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.

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 Polymerase Chain Reaction (PCR): A Short Review

2013 ◽  
Vol 4 (1) ◽  
pp. 30-36 ◽  
Author(s):  
Md Tahminur Rahman ◽  
Muhammed Salah Uddin ◽  
Razia Sultana ◽  
Arumina Moue ◽  
Muntahina Setu
Keyword(s):  
Correct Diagnosis ◽  
Medical Science ◽  
Genetic Diagnosis ◽  
High Specificity ◽  
Short Review ◽  
Laboratory Diagnosis ◽  
Molecular Techniques ◽  
Advantages And Disadvantages ◽  
Specificity And Sensitivity ◽  
Diagnostic Modalities

Diagnosis of disease now a days is mostly laboratory dependent. Due to recent advances in medical science and molecular biology, most of the diagnosis of uncommon, complicated, unusual presentation of disease has left the option of molecular diagnosis as the number one diagnostic modalities. Many molecular techniques are now being widely used throughout the  world including PCR, flow cytometry, tissue microarray, different blots, and genetic diagnosis. Among these PCR is the most widely accepted, commonly used diagnostic modalities with very high specificity and sensitivity for correct diagnosis. We have reviewed the principle, application, advantages and disadvantages of PCR in laboratory diagnosis of disease. DOI: http://dx.doi.org/10.3329/akmmcj.v4i1.13682 AKMMC J 2013: 4(1): 30-36    

scholarly journals An alternative PCR assay with high sensitivity and specificity for the detection of swine toxoplasmosis based on the GRA14 gene

2021 ◽  
Author(s):  
Xi He ◽  
Derong Zhou ◽  
Yanwu Sun ◽  
Yuan Zhang ◽  
Xiaogang Zhang ◽  
...  
Keyword(s):  
Detection Method ◽  
Southern China ◽  
Acute Infection ◽  
High Sensitivity ◽  
High Specificity ◽  
Pcr Detection ◽  
Pcr Assay ◽  
Specific Primers ◽  
Specificity And Sensitivity ◽  
Pcr Method

Abstract Background Toxoplasma gondii, an intracellular apicomplexan protozoan parasite, can infect all warm-blooded animals. Infected swine are considered one of the most important sources of T. gondii infection in humans. Rapidly and effectively diagnosing T. gondii infection in swine is essential. PCR-based diagnostic tests have been fully developed, and very sensitive and specific PCR is crucial for the diagnosis of swine toxoplasmosis. Methods To established a high specificity and sensitivity PCR detection method for swine toxoplasmosis, we used T. gondii GRA14 gene as target to design specific primers and established a PCR detection method for swine toxoplasmosis. A total of 5462 blood specimens collected from pigs in 5 provinces and autonomous regions in southern China during 2016–2017 were assessed by the newly established GRA14 gene PCR method. Result Altogether, we used T. gondii GRA14 gene as target to design specific primers and established a high specificity and sensitivity PCR detection method for swine toxoplasmosis; in particular, this PCR method could detect T. gondii tachyzoite DNA in the acute infection phase. The GRA14 gene PCR assay detected a minimum of 2.35 tachyzoites of T. gondii, and it could be used for T. gondii detection in blood, tissue, semen, urine and waste feed specimens. The overall T. gondii infection rate was 18.9% (1033/5462) by the newly established GRA14 gene PCR method. According to statistical analysis among different regions, the positive rates of swine toxoplasmosis in the Shaanxi, Fujian and Guangdong areas in China from 2016 to 2017 were the highest, at 31.7% (44/139), 21.9% (86/391) and 18.8% (874/4645), respectively (χ2 = 84.2, P < 0.0001). Specimens collected in 2017 had a higher positive rate (19.1% or 886/4639) than those collected in 2016 (16.1% or 155/963) (χ2 = 4.5, P < 0.05). Specimens collected in autumn (39.4% or 187/474), spring (22.8% or 670/2940) and winter (18.2% or 129/709) also had higher positive rates than those collected in summer (3.8% or 57/1479) (χ2 = 427.7, P < 0.0001). Conclusions These results indicate that the new PCR method based on the T. gondii GRA14 gene would be useful for the diagnosis of swine toxoplasmosis and that it would facilitate the diagnosis of toxoplasmosis in clinical laboratories.

scholarly journals Multi-peptide ELISAs overcome cross-reactivity and inadequate sensitivity of conventional Chlamydia pneumoniae serology

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kh Shamsur Rahman ◽  
Bernhard Kaltenboeck
Keyword(s):  
Chlamydia Pneumoniae ◽  
High Specificity ◽  
Cross Reactivity ◽  
Peptide Antigens ◽  
Specificity And Sensitivity ◽  
Peptide Antibody ◽  
Antibody Assays ◽  
Human Sera ◽  
Composite Reference Standard ◽  
Individual Peptide

Abstract Cross-reactivity of classical chlamydial antigens compromises Chlamydia (C.) pneumoniae serology. By testing with 185 human antisera, we expanded 18 previously discovered C. pneumoniae-specific B-cell epitopes to 48 peptide antigens from 12 C. pneumoniae immunodominant proteins. For specific detection of antibodies against C. pneumoniae, we developed novel ELISAs with strongly reactive individual peptide antigens and mixtures of these peptides. By comparison to a composite reference standard (CRS) for anti-C. pneumoniae antibody status of human sera, the top-performing CpnMixF12 peptide assay showed 91% sensitivity at 95% specificity, significantly higher than 4 commercial anti-C. pneumoniae IgG ELISAs (36-12% sensitivity at 95% specificity). Human C. pneumoniae (Cpn) and C. trachomatis (Ctr) seroreactivity was 54% biased towards co-positivity in commercial Cpn and Ctr ELISAs, but unbiased in Cpn and Ctr peptide antibody assays, suggesting severe cross-reactivity of commercial ELISAs. Using hyperimmune mouse sera against each of 11 Chlamydia spp., we confirm that commercial Cpn and Ctr ELISA antigens are cross-reactive among all Chlamydia spp., but Cpn and Ctr peptide antigens react only with antisera against the cognate chlamydial species. With simultaneously high specificity and sensitivity, and convenient use for non-specialized laboratories, these ELISAs have the potential to improve serodiagnosis of C. pneumoniae infection.

scholarly journals MANAGEMENT OF ENDOCRINE DISEASE Hyperandrogenic states in women: pitfalls in laboratory diagnosis

2018 ◽  
Vol 178 (4) ◽  
pp. R141-R154 ◽  
Author(s):  
Michel Pugeat ◽  
Ingrid Plotton ◽  
Aude Brac de la Perrière ◽  
Gérald Raverot ◽  
Henri Déchaud ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Limit Of Detection ◽  
High Specificity ◽  
Free Testosterone ◽  
Laboratory Diagnosis ◽  
Sex Hormone Binding Globulin ◽  
Total Testosterone ◽  
Adrenal Androgen ◽  
Androgen Excess ◽  
Specificity And Sensitivity

Measuring total testosterone level is the first-line approach in assessing androgen excess in women. The main pitfalls in measuring testosterone relate to its low concentration and to the structural similarity between circulating androgens and testosterone, requiring accurate techniques with high specificity and sensitivity. These goals can be achieved by immunoassay using a specific anti-testosterone monoclonal antibody, ideally after an extraction step. Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) will be commonly used for measuring testosterone, providing optimal accuracy with a low limit of detection. Yet, the pitfalls of these two techniques are well identified and must be recognized and systematically addressed. In general, laboratories using direct testosterone immunoassay and mass spectrometry need to operate within a quality framework and be actively engaged in external quality control processes and standardization, so as to ensure appropriate interpretation irrespective of the particular laboratory. Circulating testosterone is strongly bound to sex-hormone-binding globulin (SHBG), and SHBG levels are typically low in overweight hyperandrogenic patients. Thus, low SHBG may decrease circulating testosterone to normal values, which will mask androgen excess status. One way to avoid this pitfall, awaiting direct free testosterone assays that are yet to be developed, is to measure SHBG and calculate free testosterone. A few other pitfalls will be discussed in this review, including those of adrenal androgen exploration, with the aim of helping clinicians to better handle laboratory investigation of androgen excess disorders in women.

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 Application of Fisetin to the Quantitation of Serum Albumin

2020 ◽  
Vol 9 (2) ◽  
pp. 459 ◽  
Author(s):  
Jung-Min Park ◽  
Van Quan Do ◽  
Yoon-Seok Seo ◽  
Men Thi Hoai Duong ◽  
Hee-Chul Ahn ◽  
...  
Keyword(s):  
Serum Albumin ◽  
Serum Proteins ◽  
Clinical Laboratory ◽  
High Specificity ◽  
Decay Rates ◽  
High Signal ◽  
Fluorescence Signal ◽  
Specificity And Sensitivity ◽  
Wide Range ◽  
Human Sera

Fisetin (3,3′,4′,7-tetrahydroxyflavone) is a widely distributed natural flavonol. It interacts with albumin, and thereby generates a fluorescence signal quantitatively. Based on such optical characteristics, we postulated that fisetin was applicable to the quantitation of albumin as an indicator. To establish the fisetin-based albumin assay, we examined the optical properties of fisetin and fisetin–albumin complex. The assay conditions were fine-tuned to fit for the actual concentration of serum albumin and to generate an optimal signal with a high signal-to-background ratio. The reaction between fisetin and albumin was linear in a wide range of concentrations. Non-protein serum components did not interfere with the reaction. The reactivity of fisetin was apparently specific for albumin among serum proteins. Both plasma and serum were compatible with the assay. The samples could be stored in a refrigerator or a freezer without the loss of reactivity toward fisetin. The generation and decay rates of the signal were acceptable for manual handling. The recovery of fortified albumin in serum was confirmed and the assay was validated with human sera. Fisetin-based albumin assay is suitable for clinical laboratory testing, considering the simple and short procedure, high specificity and sensitivity, linearity over a wide range of albumin concentrations, and, presumably, potential automatability.

Prospective Application of Aptamer-based Assays and Therapeutics in Bloodstream Infections

2020 ◽  
Vol 20 (10) ◽  
pp. 831-840
Author(s):  
Weibin Li
Keyword(s):  
Pathogenic Bacteria ◽  
Genetic Diagnosis ◽  
Bloodstream Infections ◽  
High Specificity ◽  
Peptide Sequence ◽  
High Morbidity ◽  
Specificity And Sensitivity ◽  
Prospective Application ◽  
Small Molecule Therapeutics

Sepsis is still a severe health problem worldwide with high morbidity and mortality. Blood bacterial culture remains the gold standard for the detection of pathogenic bacteria in bloodstream infections, but it is time-consuming, and both the sophisticated equipment and well-trained personnel are required. Immunoassays and genetic diagnosis are expensive and limited to specificity and sensitivity. Aptamers are single-stranded deoxyribonucleic acid (ssDNA) and ribonucleic acid (RNA) oligonucleotide or peptide sequence generated in vitro based on the binding affinity of aptamer-target by a process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). By taking several advantages over monoclonal antibodies and other conventional small-molecule therapeutics, such as high specificity and affinity, negligible batch-to-batch variation, flexible modification and production, thermal stability, low immunogenicity and lack of toxicity, aptamers are presently becoming promising novel diagnostic and therapeutic agents. This review describes the prospective application of aptamerbased laboratory diagnostic assays and therapeutics for pathogenic bacteria and toxins in bloodstream infections.

scholarly journals SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Mikail Dogan ◽  
Lina Kozhaya ◽  
Lindsey Placek ◽  
Courtney Gunter ◽  
Mesut Yigit ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
High Specificity ◽  
Spike Protein ◽  
Humoral Immune ◽  
Specificity And Sensitivity ◽  
Wide Range ◽  
Specific Igg ◽  
Negative Controls

AbstractDevelopment of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.

scholarly journals Highly Sensitive Fluorescent Detection of Acetylcholine Based on the Enhanced Peroxidase-Like Activity of Histidine Coated Magnetic Nanoparticles

Nanomaterials ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1207
Author(s):  
Hong Jae Cheon ◽  
Quynh Huong Nguyen ◽  
Moon Il Kim
Keyword(s):  
Magnetic Nanoparticles ◽  
High Sensitivity ◽  
High Specificity ◽  
Choline Oxidase ◽  
Fluorescent Detection ◽  
One Pot ◽  
Site Structure ◽  
Specificity And Sensitivity ◽  
Highly Sensitive ◽  
Peroxidase Mimics

Inspired by the active site structure of natural horseradish peroxidase having iron as a pivotal element with coordinated histidine residues, we have developed histidine coated magnetic nanoparticles (His@MNPs) with relatively uniform and small sizes (less than 10 nm) through one-pot heat treatment. In comparison to pristine MNPs and other amino acid coated MNPs, His@MNPs exhibited a considerably enhanced peroxidase-imitating activity, approaching 10-fold higher in catalytic reactions. With the high activity, His@MNPs then were exploited to detect the important neurotransmitter acetylcholine. By coupling choline oxidase and acetylcholine esterase with His@MNPs as peroxidase mimics, target choline and acetylcholine were successfully detected via fluorescent mode with high specificity and sensitivity with the limits of detection down to 200 and 100 nM, respectively. The diagnostic capability of the method is demonstrated by analyzing acetylcholine in human blood serum. This study thus demonstrates the potential of utilizing His@MNPs as peroxidase-mimicking nanozymes for detecting important biological and clinical targets with high sensitivity and reliability.

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