIF could be a viable means of rapidly and remotely scanning surfaces for trace explosive residues.

Screening and Biosensor-Based Approaches for Lung Cancer Detection

PubMed Central

Wang, Lulu

2017-01-01

Early diagnosis of lung cancer helps to reduce the cancer death rate significantly. Over the years, investigators worldwide have extensively investigated many screening modalities for lung cancer detection, including computerized tomography, chest X-ray, positron emission tomography, sputum cytology, magnetic resonance imaging and biopsy. However, these techniques are not suitable for patients with other pathologies. Developing a rapid and sensitive technique for early diagnosis of lung cancer is urgently needed. Biosensor-based techniques have been recently recommended as a rapid and cost-effective tool for early diagnosis of lung tumor markers. This paper reviews the recent development in screening and biosensor-based techniques for early lung cancer detection. PMID:29065541

Preliminary Validation of Direct Detection of Foot-And-Mouth Disease Virus within Clinical Samples Using Reverse Transcription Loop-Mediated Isothermal Amplification Coupled with a Simple Lateral Flow Device for Detection

PubMed Central

Waters, Ryan A.; Fowler, Veronica L.; Armson, Bryony; Nelson, Noel; Gloster, John; Paton, David J.; King, Donald P.

2014-01-01

Rapid, field-based diagnostic assays are desirable tools for the control of foot-and-mouth disease (FMD). Current approaches involve either; 1) Detection of FMD virus (FMDV) with immuochromatographic antigen lateral flow devices (LFD), which have relatively low analytical sensitivity, or 2) portable RT-qPCR that has high analytical sensitivity but is expensive. Loop-mediated isothermal amplification (LAMP) may provide a platform upon which to develop field based assays without these drawbacks. The objective of this study was to modify an FMDV-specific reverse transcription–LAMP (RT-LAMP) assay to enable detection of dual-labelled LAMP products with an LFD, and to evaluate simple sample processing protocols without nucleic acid extraction. The limit of detection of this assay was demonstrated to be equivalent to that of a laboratory based real-time RT-qPCR assay and to have a 10,000 fold higher analytical sensitivity than the FMDV-specific antigen LFD currently used in the field. Importantly, this study demonstrated that FMDV RNA could be detected from epithelial suspensions without the need for prior RNA extraction, utilising a rudimentary heat source for amplification. Once optimised, this RT-LAMP-LFD protocol was able to detect multiple serotypes from field epithelial samples, in addition to detecting FMDV in the air surrounding infected cattle, pigs and sheep, including pre-clinical detection. This study describes the development and evaluation of an assay format, which may be used as a future basis for rapid and low cost detection of FMDV. In addition it provides providing “proof of concept” for the future use of LAMP assays to tackle other challenging diagnostic scenarios encompassing veterinary and human health. PMID:25165973

[Development and comparative evaluation of up-converting phosphor technology based lateral flow assay for rapid detection of Yersinia pestis, Bacillus anthracis spore and Brucella spp].

PubMed

Li, Chunfeng; Zhang, Pingping; Wang, Xiaoying; Liu, Xiao; Zhao, Yong; Sun, Chongyun; Wang, Chengbin; Yang, Ruifu; Zhou, Lei

2015-01-01

To develop an up-converting phosphor technology based lateral flow (UPT-LF) assay for rapid and quantitative detection of Yersinia pestis, Bacillus anthracis spore and Brucella spp.and make the comparison with BioThreat Alert (BTA) test strips (Tetracore Inc., USA). Using up-converting phosphor nano-particles (UCP-NPs) as the bio-marker, three double-antibody-sandwich model based UPT-LF strips including Plague-UPT-LF, Anthrax-UPT-LF, Brucella-UPT-LF were prepared and its sensitivity, accuracy, linearity and specificity were determined by detecting 10(10), 10(9), 10(8), 10(7), 10(6), 10(5) and 0 CFU/ml series of concentrations of Y.pestis, B.anthracis, Brucella standards and other 27 kinds of 10(9) CFU/ml series of contrations of bacteria strains.Furthermore, the speed, sensitivity and accuracy of bacteria standards and simulated sample detection were compared between UPT-LF and BTA system. The detection limit of Plague-UPT-LF, Anthrax-UPT-LF and Brucella-LF was 10(5) CFU/ml. The CV of series of bacteria concentrations was ≤ 15%, and the r between lg (T/C-cut-off) and lg (concentration) was 0.996,0.998 and 0.999 (F values were 1 647.57, 743.51 and 1 822.17. All the P values were <0.001), respectively. The specificity of Plague-UPT-LF and Brucella-LF were excellent, while that of Anthrax-UPT-LF was a little bit regretful because of non-specific reaction with two isolates of B. subtilis and one B.cereus. On-site evaluation showed the detection time of UPT-LF for all Y.pestis, B.anthracis spore and Brucella spp.was 33, 36 and 37 min, while BTA was 115, 115 and 111 min, which revealed the higher detection speed and sensitivity of UPT-LF comparing with BTA. The negative rate of two methods for blank standard was both 5/5, the sensitivity of UPT-LF for Y.pestis,B.anthracis spore and Brucella spp. was all 10(5) CFU/ml, then BTA was 10(6), 10(6) and 10(5) CFU/ml, respectively. The detection rate of UPT-LF for all three bacteria analog positive samples was 16/16, while BTA for B.anthracis was 7/16 only. The good performance including rapidness, simplicity and high sensitivity will bring the bright future of UPT-LF to be broadly used on-site as first response to bio-terrorism.

Fluorescent-Magnetic-Biotargeting Multifunctional Nanobioprobes for Detecting and Isolating Multiple Types of Tumor Cells

PubMed Central

Song, Er-Qun; Hu, Jun; Wen, Cong-Ying; Tian, Zhi-Quan; Yu, Xu; Zhang, Zhi-Ling; Shi, Yun-Bo; Pang, Dai-Wen

2011-01-01

Fluorescent-magnetic-biotargeting multifunctional nanobioprobes (FMBMNs) have attracted great attention in recent years due to their increasing, important applications in biomedical research, clinical diagnosis, and biomedicine. We have previously developed such nanobioprobes for the detection and isolation of a single kind of tumor cells. Detection and isolation of multiple tumor markers or tumor cells from complex samples sensitively and with high efficiency is critical for the early diagnosis of tumors, especially malignant tumors or cancers, which will improve clinical diagnosis outcomes and help to select effective treatment approaches. Here, we expanded the application of the monoclonal antibody (mAb)-coupled FMBMNs for multiplexed assays. Multiple types of cancer cells, such as leukemia cells and prostate cancer cells, were detected and collected from mixed samples within 25 minutes by using a magnet and an ordinary fluorescence microscope. The capture efficiencies of mAb-coupled FMBMNs for the above mentioned two types of cells were 96% and 97% respectively. Furthermore, by using the mAb-coupled FMBMNs, specific and sensitive detection and rapid separation of a small number of spiked leukemia cells and prostate cancer cells in a large population of cultured normal cells (about 0.01% were tumor cells) were achieved simply and inexpensively without any sample pretreatment before cell analysis. Therefore, mAb-coupled multicolour FMBMNs may be used for very sensitive detection and rapid isolation of multiple cancer cells in biomedical research and medical diagnostics. PMID:21250650

An ATMND/SGI based label-free and fluorescence ratiometric aptasensor for rapid and highly sensitive detection of cocaine in biofluids.

PubMed

Wang, Jiamian; Song, Jie; Wang, Xiuyun; Wu, Shuo; Zhao, Yanqiu; Luo, Pinchen; Meng, Changgong

2016-12-01

A label-free ratiometric fluorescence aptasensor has been developed for the rapid and sensitive detection of cocaine in complex biofluids. The fluorescent aptasensor is composed of a non-labeled GC-38 cocaine aptamer which serves as a basic sensing unit and two fluorophores, 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) and SYBR Green I (SGI) which serves as a signal reporter and a build-in reference, respectively. The detection principle is based on a specific cocaine mediated ATMND displacement reaction and the corresponding change in the fluorescence ratio of ATMND to SGI. Due to the high affinity of the non-labeled aptamer, the good precision originated from the ratiometric method, and the good fluorescence quantum yield of the fluorophore, the aptasensor shows good analytical performance with respect to cocaine detection. Under optimal conditions, the aptasensor shows a linear range of 0.10-10μM and a low limit of detection of 56nM, with a fast response of 20s. The low limit of detection is comparable to most of the fluorescent aptasensors with signal amplification strategies and much lower than all of the unamplified cocaine aptasensors. Practical sample analysis in a series of complex biofluids, including urine, saliva and serum, also indicates the good precision, stability, and high sensitivity of the aptasensor, which may have great potential for the point-of-care screening of cocaine in complex biofluids. Copyright © 2016 Elsevier B.V. All rights reserved.

Visual Detection of Canine Parvovirus Based on Loop-Mediated Isothermal Amplification Combined with Enzyme-Linked Immunosorbent Assay and with Lateral Flow Dipstick

PubMed Central

SUN, Yu-Ling; YEN, Chon-Ho; TU, Ching-Fu

2013-01-01

ABSTRACT Loop-mediated isothermal amplification (LAMP) combined with enzyme-linked immunosorbent assay (LAMP–ELISA) and with lateral flow dipstick (LAMP–LFD) are rapid, sensitive and specific methods for the visual detection of clinical pathogens. In this study, LAMP–ELISA and LAMP–LFD were developed for the visual detection of canine parvovirus (CPV). For LAMP, a set of four primers (biotin-labeled forward inner primers) was designed to specifically amplify a region of the VP2 gene of CPV. The optimum time and temperature for LAMP were 60 min and 65°C, respectively. The specific capture oligonucleotide probes, biotin-labeled CPV probe for LAMP–ELISA and fluorescein isothiocyanate-labeled CPV probe for LAMP–LFD were also designed for hybridization with LAMP amplicons on streptavidin-coated wells and LFD strips, respectively. For the comparison of detection sensitivity, conventional PCR and LAMP for CPV detection were also performed. The CPV detection limits by PCR, PCR–ELISA, LAMP, LAMP–ELISA and LAMP–LFD were 102, 102, 10−1, 10−1 and 10−1 TCID50/ml, respectively. In tests using artificially contaminated dog fecal samples, the samples with CPV inoculation levels of ≥1 TCID50/ml gave positive results by both LAMP–ELISA and LAMP–LFD. Our data indicated that both LAMP–ELISA and LAMP–LFD are promising as rapid, sensitive and specific methods for an efficient diagnosis of CPV infection. PMID:24334855

Rapid antigen tests for dengue virus serotypes and Zika virus in patient serum.

PubMed

Bosch, Irene; de Puig, Helena; Hiley, Megan; Carré-Camps, Marc; Perdomo-Celis, Federico; Narváez, Carlos F; Salgado, Doris M; Senthoor, Dewahar; O'Grady, Madeline; Phillips, Elizabeth; Durbin, Ann; Fandos, Diana; Miyazaki, Hikaru; Yen, Chun-Wan; Gélvez-Ramírez, Margarita; Warke, Rajas V; Ribeiro, Lucas S; Teixeira, Mauro M; Almeida, Roque P; Muñóz-Medina, José E; Ludert, Juan E; Nogueira, Mauricio L; Colombo, Tatiana E; Terzian, Ana C B; Bozza, Patricia T; Calheiros, Andrea S; Vieira, Yasmine R; Barbosa-Lima, Giselle; Vizzoni, Alexandre; Cerbino-Neto, José; Bozza, Fernando A; Souza, Thiago M L; Trugilho, Monique R O; de Filippis, Ana M B; de Sequeira, Patricia C; Marques, Ernesto T A; Magalhaes, Tereza; Díaz, Francisco J; Restrepo, Berta N; Marín, Katerine; Mattar, Salim; Olson, Daniel; Asturias, Edwin J; Lucera, Mark; Singla, Mohit; Medigeshi, Guruprasad R; de Bosch, Norma; Tam, Justina; Gómez-Márquez, Jose; Clavet, Charles; Villar, Luis; Hamad-Schifferli, Kimberly; Gehrke, Lee

2017-09-27

The recent Zika virus (ZIKV) outbreak demonstrates that cost-effective clinical diagnostics are urgently needed to detect and distinguish viral infections to improve patient care. Unlike dengue virus (DENV), ZIKV infections during pregnancy correlate with severe birth defects, including microcephaly and neurological disorders. Because ZIKV and DENV are related flaviviruses, their homologous proteins and nucleic acids can cause cross-reactions and false-positive results in molecular, antigenic, and serologic diagnostics. We report the characterization of monoclonal antibody pairs that have been translated into rapid immunochromatography tests to specifically detect the viral nonstructural 1 (NS1) protein antigen and distinguish the four DENV serotypes (DENV1-4) and ZIKV without cross-reaction. To complement visual test analysis and remove user subjectivity in reading test results, we used image processing and data analysis for data capture and test result quantification. Using a 30-μl serum sample, the sensitivity and specificity values of the DENV1-4 tests and the pan-DENV test, which detects all four dengue serotypes, ranged from 0.76 to 1.00. Sensitivity/specificity for the ZIKV rapid test was 0.81/0.86, respectively, using a 150-μl serum input. Serum ZIKV NS1 protein concentrations were about 10-fold lower than corresponding DENV NS1 concentrations in infected patients; moreover, ZIKV NS1 protein was not detected in polymerase chain reaction-positive patient urine samples. Our rapid immunochromatography approach and reagents have immediate application in differential clinical diagnosis of acute ZIKV and DENV cases, and the platform can be applied toward developing rapid antigen diagnostics for emerging viruses. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

PubMed Central

Ghelardini, Luisa; Belbahri, Lassaâd; Quartier, Marion; Santini, Alberto

2013-01-01

Ceratocystis platani is the causal agent of canker stain of plane trees, a lethal disease able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread. The goal of this study was to design a rapid, real-time quantitative PCR assay to detect a C. platani airborne inoculum. Airborne inoculum traps (AITs) were placed in an urban setting in the city of Florence, Italy, where the disease was present. Primers and TaqMan minor groove binder (MGB) probes were designed to target cerato-platanin (CP) and internal transcribed spacer 2 (ITS2) genes. The detection limits of the assay were 0.05 pg/μl and 2 fg/μl of fungal DNA for CP and ITS, respectively. Pathogen detection directly from AITs demonstrated specificity and high sensitivity for C. platani, detecting DNA concentrations as low as 1.2 × 10−2 to 1.4 × 10−2 pg/μl, corresponding to ∼10 conidia per ml. Airborne inoculum traps were able to detect the C. platani inoculum within 200 m of the closest symptomatic infected plane tree. The combination of airborne trapping and real-time quantitative PCR assay provides a rapid and sensitive method for the specific detection of a C. platani inoculum. This technique may be used to identify the period of highest risk of pathogen spread in a site, thus helping disease management. PMID:23811499

Rapid and sensitive detection of canine parvovirus type 2 by recombinase polymerase amplification.

PubMed

Wang, Jianchang; Liu, Libing; Li, Ruiwen; Wang, Jinfeng; Fu, Qi; Yuan, Wanzhe

2016-04-01

A novel recombinase polymerase amplification (RPA)-based method for detection of canine parvovirus type 2 (CPV-2) was developed. Sensitivity analysis showed that the detection limit of RPA was 10 copies of CPV-2 genomic DNA. RPA amplified both CPV-2a and -2b DNA but did not amplify the template of other important dog viruses (CCoV, PRV or CDV), demonstrating high specificity. The method was further validated with 57 canine fecal samples. An outstanding advantage of RPA is that it is an isothermal reaction and can be performed in a water bath, making RPA a potential alternative method for CPV-2 detection in resource-limited settings.

Rapid detection of contagious ecthyma by loop-mediated isothermal amplification and epidemiology in Jilin Province China.

PubMed

Wang, Kai; Shao, Hongze; Pei, Zhihua; Hu, Guixue

2016-01-01

The aim of this experiment was to develop a loop-mediated isothermal amplification (LAMP) assay and to research the recent epidemiology of contagious ecthyma in Jilin Province, China, using the assay. A LAMP assay targeting a highly conserved region of the F1L gene was developed to detect contagious ecthyma virus (CEV). Three hundred and sixty-five cases from 64 flocks in 9 different areas of Jilin Province, China, from 2011 to 2014 were tested using the LAMP assay. The results showed that the sensitivity of the LAMP assay was 100 copies of the standard plasmid, which is 100-fold higher than the sensitivity of PCR. No cross-reactivity was observed with capripoxvirus, fowlpox virus, foot-and-mouth disease virus serotype O, foot-and-mouth disease virus serotype Asia I and bluetongue virus. The average positive rate was 19.73% (72/365), and the positive rate was highest in lambs aged 1-6 months. Our results demonstrated that CEV infection was very widespread in the flocks of Jilin Province and that the LAMP assay allows for easy, rapid, accurate and sensitive detection of CEV infection.

Development of a polymerase chain reaction applicable to rapid and sensitive detection of Clonorchis sinensis eggs in human stool samples

PubMed Central

Cho, Pyo Yun; Na, Byoung-Kuk; Mi Choi, Kyung; Kim, Jin Su; Cho, Shin-Hyeong; Lee, Won-Ja; Lim, Sung-Bin; Cha, Seok Ho; Park, Yun-Kyu; Pak, Jhang Ho; Lee, Hyeong-Woo; Hong, Sung-Jong; Kim, Tong-Soo

2013-01-01

Microscopic examination of eggs of parasitic helminths in stool samples has been the most widely used classical diagnostic method for infections, but tiny and low numbers of eggs in stool samples often hamper diagnosis of helminthic infections with classical microscopic examination. Moreover, it is also difficult to differentiate parasite eggs by the classical method, if they have similar morphological characteristics. In this study, we developed a rapid and sensitive polymerase chain reaction (PCR)-based molecular diagnostic method for detection of Clonorchis sinensis eggs in stool samples. Nine primers were designed based on the long-terminal repeat (LTR) of C. sinensis retrotransposon1 (CsRn1) gene, and seven PCR primer sets were paired. Polymerase chain reaction with each primer pair produced specific amplicons for C. sinensis, but not for other trematodes including Metagonimus yokogawai and Paragonimus westermani. Particularly, three primer sets were able to detect 10 C. sinensis eggs and were applicable to amplify specific amplicons from DNA samples purified from stool of C. sinensis-infected patients. This PCR method could be useful for diagnosis of C. sinensis infections in human stool samples with a high level of specificity and sensitivity. PMID:23916334

Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

PubMed

Agasti, Sarit S; Liong, Monty; Peterson, Vanessa M; Lee, Hakho; Weissleder, Ralph

2012-11-14

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

Simultaneous point-of-care detection of anemia and sickle cell disease in Tanzania: the RAPID study.

PubMed

Smart, Luke R; Ambrose, Emmanuela E; Raphael, Kevin C; Hokororo, Adolfine; Kamugisha, Erasmus; Tyburski, Erika A; Lam, Wilbur A; Ware, Russell E; McGann, Patrick T

2018-02-01

Both anemia and sickle cell disease (SCD) are highly prevalent across sub-Saharan Africa, and limited resources exist to diagnose these conditions quickly and accurately. The development of simple, inexpensive, and accurate point-of-care (POC) assays represents an important advance for global hematology, one that could facilitate timely and life-saving medical interventions. In this prospective study, Robust Assays for Point-of-care Identification of Disease (RAPID), we simultaneously evaluated a POC immunoassay (Sickle SCAN™) to diagnose SCD and a first-generation POC color-based assay to detect anemia. Performed at Bugando Medical Center in Mwanza, Tanzania, RAPID tested 752 participants (age 1 day to 20 years) in four busy clinical locations. With minimally trained medical staff, the SCD POC assay diagnosed SCD with 98.1% sensitivity and 91.1% specificity. The hemoglobin POC assay had 83.2% sensitivity and 74.5% specificity for detection of severe anemia (Hb ≤ 7 g/dL). Interobserver agreement was excellent for both POC assays (r = 0.95-0.96). Results for the hemoglobin POC assay have informed the second-generation assay design to be more suitable for low-resource settings. RAPID provides practical feasibility data regarding two novel POC assays for the diagnosis of anemia and SCD in real-world field evaluations and documents the utility and potential impact of these POC assays for sub-Saharan Africa.

Epitope-blocking enzyme-linked immunosorbent assay for detection of antibodies to Ross River virus in vertebrate sera.

PubMed

Oliveira, Nidia M M; Broom, Annette K; Mackenzie, John S; Smith, David W; Lindsay, Michael D A; Kay, Brian H; Hall, Roy A

2006-07-01

We describe the development of an epitope-blocking enzyme-linked immunosorbent assay (ELISA) for the sensitive and rapid detection of antibodies to Ross River virus (RRV) in human sera and known vertebrate host species. This ELISA provides an alternative method for the serodiagnosis of RRV infections.

Differentiation of live and dead salmonella cells using fourier transform infrared (FTIR) spectroscopy and principle component analysis (PCA) technique

USDA-ARS?s Scientific Manuscript database

Various technologies have been developed for pathogen detection using optical, electrochemical, biochemical and physical properties. Conventional microbiological methods need time from days to week to get the result. Though this method is very sensitive and accurate, a rapid detection of pathogens i...

Loop-mediated isothermal amplification for detection of the tomato and potato late blight pathogen, Phytophthora infestans

USDA-ARS?s Scientific Manuscript database

Aims: To design and validate a colorimetric loop-mediated isothermal amplification assay for rapid detection of P. infestans DNA. Methods and Results: Two sets of LAMP primers were designed and evaluated for their sensitivity and specificity for P. infestans. ITSII primers targeted a portion of the ...

Development of one-step Loop-Mediated Isothermal Amplification (LAMP) for the detection of norovirus in oysters

USDA-ARS?s Scientific Manuscript database

The aim of this study was to develop a simple and rapid technique for detecting human norovirus (NoV). The loop-mediated isothermal amplification (LAMP) technique was evaluated and found to be sensitive, highly specific, and useful for routine oyster testing. Reverse transcription-LAMP (RT-LAMP) pri...

Avian influenza virus detection and quantitation by real-time RT-PCR

USDA-ARS?s Scientific Manuscript database

Real-time RT-PCR (rRT-PCR) has been used for avian influenza virus (AIV) detection since the early 2000’s for routine surveillance, during outbreaks and for research. Some of the advantages of rRT-PCR are: high sensitivity, high specificity, rapid time-to-result, scalability, cost, and its inherentl...

Rapid microfluidic assay for the detection of botulinum neurotoxin in animal sera

USDA-ARS?s Scientific Manuscript database

The potent botulinum neurotoxins (BoNTs) represent a threat to public health and safety. Botulism is a disease caused by BoNT intoxication that results in muscle paralysis that can be fatal. Sensitive assays capable of detecting BoNTs from different substrates and settings are essential to limit f...

Rapid and accurate identification by real-time PCR of biotoxin-producing dinoflagellates from the family gymnodiniaceae.

PubMed

Smith, Kirsty F; de Salas, Miguel; Adamson, Janet; Rhodes, Lesley L

2014-03-07

The identification of toxin-producing dinoflagellates for monitoring programmes and bio-compound discovery requires considerable taxonomic expertise. It can also be difficult to morphologically differentiate toxic and non-toxic species or strains. Various molecular methods have been used for dinoflagellate identification and detection, and this study describes the development of eight real-time polymerase chain reaction (PCR) assays targeting the large subunit ribosomal RNA (LSU rRNA) gene of species from the genera Gymnodinium, Karenia, Karlodinium, and Takayama. Assays proved to be highly specific and sensitive, and the assay for G. catenatum was further developed for quantification in response to a bloom in Manukau Harbour, New Zealand. The assay estimated cell densities from environmental samples as low as 0.07 cells per PCR reaction, which equated to three cells per litre. This assay not only enabled conclusive species identification but also detected the presence of cells below the limit of detection for light microscopy. This study demonstrates the usefulness of real-time PCR as a sensitive and rapid molecular technique for the detection and quantification of micro-algae from environmental samples.

High-throughput label-free microcontact printing graphene-based biosensor for valley fever.

PubMed

Tsai, Shih-Ming; Goshia, Tyler; Chen, Yen-Chang; Kagiri, Agnes; Sibal, Angelo; Chiu, Meng-Hsuen; Gadre, Anand; Tung, Vincent; Chin, Wei-Chun

2018-06-18

The highly prevalent and virulent disease in the Western Hemisphere Coccidioidomycosis, also known as Valley Fever, can cause serious illness such as severe pneumonia with respiratory failure. It can also take on a disseminated form where the infection spreads throughout the body. Thus, a serious impetus exists to develop effective detection of the disease that can also operate in a rapid and high-throughput fashion. Here, we report the assembly of a highly sensitive biosensor using reduced graphene oxide (rGO) with Coccidioides(cocci) antibodies as the target analytes. The facile design made possible by the scalable microcontact printing (μCP) surface patterning technique which enables rapid, ultrasensitive detection. It provides a wide linear range and sub picomolar (2.5 pg/ml) detection, while also delivering high selectivity and reproducibility. This work demonstrates an important advancement in the development of a sensitive label-free rGO biosensor for Coccidioidomycosis detection. This result also provides the potential application of direct pathogen diagnosis for the future biosensor development. Copyright © 2018 Elsevier B.V. All rights reserved.

Validation of caffeine dehydrogenase from Pseudomonas sp. strain CBB1 as a suitable enzyme for a rapid caffeine detection and potential diagnostic test.

PubMed

Mohanty, Sujit K; Yu, Chi Li; Gopishetty, Sridhar; Subramanian, Mani

2014-08-06

Excess consumption of caffeine (>400 mg/day/adult) can lead to adverse health effects. Recent introduction of caffeinated products (gums, jelly beans, energy drinks) might lead to excessive consumption, especially among children and nursing mothers, hence attracting the Food and Drug Administration's attention and product withdrawals. An "in-home" test will aid vigilant consumers in detecting caffeine in beverages and milk easily and quickly, thereby restricting its consumption. Known diagnostic methods lack speed and sensitivity. We report a caffeine dehydrogenase (Cdh)-based test which is highly sensitive (1-5 ppm) and detects caffeine in beverages and mother's milk in 1 min. Other components in these complex test samples do not interfere with the detection. Caffeine-dependent reduction of the dye iodonitrotetrazolium chloride results in shades of pink proportional to the levels in test samples. This test also estimates caffeine levels in pharmaceuticals, comparable to high-performance liquid chromatography. The Cdh-based test is the first with the desired attributes of a rapid and robust caffeine diagnostic kit.

[Demand for and the Development of Detection Techniques for Source of Schistosome Infection in China].

PubMed

Wang, Shi-ping; He, Xin; Zhou, Yun-fei

2015-12-01

Schistosomiasis is a type of zoonotic parasitosis that severely impairs human health. Rapid detection of infection sources is a key to the control of schistosomiasis. With the effective control of schistosomiasis in China, the detection techniques for infection sources have also been developed. The rate and the intensity of infection among humans and livestocks have been significantly decreased in China, as the control program has entered the transmission control stage in most of the endemic areas. Under this situation, the traditional etiological diagnosing techniques and common immunological methods can not afford rapid detection of infection sources of schistosomiasis. Instead, we are calling for detection methods with higher sensitivity, specificity and stability while being less time-consuming, more convenient and less costing. In recent years, many improved or novel detection methods have been applied for the epidemiological surveillance of schistosomiasis, such as the automatic scanning microscopic image acquisition system, PCR-ELISA, immunosensors, loop-mediated isothermal amplification, etc. The development of new monitoring techniques can facilitate rapid detection of schistosome infection sources in endemic areas.

Current and Developing Technologies for Monitoring Agents of Bioterrorism and Biowarfare

PubMed Central

Lim, Daniel V.; Simpson, Joyce M.; Kearns, Elizabeth A.; Kramer, Marianne F.

2005-01-01

Recent events have made public health officials acutely aware of the importance of rapidly and accurately detecting acts of bioterrorism. Because bioterrorism is difficult to predict or prevent, reliable platforms to rapidly detect and identify biothreat agents are important to minimize the spread of these agents and to protect the public health. These platforms must not only be sensitive and specific, but must also be able to accurately detect a variety of pathogens, including modified or previously uncharacterized agents, directly from complex sample matrices. Various commercial tests utilizing biochemical, immunological, nucleic acid, and bioluminescence procedures are currently available to identify biological threat agents. Newer tests have also been developed to identify such agents using aptamers, biochips, evanescent wave biosensors, cantilevers, living cells, and other innovative technologies. This review describes these current and developing technologies and considers challenges to rapid, accurate detection of biothreat agents. Although there is no ideal platform, many of these technologies have proved invaluable for the detection and identification of biothreat agents. PMID:16223949

A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots.

PubMed

Chen, Rui; Huang, Xiaolin; Li, Juan; Shan, Shan; Lai, Weihua; Xiong, Yonghua

2016-12-01

Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H 2 O 2 ) and H 2 O 2 -sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H 2 O 2 and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H 2 O 2 , as well as the incubation time between H 2 O 2 and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 10 2  CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 10 3  CFU/mL to 1.18 × 10 6  CFU/mL were in the range of 65.88%-105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control. Copyright © 2016 Elsevier B.V. All rights reserved.

Immunochromatographic strip assay for the rapid and sensitive detection of Salmonella Typhimurium in artificially contaminated tomato samples.

PubMed

Shukla, Shruti; Leem, Hyerim; Lee, Jong-Suk; Kim, Myunghee

2014-06-01

This study was designed to confirm the applicability of a liposome-based immunochromatographic assay for the rapid detection of Salmonella enterica subsp. enterica serovar Typhimurium (Salmonella Typhimurium) in artificially contaminated tomato samples. To determine the detection limit and pre-enrichment incubation time (10, 12, and 18 h pre-enrichment in 1% buffered peptone water), the tests were performed with different cell numbers of Salmonella Typhimurium (3 × 10(0), 3 × 10(1), 3 × 10(2), and 3 × 10(3) CFU·mL(-1)) inoculated into 25 g of crushed tomato samples. The assay was able to detect as few as 30 Salmonella Typhimurium cells per 25 g of tomato samples (1.2 cells·g(-1)) after 12 h pre-enrichment incubation. Moreover, when the developed assay was compared with traditional morphological and biochemical culture-based methods as well as colloidal gold nanoparticle-based commercial test strips, the developed assay yielded positive results for the detection of Salmonella Typhimurium within a shorter period time. These findings confirm that the developed assay may have practical application for the sensitive detection of Salmonella Typhimurium in various food samples, including raw vegetables, with a relatively low detection limit and shorter analysis time.

Rapid Detection of Cyprinid Herpesvirus 3 in Latently Infected Koi (Cyprinus carpio) by Recombinase Polymerase Amplification

PubMed Central

Prescott, Meagan A.; Reed, Aimee N.; Jin, Ling; Pastey, Manoj K.

2018-01-01

Since the emergence of cyprinid herpes virus 3 (CyHV-3), outbreaks have been devastating to koi and common carp leading to high economic losses. Current diagnostics for detecting CyHV-3 are limited in sensitivity and are further complicated by latency. Here we describe the detection of CyHV-3 by recombinase polymerase amplification (RPA). The RPA assay can detect as low as 10 copies of CyHV-3 genome by an isothermal reaction and yields results in approximately 20 minutes. Using the RPA assay, CyHV-3 genome can be detected in total DNA of white blood cells isolated from koi latently infected with CyHV-3, while less than 10% of the latently infected koi can be detected by a real-time PCR assay in total DNA of white blood cells. In addition, RPA products can be detected in a lateral flow device that is cheap, fast, and can be used outside of the diagnostic lab. The RPA assay and lateral flow device provide for the rapid, sensitive, and specific amplification of CyHV-3 that with future modifications for field use and validation could lead to enhanced surveillance and early diagnosis of CyHV-3 in the laboratory and field. PMID:27485254