Touchdown PCR for increased specificity and sensitivity in PCR amplification

2008 ◽  
Vol 3 (9) ◽  
pp. 1452-1456 ◽  
Author(s):  
Darren J Korbie ◽  
John S Mattick
Keyword(s):  
Pcr Amplification ◽  
Specificity And Sensitivity ◽  
Touchdown Pcr

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.

Detection of adenovirus and enterovirus by PCR amplification in polluted waters

1995 ◽  
Vol 31 (5-6) ◽  
pp. 351-357 ◽  
Author(s):  
R. Girones ◽  
M. Puig ◽  
A. Allard ◽  
F. Lucena ◽  
G. Wadell ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Environmental Samples ◽  
Hepatitis A Virus ◽  
Pcr Amplification ◽  
Human Adenovirus ◽  
Acid Extraction ◽  
Specific Primers ◽  
Specificity And Sensitivity ◽  
High Prevalence ◽  
Virus Recovery

Several systems for virus recovery from environmental samples and extraction of nucleic acid were tested by adding adenovirus 2 and poliovirus 1 to different sewage samples. The most promising method involved: concentration of viruses by centrifugation, and elution of the pelleted viruses by treatment with 0.25 N glycine buffer pH 9.5. The nucleic acids were extracted by adsorption of RNA and DNA to silica particles. One aliquot was directly used for a two-step PCR in a nested fashion, with specific primers for all adenoviruses; the other aliquot was used to synthesize cDNA and a nested two-step PCR with specific primers for enteroviruses. The specificity and sensitivity of the selected primers were evaluated, the 47 human adenovirus serotypes were identified and 24 different enterovirus strains were recognized. The sensitivity of the nucleic acid extraction, cDNA synthesis and nested PCR amplification was estimated to be between 1 and 10 viral particles. Sewage and polluted river samples were analyzed showing, as expected, a much higher number of positive samples by the method described than by tissue culture analysis, a high prevalence of hepatitis A virus in sewage and the adenoviruses as the most commonly detected virus in the environmental samples analyzed.

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 Optimization of PCR amplification of maize microsatellite loci

2000 ◽  
Vol 23 (2) ◽  
pp. 395-398 ◽  
Author(s):  
Juliana Bernardi Ogliari ◽  
Raquel L. Boscariol ◽  
Luis E.A. Camargo
Keyword(s):  
Microsatellite Loci ◽  
Annealing Temperature ◽  
Zea Mays L ◽  
Pcr Amplification ◽  
Nucleotide Composition ◽  
Nucleic Acid Content ◽  
Alternative Programs ◽  
Annealing Temperatures ◽  
Opposite Situation ◽  
Touchdown Pcr

Maize (Zea mays L.) microsatellite loci are useful as genetic markers because they are numerous, occur in every chromosome, and have a high content of polymorphism information. Furthermore, they can be amplified by PCR and the resulting fragments resolved on agarose gels. A major problem, however, is that the primers used in their amplification often have different length and nucleic acid content, requiring optimization of PCR amplification programs for each locus. We developed "touchdown" PCR programs successfully used in the amplification of a set of 125-maize microsatellite loci, chosen as representative of all chromosomes. The programs varied both in relation to the annealing temperature (Tm) and primer concentration. Primers could be divided into four groups. A large group included primers that amplified well in a basic previously published amplification program but with different primer concentrations. A second group amplified in alternative programs in which the annealing temperatures were changed so as to include the Tm of either both primers or the highest Tm of the primer pair estimated based on their nucleotide composition. A third group included primers that amplified in programs with Tm higher than those estimated, and in a fourth group, with just two pairs of primers, the opposite situation prevailed.

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 Strategy for DNA extraction and detection from insect pests in stored home grain samples RESEARCH

2021 ◽  
Vol 01 (1) ◽  
pp. 24-32
Author(s):  
Ibrahim Abbasi
Keyword(s):  
Dna Extraction ◽  
Insect Pests ◽  
Pcr Amplification ◽  
Extraction Methods ◽  
Alternative Methods ◽  
Coi Gene ◽  
Pest Species ◽  
Detection Techniques ◽  
Specificity And Sensitivity ◽  
Washing Method

Stored grains are subjected to infestations with more than 60 species of insects, that responsible for millions of dollars’ loss and cause several health problems including allergies and gastrointestinal disorders. Traditional detection techniques are laborious, expensive and not sensitive to detect insect contamination at the egg and larvae stages. Therefore, alternative methods are needed for rapid and sensitive detection. One obvious approach is to develop a molecular approach utilizing genetic information of the potential insect species that infest grains for amplification of specific target gene fragment utilizing polymerase chain reaction [PCR]. In the present study, a number of known infested grain samples were used in standardizing a method to isolate larvae and adult insects that were based on centrifugation washing method and a filtration washing method. The isolated insects were subjected to DNA extraction and PCR amplification of defined regions of cytochrome oxidase I (COI) gene followed by sequencing to identify the di"erent pest species. For PCR amplification new primers were designed and for this purpose the obtained COI sequences from di"erent insects were aligned to design two sets of primers (named: COI-PCR4 and COI-PCR5) specific for the indicated insect mitochondrial COI gene. The designed primers were tested for their specificity and sensitivity. The suitability of PCR primers and DNA extraction methods were evaluated on eleven samples of commercial grains utilizing each primer set with the two extraction methods.

scholarly journals Massively parallel DNA target capture using Long Adapter Single Stranded Oligonucleotide (LASSO) probes assembled through a novel DNA recombinase mediated methodology

2021 ◽  
Author(s):  
LORENZO TOSI ◽  
Lamia Chkaiban ◽  
H. Benjamin Larman ◽  
Jeffrey Rosenfeld ◽  
Biju Parekkadan
Keyword(s):  
Pcr Amplification ◽  
Plasmid Vector ◽  
Open Reading Frames ◽  
Dna Oligonucleotides ◽  
Target Capture ◽  
Specificity And Sensitivity ◽  
Long Read ◽  
Dna Template ◽  
Dna Recombinase ◽  
Reading Frames

In the attempt to bridge the widening gap from DNA sequence to biological function, we developed a novel methodology to assemble Long-Adapter Single-Strand Oligonucleotide (LASSO) probe libraries that enabled the massively multiplexed capture of kilobase-sized DNA fragments for downstream long read DNA sequencing or expression. This method uses short DNA oligonucleotides (pre-LASSO probes) and a plasmid vector that supplies the backbone for the mature LASSO probe through Cre-Loxp intramolecular recombination. This strategy generates high quality LASSO probes libraries (~46% of probes). We performed NGS analysis of the post-capture PCR amplification of DNA circles obtained from the LASSO capture of 3087 E.coli ORFs spanning from 400- to 4,000 bp. The median enrichment of all targeted ORFs versus untargeted ORFs was 30 times. For ORFs up to 1kb in size, targeted ORFs were enriched up to a median of 260-fold. Here, we show that LASSO probes obtained in this manner, are able to capture full-length open reading frames from total human cDNA. Furthermore, we show that the LASSO capture specificity and sensitivity is sufficient for target capture from total human genomic DNA template. This technology can be used for the preparation of long-read sequencing libraries and for massively multiplexed cloning of human sequences.

scholarly journals Evaluation of Cryptosporidium parvumGenotyping Techniques

1999 ◽  
Vol 65 (10) ◽  
pp. 4431-4435 ◽  
Author(s):  
Irshad M. Sulaiman ◽  
Lihua Xiao ◽  
Altaf A. Lal
Keyword(s):  
Genomic Dna ◽  
Giardia Duodenalis ◽  
Pcr Amplification ◽  
Small Subunit ◽  
Species Differentiation ◽  
Small Subunit Rrna ◽  
Specificity And Sensitivity ◽  
Genotype 2 ◽  
Pcr Rflp ◽  
Sensitivity Studies

ABSTRACT We evaluated the specificity and sensitivity of 11 previously described species differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. Genomic DNA from three species of Cryptosporidium parasites (genotype 1 and genotype 2 of C. parvum, C. muris, and C. serpentis), two Eimeria species (E. neischulzi and E. papillata), and Giardia duodenalis were used to evaluate the specificity of primers. Furthermore, the sensitivity of the genotyping primers was tested by using genomic DNA isolated from known numbers of oocysts obtained from a genotype 2 C. parvum isolate. PCR amplification was repeated at least three times with all of the primer pairs. Of the 11 protocols studied, 10 amplified C. parvum genotypes 1 and 2, and the expected fragment sizes were obtained. Our results indicate that two species-differentiating protocols are notCryptosporidium specific, as the primers used in these protocols also amplified the DNA of Eimeria species. The sensitivity studies revealed that two nested PCR-restriction fragment length polymorphism (RFLP) protocols based on the small-subunit rRNA and dihydrofolate reductase genes are more sensitive than single-round PCR or PCR-RFLP protocols.

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