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 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 Laboratory diagnosis ofMycoplasma pneumoniaeinfection: 2. Comparison of methods for the direct detection of specific antigen or nucleic acid sequences in respiratory exudates

1988 ◽  
Vol 101 (3) ◽  
pp. 685-694 ◽  
Author(s):  
R. Harris ◽  
B. P. Marmion ◽  
G. Varkanis ◽  
T. Kok ◽  
B. Lunn ◽  
...  
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Direct Detection ◽  
Cdna Probe ◽  
Specific Antigen ◽  
Diagnostic System ◽  
Laboratory Diagnosis ◽  
Mycoplasma Species ◽  
Rna Sequences ◽  
Current Infection ◽  
Probe Assay

SUMMARYThe efficiency of the direct detection ofMycoplasma pneumoniaein respiratory exudates by an antigen capture, indirect enzyme immunoassay (Ag-EIA), has been compared with its detection with a cDNA probe (‘Gen-Probe assay’) directed against the specific ribosomal RNA sequences of the organism (‘Mycoplasma pneumoniaeRapid Diagnostic System’, Gen-Probe, San Diego, California).Both assays showed excellent specificity against a range of mycoplasma species suspended in negative nasopharyngeal aspirates; onlyM. pneumoniaeandM. genitaliumreacted. In experiments with graded doses of viableM. pneumoniaecells suspended in negative nasopharyngeal aspirate, the Gen-Probe assay was more sensitive than Ag-EIA; detection limits were respectively 2 × 103 c.f.u./ml (3·2 × 105genomes) and 2·5 × 104c.f.u./ml (4 × 106genomes); detection levels 10–100 times less sensitive than culture.The two assays were also tested on nasopharyngeal aspirates or sputum specimens from 90 patients with respiratory infection; 67 of these were culture-or seronegative forM. pneumoniaeand 23 were culture-or seropositive. Ag-EIA detected 21 (91%) of the latter but the Gen-Probe assay detected only 5 (22%). Both assays were negative with the 67 culture-/sero-negatives; there were no Gen-Probe assay positive/Ag-EIA negatives.Overall, it is concluded that although Ag-EIA and the Gen-Probe assay are effective substitutes for culture as a diagnostic procedure, there is a significant problem with samples which are culture-negative and from patients who have good serological evidence of current infection. Possible reasons for the disparity between the two assays are advanced.

scholarly journals Laboratory diagnosis ofMycoplasma pneumoniaeinfection: 1. Direct detection of antigen in respiratory exudates by enzyme immunoassay.

1988 ◽  
Vol 101 (3) ◽  
pp. 669-684 ◽  
Author(s):  
T. W. Kok ◽  
G. Varkanis ◽  
B. P. Marmion ◽  
J. Martin ◽  
A. Esterman
Keyword(s):  
Indirect Method ◽  
Direct Detection ◽  
Culture Method ◽  
Laboratory Diagnosis ◽  
Current Level ◽  
Serological Examination ◽  
Antigen Capture ◽  
Colony Forming Units ◽  
Wide Range ◽  
Culture Negative

SUMMARYDirect and indirect antigen capture enzyme immunoassays (Ag-EIA) have been developed for the detection ofMycoplasma pneumoniaein nasopharyngeal aspirates or sputum from respiratory infection. The sensitivity of the two Ag-EIA were similar, but the indirect method using polyclonal rabbit and guinea-pig antisera was more convenient. The Ag-EIA had a detection limit of 104–4·5colony-forming units/ml of sample. It was specific for M. pneumoniae and gave a low level response with M. genitalium. There were no cross-reactions with 10 other species of mycoplasmas. Tests with a wide range of bacteria and chlamydia group antigen, representing agents sometimes found in the respiratory tract, were also negative. At the current level of development, the Ag-EIA detected about 90% of specimens that were also positive for culture; 43% of specimens from culture-negative-seropositive patients gave a positive result. The overall pattern of results indicated that while antigen detection is a quick and effective substitute for the slow culture method, serological examination for specific IgM antibody is also necessary to give a complete diagnostic coverage.

scholarly journals SARS-CoV-2 infection is at herd immunity in the majority segment of the population of Qatar

2021 ◽  
Author(s):  
Mohamed H Al-Thani ◽  
Elmoubasher Farag ◽  
Roberto Bertollini ◽  
Hamad Eid Al Romaihi ◽  
Sami Abdeen ◽  
...  
Keyword(s):  
Total Population ◽  
Herd Immunity ◽  
Geographic Location ◽  
Population Based ◽  
Infection Prevalence ◽  
Cross Sectional ◽  
Previous Infection ◽  
Current Infection ◽  
Polymerase Chain ◽  
Population Based Survey

Abstract Background Qatar experienced a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic that disproportionately affected the craft and manual worker (CMW) population who comprise 60% of the total population. This study aimed to assess ever and/or current infection prevalence in this population. Methods A cross-sectional population-based survey was conducted during July 26-September 09, 2020 to assess both anti-SARS-CoV-2 positivity through serological testing and current infection positivity through polymerase chain reaction (PCR) testing. Associations with antibody and PCR positivity were identified through regression analyses. Results Study included 2,641 participants, 69.3% of whom were <40 years of age. Anti-SARS-CoV-2 positivity was 55.3% (95% CI: 53.3-57.3%) and was significantly associated with nationality, geographic location, educational attainment, occupation, and previous infection diagnosis. PCR positivity was 11.3% (95% CI: 9.9-12.8%) and was significantly associated with nationality, geographic location, occupation, contact with an infected person, and reporting two or more symptoms. Infection positivity (antibody and/or PCR positive) was 60.6% (95% CI: 58.6-62.5%). The proportion of antibody-positive CMWs that had a prior SARS-CoV-2 diagnosis was 9.3% (95% CI: 7.9-11.0%). Only seven infections were ever severe and one was ever critical—an infection severity rate of 0.5% (95% CI: 0.2-1.0%). Conclusions Six in every 10 CMWs have been infected, suggestive of reaching the herd immunity threshold. Infection severity was low with only one in every 200 infections progressing to be severe or critical. Only one in every 10 infections had been previously diagnosed suggestive of mostly asymptomatic or mild infections.

scholarly journals Diagnosis of malaria by detection of Plasmodium falciparum HRP-2 antigen with a rapid dipstick antigen-capture assay

The Lancet ◽  
1994 ◽  
Vol 343 (8897) ◽  
pp. 564-568 ◽  
Author(s):  
C Beadle ◽  
G.W Long ◽  
P.D McElroy ◽  
S.L Hoffman ◽  
G.W Long ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Capture Assay ◽  
Antigen Capture

ACIDIFICATION MODIFIED P24 ANTIGEN CAPTURE ASSAY IN HIV-SEROPOSITIVE PATIENTS

1992 ◽  
Vol 85 (Supplement) ◽  
pp. 3S-42
Author(s):  
David Ascher ◽  
Chet Roberts ◽  
Arnold Fowler
Keyword(s):  
Capture Assay ◽  
Antigen Capture ◽  
P24 Antigen ◽  
Hiv Seropositive

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.

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