Application of Concanavalin A–Linked Magnetic Beads for the Detection of Hepatitis A Virus

2018 ◽  
Vol 81 (12) ◽  
pp. 1997-2002
Author(s):  
SANG-MU KO ◽  
SE-YOUNG CHO ◽  
MYUNG-JOO OH ◽  
JOSEPH KWON ◽  
BIPIN VAIDYA ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Binding Affinity ◽  
Concanavalin A ◽  
Hepatitis A ◽  
Magnetic Beads ◽  
Hepatitis A Virus ◽  
Immunomagnetic Separation ◽  
Carbohydrate Binding ◽  
Con A ◽  
Reverse Transcriptase Pcr

ABSTRACT Prompt and inexpensive detection of hepatitis A virus (HAV) is essential to control acute hepatitis outbreaks associated with the consumption of contaminated raw or minimally processed food. In this study, various carbohydrate-binding lectins, including concanavalin A (Con A), wheat germ agglutinin, and soybean agglutinin, were compared for their binding affinity to HAV. Con A, which showed significantly higher binding affinity than other lectins, was used to develop an alternative and affordable method to conventional antibody-linked immunomagnetic separation prior to detection of HAV using reverse transcriptase PCR. This method, Con A–linked immunomagnetic separation combined with reverse transcriptase PCR, can detect HAV at a dilution concentration of 10−4 of the virus stock (titer: 104 median tissue culture infective dose per mL), indicating that Con A could be a promising candidate for concentrating HAV.

scholarly journals Reverse Transcriptase PCR Detection of Astrovirus, Hepatitis A Virus, and Poliovirus in Experimentally Contaminated Mussels: Comparison of Several Extraction and Concentration Methods

1998 ◽  
Vol 64 (8) ◽  
pp. 3118-3122 ◽  
Author(s):  
Ousmane Traore ◽  
Charlotte Arnal ◽  
Berengere Mignotte ◽  
Armand Maul ◽  
Henri Laveran ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Extraction Methods ◽  
Borate Buffer ◽  
Rt Pcr ◽  
Peg 6000 ◽  
Reverse Transcriptase Pcr ◽  
Glycine Solution ◽  
Concentration Methods

ABSTRACT Four methods of extraction and three methods of concentration of three enteric viruses from mussels were comparatively evaluated by reverse transcriptase PCR (RT-PCR). Shellfish were experimentally contaminated by immersion in seawater seeded with astrovirus, hepatitis A virus, or poliovirus. Sixty-gram samples of mussel tissues were processed by using borate buffer, glycine solution, saline beef, and saline beef-Freon extraction methods. The viruses were concentrated by precipitation with polyethylene glycol 6000 (PEG 6000) or PEG 8000 or by organic flocculation. RT-PCR was performed with RNA extracts from crude shellfish extracts and concentrates with and without Sephadex LH20 filtration. The glycine solution and borate buffer extraction methods resulted in significantly more RT-PCR-positive samples than the saline beef extraction method. We assessed the efficiency of 20 combinations of extraction and concentration methods. The borate buffer-organic flocculation, borate buffer-PEG 6000, and glycine solution-PEG 6000 combinations gave RT-PCR-positive results for all 27 samples analyzed for the three viruses. Detoxification of the samples by Sephadex LH20 filtration significantly decreased the efficiency of RT-PCR virus detection.

scholarly journals Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus

2005 ◽  
Vol 71 (11) ◽  
pp. 7113-7116 ◽  
Author(s):  
Khaled H. Abd El Galil ◽  
M. A. El Sokkary ◽  
S. M. Kheira ◽  
Andre M. Salazar ◽  
Marylynn V. Yates ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Hepatitis A ◽  
Molecular Beacon ◽  
Hepatitis A Virus ◽  
Immunomagnetic Separation ◽  
Nucleic Acid Sequence ◽  
The Real ◽  
Nasba Assay ◽  
Environmental Pathogens

ABSTRACT A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.

scholarly journals Detection of Viral Pathogens by Reverse Transcriptase PCR and of Microbial Indicators by Standard Methods in the Canals of the Florida Keys

1999 ◽  
Vol 65 (9) ◽  
pp. 4118-4125 ◽  
Author(s):  
Dale W. Griffin ◽  
Charles J. Gibson ◽  
Erin K. Lipp ◽  
Kelley Riley ◽  
John H. Paul ◽  
...  
Keyword(s):  
Water Quality ◽  
Reverse Transcriptase ◽  
Hepatitis A ◽  
Treatment Strategies ◽  
Enteric Viruses ◽  
Florida Keys ◽  
Fecal Coliforms ◽  
Microbial Water Quality ◽  
Reverse Transcriptase Pcr ◽  
Human Enteric Viruses

ABSTRACT In order to assess the microbial water quality in canal waters throughout the Florida Keys, a survey was conducted to determine the concentration of microbial fecal indicators and the presence of human pathogenic microorganisms. A total of 19 sites, including 17 canal sites and 2 nearshore water sites, were assayed for total coliforms, fecal coliforms, Escherichia coli, Clostridium perfringens, enterococci, coliphages, F-specific (F+) RNA coliphages, Giardia lamblia, Cryptosporidium parvum, and human enteric viruses (polioviruses, coxsackie A and B viruses, echoviruses, hepatitis A viruses, Norwalk viruses, and small round-structured viruses). Numbers of coliforms ranged from <1 to 1,410, E. coli organisms from <1 to 130,Clostridium spp. from <1 to 520, and enterococci from <1 to 800 CFU/100 ml of sample. Two sites were positive for coliphages, but no F+ phages were identified. The sites were ranked according to microbial water quality and compared to various water quality standards and guidelines. Seventy-nine percent of the sites were positive for the presence of enteroviruses by reverse transcriptase PCR (polioviruses, coxsackie A and B viruses, and echoviruses). Sixty-three percent of the sites were positive for the presence of hepatitis A viruses. Ten percent of the sites were positive for the presence of Norwalk viruses. Ninety-five percent of the sites were positive for at least one of the virus groups. These results indicate that the canals and nearshore waters throughout the Florida Keys are being impacted by human fecal material carrying human enteric viruses through current wastewater treatment strategies such as septic tanks. Exposure to canal waters through recreation and work may be contributing to human health risks.

scholarly journals Detection of both Hepatitis A Virus and Norwalk-Like Virus in Imported Clams Associated with Food-Borne Illness

2002 ◽  
Vol 68 (8) ◽  
pp. 3914-3918 ◽  
Author(s):  
David H. Kingsley ◽  
Gloria K. Meade ◽  
Gary P. Richards
Keyword(s):  
Hepatitis A ◽  
Magnetic Beads ◽  
Hepatitis A Virus ◽  
New York State ◽  
Rna Extraction ◽  
The United States ◽  
Fecal Coliforms ◽  
Most Probable Number ◽  
Genotype I ◽  
Probable Number

ABSTRACT Hepatitis A virus (HAV) and Norwalk-like virus (NLV) were detected by reverse transcription-PCR in clams imported into the United States from China. An epidemiological investigation showed that these clams were associated with five cases of Norwalk-like gastroenteritis in New York State in August 2000 (Food and Drug Administration Import Alert 16-50). They were labeled “cooked” but appeared raw. Viral RNA extraction was performed by using dissected digestive tissues rather than whole shellfish meats; this was followed by glycine buffer elution, polyethylene glycol precipitation, Tri-Reagent treatment, and purification of poly(A) RNA with magnetic beads coupled to poly(dT) oligonucleotides. We identified HAV and NLV as genotype I and genogroup II strains, respectively. Both viruses have high levels of homology to Asian strains. An analysis of fecal coliforms revealed a most-probable number of 93,000/100 g of clam meat, which is approximately 300-fold higher than the hygienic standard for shellfish meats.

scholarly journals Concanavalin-A Induces Granulosa Cell Death and Inhibits FSH-Mediated Follicular Growth and Ovarian Maturation in Female Rats

Endocrinology ◽  
2013 ◽  
Vol 154 (5) ◽  
pp. 1885-1896 ◽  
Author(s):  
Ethel V. Velasquez ◽  
Mariana Ríos ◽  
María Elena Ortiz ◽  
Carlos Lizama ◽  
Elizabeth Nuñez ◽  
...  
Keyword(s):  
Cell Death ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Concanavalin A ◽  
Ex Vivo ◽  
Female Rats ◽  
Carbohydrate Binding ◽  
Con A ◽  
Follicular Maturation

Abstract Reproductive success stems from a finely regulated balance between follicular maturation and atresia, in which the role of carbohydrate structure is poorly understood. Here, we describe for the first time a fraction of purified recombinant human FSH that is capable of bringing about the cell death of granulosa cells and preventing follicular maturation in a rat model. Further analysis by mass spectrometry revealed the presence of the lectin Concanavalin-A (Con-A) within this fraction of recombinant FSH. Using both the fractionated FSH and Con-A, the observed cell death was predominantly located to the granulosa cells. Ex vivo culture of rat follicles demonstrated that follicle degeneration occurred and resulted in the release of a denuded and deteriorated oocyte. Moreover, in vivo experiments confirmed an increase in atresia and a corresponding reduction confined to follicle in early antral stage. As a mechanism of action, Con-A reduces ovarian proliferation, Von Willebrand staining, and angiogenesis. Based on the observation that Con-A may induce granulosa cell death followed by follicle death, our results further demonstrate that follicular carbohydrate moiety is changing under the influence of FSH, which may allow a carbohydrate-binding lectin to increase granulosa cell death. The physiological consequences of circulating lectin-like molecules remain to be determined. However, our results suggest a potential exploitation of carbohydrate binding in fertility and ovarian cancer treatment. This work may shed light on a key role of carbohydrates in the still obscure physiological process of follicular selection and atresia.

scholarly journals Preparation of optimized concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259846
Author(s):  
Yasuhiro Fujiwara ◽  
Yuji Tanno ◽  
Hiroki Sugishita ◽  
Yusuke Kishi ◽  
Yoshinori Makino ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Concanavalin A ◽  
Magnetic Particles ◽  
Magnetic Beads ◽  
Protein A ◽  
Con A ◽  
Established Method ◽  
Genomic Location ◽  
Tn5 Transposase ◽  
Selection Of

Epigenome research has employed various methods to identify the genomic location of proteins of interest, such as transcription factors and histone modifications. A recently established method called CUT&Tag uses a Protein-A Tn5 transposase fusion protein, which cuts the genome and inserts adapter sequences nearby the target protein. Throughout most of the CUT&Tag procedure, cells are held on concanavalin A (con A)-conjugated magnetic beads. Proper holding of cells would be decisive for the accessibility of Tn5 to the chromatin, and efficacy of the procedure of washing cells. However, BioMag®Plus ConA magnetic beads, used in the original CUT&Tag protocol, often exhibit poor suspendability and severe aggregation. Here, we compared the BioMag beads and Dynabeads® magnetic particles of which conjugation of con A was done by our hands, and examined the performance of these magnetic beads in CUT&Tag. Among tested, one of the Dynabeads, MyOne-T1, kept excessive suspendability in a buffer even after overnight incubation. Furthermore, the MyOne-T1 beads notably improved the sensitivity in CUT&Tag assay for H3K4me3. In conclusion, the arrangement and the selection of MyOne-T1 refine the suspendability of beads, which improves the association of chromatin with Tn5, which enhances the sensitivity in CUT&Tag assay.

Effects of an Oxidative Agent and Lectins on the Binding Inhibition of Recombinant Hepatitis A Virus Proteins to Oyster Digestive Tissues

2011 ◽  
Vol 74 (1) ◽  
pp. 157-160 ◽  
Author(s):  
SANG-MU KO ◽  
JONG-OH KIM ◽  
MYUNG-JOO OH ◽  
DUWOON KIM
Keyword(s):  
Binding Sites ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Helix Pomatia ◽  
Control Group ◽  
Carbohydrate Binding ◽  
Dolichos Biflorus ◽  
Ulex Europaeus ◽  
Binding Inhibition ◽  
Viral Contaminants

While the exact mechanism of hepatitis A virus (HAV) accumulation remains unclear, it has been demonstrated that viruses related to shellfish-borne gastroenteritis can bind to carbohydrates of oyster tissues. We investigated carbohydrate-binding sites to determine if they were related to the binding of HAV to carbohydrate moieties on oyster digestive tissues (DTs) using recombinant HAV proteins (rHAVPs). In addition, we evaluated lectins to determine if they influenced the inhibition of binding of rHAVPs to carbohydrates present in DT. DT that was treated with 0.5% potassium periodate allowed only 23% ± 3.6% and 33% ± 7.8% binding of VP1-P2A and VP1 rHAVPs, respectively, when compared with a control group (100%) treated with distilled water, indicating that carbohydrate-binding sites are strongly related to the binding of HAV. Soybean agglutinin (SBA) led to the greatest decrease in the binding affinity among six lectins (Helix pomatia, Dolichos biflorus, Ulex europaeus, SBA, Triticum vulgaris, and Arachis hypogaea) tested for inhibition of the binding of rHAVPs to DT, indicating that exposing the virus-contaminated DT to SBA might have the potential to depurate viral contaminants found in shellfish food products by high-affinity binding between SBA and rHAVPs, thus improving food safety.

Concanavalin A targeting N-linked glycans in spike proteins influence viral interactions

2020 ◽  
Vol 49 (39) ◽  
pp. 13538-13543 ◽  
Author(s):  
Hara Jang ◽  
Dong-Heon Lee ◽  
Hyun Goo Kang ◽  
Seung Jae Lee
Keyword(s):  
Binding Affinity ◽  
Concanavalin A ◽  
Carbohydrate Binding ◽  
Binding Domains ◽  
Viral Interactions ◽  
Spike Proteins

The N-linked glycans of spike proteins in viruses are a target of the carbohydrate-binding domains of lectins having higher binding affinity and low specificity.

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