SELEX-Based Direct Enzyme-Linked Aptamer Assay(DELAA) for Diagnosis of Toxoplasmosis by Detection of SAG1 Antigen in Sera of Mice and Humans

2020 ◽  
Author(s):  
Xuhang Shen ◽  
Wen Cui ◽  
Cong Wang ◽  
Obed Cudjoe ◽  
Liang Zhao ◽  
...  
Keyword(s):  
Acute Infection ◽  
Pcr Amplification ◽  
Laboratory Diagnosis ◽  
Specificity And Sensitivity ◽  
Recognition Probe ◽  
Circulating Antigen ◽  
Human Sera ◽  
Aptamer Assay ◽  
Oligonucleotide Library ◽  
Major Surface

Abstract Background: Toxoplasma gondii is a single-celled parasite commonly found in mammals. Diagnosis of toxoplasmosis largely depends on measurements of the antibody and/or antigen and Toxoplasma-derived DNAs due to the presence of tissue dwelling quiescent cysts and latent infection of the parasite. As a major surface antigen of T.gondii tachyzoites, SAG1 is a key marker for laboratory diagnosis. However, at present, there are no methods available for SAG1 detection using aptamer-based technology.Methods: Recombinant truncated SAG1（r-tSAG1）of Toxoplasma WH3 strain (type Chinese 1) was prokaryotically expressed and subjected to the synthetic oligonucleotide library for selection of nucleic acid aptamer which targets the r-tSAG1, with systematic evolution of ligands by exponential enrichment (SELEX) strategy. The screened specific aptamer-2 was used in direct enzyme-linked aptamer assay (DELAA) to detect native SAG1 obtained from tachyzoite lysates, mouse sera of acute infection, and human sera that had been verified to be positive for ToxoDNAs by PCR amplification. Results: The soluble r-tSAG1 protein was obtained from E.coli lysates by using 0.01M Tris-Cl in PBS, and was purified and identified by immunoblotting, and then labelled with biotin. The screened aptamers were amplified by PCR, followed by DNA sequencing. The results showed that the aptamer-2, with the highest affinity to nSAG1 among the four aptamer candidates, has a higher specificity and sensitivity when used in detection of nSAG1 in the sera of both animals and humans when compared with the commercial Toxoplasma circulating antigen testing kit.Conclusions: A new direct enzyme-linked aptamer assay (DELAA), with aptamer-2 as the recognition probe, was developed for detection of native SAG1 protein secreted by T.gondii. With increased sensitivity and specificity, stability during storage, easy and cheaper production, the aptamer-based technique is considered as a efficient method for the diagnosis of active and reactivated toxoplasmosis.

scholarly journals SELEX-Based Direct Enzyme-Linked Aptamer Assay (DELAA) for Diagnosis of Toxoplasmosis by Detection of SAG1 Protein in Mice and Humans

2021 ◽  
Author(s):  
Jilong Shen ◽  
Wei Wang ◽  
Yuanhong Xu ◽  
Xuhang Shen ◽  
Wen Cui ◽  
...  
Keyword(s):  
Acute Infection ◽  
Pcr Amplification ◽  
High Specificity ◽  
Laboratory Diagnosis ◽  
Specificity And Sensitivity ◽  
Recognition Probe ◽  
Human Sera ◽  
Aptamer Assay ◽  
Oligonucleotide Library ◽  
Major Surface

Abstract Background: Toxoplasma gondii is a single-celled parasite commonly found in mammals. Diagnosis of toxoplasmosis largely depends on measurements of the antibody and/or antigen and Toxoplasma-derived DNAs due to the presence of tissue dwelling duplicating tachyzoites, or quiescent cysts in latent infection of the parasite. As a major surface antigen of T.gondii tachyzoites, SAG1 is a key marker for laboratory diagnosis. However, there are no methods available yet for SAG1 detection using aptamer-based technology.Methods: Recombinant truncated SAG1（r-SAG1）of Toxoplasma WH3 strain (type Chinese 1) was prokaryotically expressed and subjected to the synthetic oligonucleotide library for selection of nucleic acid aptamers which target the r-SAG1, with systematic evolution of ligands by exponential enrichment (SELEX) strategy. The specific aptamer-2 was screened out and used in direct enzyme-linked aptamer assay (DELAA) for detection of native SAG1 obtained from tachyzoite lysates (n-SAG1), mouse sera of acute infection, and human sera that had been verified to be positive for Toxoplasma DNAs by PCR amplification. Results: The soluble r-SAG1 protein was obtained from E.coli lysates by purification and identification with immunoblotting, and then labelled with biotin. The selected aptamers were amplified by PCR, followed by DNA sequencing. The results showed that the aptamer-2, with the highest affinity to n-SAG1 in the sera of animals in the four aptamer candidates, has a high specificity and sensitivity when used in detection of n-SAG1 in the sera of humans when compared with the commercial kit of ELISA for Toxoplasma circulating antigen test.Conclusions: A new direct enzyme-linked aptamer assay (DELAA), with aptamer-2 as the recognition probe, was developed for detection of native SAG1 protein of Toxoplasma. With increased sensitivity and specificity, stability, easy and cheap preparation, the aptamer-based technology is considered as a efficient method for the diagnosis of active and reactivated toxoplasmosis.

scholarly journals Laboratory diagnosis ofMycoplasma pneumoniaeinfection. 4. Antigen capture and PCR-gene amplification for detection of the mycoplasma: problems of clinical correlation

1992 ◽  
Vol 109 (3) ◽  
pp. 519-537 ◽  
Author(s):  
J. Williamson ◽  
B. P. Marmion ◽  
D. A. Worswick ◽  
T. -W. Kok ◽  
G. Tannock ◽  
...  
Keyword(s):  
Direct Detection ◽  
Pcr Amplification ◽  
Laboratory Diagnosis ◽  
Capture Assay ◽  
Antigen Capture ◽  
Specificity And Sensitivity ◽  
Antibody Assays ◽  
Clinical Episode ◽  
Previous Infection ◽  
Current Infection

SUMMARYDirect detection assays forMycoplasma pneumoniaewere established by PCR amplification of short sequences within the foot protein/adhesin (P1) gene and the 16S ribosomal RNA gene.Specificity and sensitivity was excellent, no hybridization was observed withM. genitaliumand other humanMycoplasmaspecies. In nose and throat washings from subjects with respiratory infection a pattern of high counts (c.f.u./ml) ofM. pneumoniae(deduced from the amount of amplified PCR product), and a positive antigen capture assay, was found in 83% of subjects with serological evidence of current infection withM. pneumoniae.A small proportion of subjects with serological patterns suggesting infection in the more distant past had positive PCR assays. This was considered to represent either persistence of the organism from a previous infection or perhaps transient carriage during a reinfection, without substantial change in antibody response.PCR-based assay ofM. pneumoniaeoffers a powerful, rapid, and sensitive substitute for culture of the mycoplasma. Antigen capture, while less sensitive than PCR, offers the advantage that it is more often positive with samples from current infection and requires less stringent laboratory organization to contain false positive results. We conclude however that the laboratory diagnosis of a chosen clinical episode should not rest on the PCR or Ag-EIA assays alone, but must also include antibody assays to confirm whether infection is current or represents persistence from past exposure.

scholarly journals Mapping person-to-person variation in viral mutations that escape polyclonal serum targeting influenza hemagglutinin

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Juhye M Lee ◽  
Rachel Eguia ◽  
Seth J Zost ◽  
Saket Choudhary ◽  
Patrick C Wilson ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Viral Evolution ◽  
Surface Protein ◽  
Viral Escape ◽  
Viral Neutralization ◽  
Influenza Hemagglutinin ◽  
Human Sera ◽  
Order Of Magnitude ◽  
Amino Acid Mutations ◽  
Major Surface

A longstanding question is how influenza virus evolves to escape human immunity, which is polyclonal and can target many distinct epitopes. Here, we map how all amino-acid mutations to influenza’s major surface protein affect viral neutralization by polyclonal human sera. The serum of some individuals is so focused that it selects single mutations that reduce viral neutralization by over an order of magnitude. However, different viral mutations escape the sera of different individuals. This individual-to-individual variation in viral escape mutations is not present among ferrets that have been infected just once with a defined viral strain. Our results show how different single mutations help influenza virus escape the immunity of different members of the human population, a phenomenon that could shape viral evolution and disease susceptibility.

scholarly journals Selection and Identification of Novel Aptamers Specific for Clenbuterol Based on ssDNA Library Immobilized SELEX and Gold Nanoparticles Biosensor

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2337 ◽  
Author(s):  
Xixia Liu ◽  
Qi Lu ◽  
Sirui Chen ◽  
Fang Wang ◽  
Jianjun Hou ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Retention Rate ◽  
Pcr Amplification ◽  
Cross Reactivity ◽  
Enriched Library ◽  
Amplification Curve ◽  
Specificity And Sensitivity

We describe a multiple combined strategy to discover novel aptamers specific for clenbuterol (CBL). An immobilized ssDNA library was used for the selection of specific aptamers using the systematic evolution of ligands by exponential enrichment (SELEX). Progress was monitored using real-time quantitative PCR (Q-PCR), and the enriched library was sequenced by high-throughput sequencing. Candidate aptamers were picked and preliminarily identified using a gold nanoparticles (AuNPs) biosensor. Bioactive aptamers were characterized for affinity, circular dichroism (CD), specificity and sensitivity. The Q-PCR amplification curve increased and the retention rate was about 1% at the eighth round. Use of the AuNPs biosensor and CD analyses determined that six aptamers had binding activity. Affinity analysis showed that aptamer 47 had the highest affinity (Kd = 42.17 ± 8.98 nM) with no cross reactivity to CBL analogs. Indirect competitive enzyme linked aptamer assay (IC-ELAA) based on a 5′-biotin aptamer 47 indicated the limit of detection (LOD) was 0.18 ± 0.02 ng/L (n = 3), and it was used to detect pork samples with a mean recovery of 83.33–97.03%. This is the first report of a universal strategy including library fixation, Q-PCR monitoring, high-throughput sequencing, and AuNPs biosensor identification to select aptamers specific for small molecules.

Application of a circulating antigen detection immunoassay for laboratory diagnosis of extra-pulmonary and pulmonary tuberculosis

2005 ◽  
Vol 356 (1-2) ◽  
pp. 58-66 ◽  
Author(s):  
Abdelfattah M. Attallah ◽  
Sanaa Osman ◽  
Amr Saad ◽  
Mohamed Omran ◽  
Hisham Ismail ◽  
...  
Keyword(s):  
Pulmonary Tuberculosis ◽  
Laboratory Diagnosis ◽  
Antigen Detection ◽  
Circulating Antigen ◽  
Extra Pulmonary

scholarly journals Update on Malaria Diagnostics and Test Utilization

2017 ◽  
Vol 55 (7) ◽  
pp. 2009-2017 ◽  
Author(s):  
Blaine A. Mathison ◽  
Bobbi S. Pritt
Keyword(s):  
Gold Standard ◽  
Acute Infection ◽  
Laboratory Diagnosis ◽  
Rapid Diagnosis ◽  
Diagnostic Methods ◽  
Nucleic Acid Amplification ◽  
Advantages And Disadvantages ◽  
Life Threatening ◽  
Antigen Tests ◽  
Malaria Test

ABSTRACT Malaria is a potentially life-threatening disease requiring rapid diagnosis and treatment. Although microscopic examination of thick and thin blood films remains the gold standard for laboratory diagnosis, rapid antigen tests and nucleic acid amplification methods may also play a useful role in detection of acute infection. This review discusses the advantages and disadvantages of the commonly used diagnostic methods and provides important practice points for optimal malaria test utilization.

Identification and Comparative Phylogeny of Sheep and Goat Pox Isolates from Jammu, India

2020 ◽  
Author(s):  
Sankalp Sharma ◽  
Nawab Nashiruddullah ◽  
Anil Taku ◽  
Parimal Roychoudhury ◽  
Jafrin Ara Ahmed
Keyword(s):  
Core Protein ◽  
Small Ruminants ◽  
Pcr Amplification ◽  
Vaccination Strategy ◽  
Laboratory Diagnosis ◽  
Lumpy Skin Disease ◽  
P32 Gene ◽  
Cross Transmission ◽  
Clinical Survey ◽  
Comparative Phylogeny

Background: In view of the limited documentation of sheep and goat pox disease in animals of Jammu region, it was felt necessary to conduct a blanket screening of the small ruminant populations in areas where they are largely reared. Methods: The investigation was conducted to study the occurrence of capripox infection amongst small ruminants through clinical survey and confirmatory laboratory diagnosis. PCR amplification of a part of the P32 core protein gene successfully confirmed Capripoxvirus (CaPV) in clinical scab samples from both sheep and goats and from inoculated CAM. Positive amplified samples yielded a predicted 192 bp product. Result: Sequencing of the partial P32 gene of one isolate each from sheep (Sheeppoxvirus, SPPV) and goat origin (Goatpoxvirus, GTPV) revealed that each isolate were distinct and showed 97.8% identity with each other. The SPPV clustered with respective sheeppox cluster with 100% homology to with other SPPV strains reported within India and abroad and also to vaccine strains Srinagar and Rumanian-Fanar (RF). The GTPV was closely identical (~98-99%) with other strains from India and abroad with some unique residues of its own. Both the SPPV and GTPV were divergent from Lumpy Skin Disease Virus (LSDV) strains. The sequence of SPPV strain being similar to circulating strains and prevalent vaccines in the country favours a common vaccination strategy. However, specific GTPV vaccine is not available in the country. The circulating CaPV isolates were found to be host specific. The possibility of vaccination failures or disease caused by vaccines itself, or cross transmission between hosts cannot be ruled out and in such instances it becomes difficult to distinguish with natural disease.

scholarly journals Immunodetection of Fasciola gigantica Circulating Antigen in Sera of Infected Individuals for Laboratory Diagnosis of Human Fascioliasis

2013 ◽  
Vol 20 (10) ◽  
pp. 1569-1577 ◽  
Author(s):  
Abdelfattah M. Attallah ◽  
Faisal A. Bughdadi ◽  
Atef M. El-Shazly ◽  
Hisham Ismail
Keyword(s):  
Laboratory Diagnosis ◽  
Fasciola Gigantica ◽  
Antigen Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Target Antigen ◽  
Immunoperoxidase Staining ◽  
Serum Samples ◽  
Content Type ◽  
Human Fascioliasis ◽  
Circulating Antigen

ABSTRACTCurrently, the laboratory diagnosis of human fascioliasis is based on the parasitological examination of parasite eggs in stool specimens and serological detection of specific antibodies in serum samples, which are often unreliable diagnostic approaches. Ideally, a sensitive and specific diagnostic test forFasciolainfection should be based on the detection of circulatingFasciolaantigen, which implies active infection. Here, a 27-kDa-molecular-mass antigen was identified in aFasciola giganticaadult worm antigen preparation, excretory-secretory products, and sera fromF. gigantica-infected individuals, and it was not detected in antigenic extracts of other parasites and sera from noninfected individuals. The target antigen was isolated and partially characterized as a protein. Immunoperoxidase staining located the target epitope within teguments and guts ofF. giganticaadult worms. The performance characteristics of a newly developed enzyme-linked immunosorbent assay (ELISA) based onF. giganticacirculating antigen detection in serum (FgCA-27 ELISA) were investigated using sera of 120 parasitologically diagnosedF. gigantica-infected individuals and 80 noninfected individuals. The area under the receiving operating characteristic (ROC) curve (AUC) for ELISA was significantly high (AUC = 0.961,P< 0.0001) for discriminatingFasciola-infected and noninfected individuals. The developed assay showed high degrees of sensitivity, specificity, and efficiency (>93%), and a significant correlation (r= 0.715,P< 0.0001) between antigen level and parasite egg count was shown. In conclusion, a 27-kDaFasciolaantigen was identified in sera ofF. gigantica-infected individuals. A highly sensitive and specificFasciolaantigen detection assay, FgCA-27 ELISA, was developed for laboratory diagnosis of human fascioliasis.

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