DNA Sequence Signatures for Rapid Detection of Six Target Bacterial Pathogens Using PCR Assays

Using Streptococcus pyogenes as a model, we previously established a stepwise computational workflow to effectively identify species-specific DNA signatures that could be used as PCR primer sets to detect target bacteria with high specificity and sensitivity. In this study, we extended the workflow for the rapid development of PCR assays targeting Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Clostridium difficile, Clostridium tetani, and Staphylococcus aureus, which are of safety concern for human tissue intended for transplantation. Twenty-one primer sets that had sensitivity of detecting 5–50 fg DNA from target bacteria with high specificity were selected. These selected primer sets can be used in a PCR array for detecting target bacteria with high sensitivity and specificity. The workflow could be widely applicable for the rapid development of PCR-based assays for a wide range of target bacteria, including those of biothreat agents.

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Novel Primer Sets for Rapid Detection of Zymoseptoria tritici in Wheat

Plant Disease ◽

10.1094/pdis-02-20-0318-sc ◽

2020 ◽

pp. PDIS-02-20-0318

Author(s):

Adam Kuzdraliński ◽

Justyna Leśniowska-Nowak ◽

Michał Nowak ◽

Magdalena Kawęcka ◽

Anna Kot ◽

...

Keyword(s):

High Specificity ◽

Septoria Tritici Blotch ◽

Septoria Tritici ◽

In Vitro Tests ◽

Zymoseptoria Tritici ◽

Specificity And Sensitivity ◽

Wheat Leaf ◽

Primer Sets ◽

Species Specific

Zymoseptoria tritici is a fungal pathogen causing losses in wheat yields. Here, we present new primer sets for species-specific identification of this microorganism in wheat leaf samples using conventional PCR. Primer sets were validated in silico using tools available in genetic databases. Furthermore, in vitro tests were also carried out on 190 common wheat samples with visual symptoms of Septoria tritici blotch (STB) collected in Poland in three growing seasons (2015, 2016, 2017). The designed primer sets showed full hybridization to the available genetic resources deposited in the NCBI GenBank database, and their high specificity and sensitivity were demonstrated on wheat leaf samples and selected fungal strains.

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

Communications Biology ◽

10.1038/s42003-021-01649-6 ◽

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.

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

Nanomaterials ◽

10.3390/nano11051207 ◽

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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Development and Application of a New PCR Method for Detection of Blumeria graminis f. sp. tritici

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000494432 ◽

2018 ◽

Vol 28 (3) ◽

pp. 137-146 ◽

Cited By ~ 1

Author(s):

Adam Kuzdraliński ◽

Hubert Szczerba ◽

Anna Kot ◽

Agnieszka Ostrowska ◽

Michał Nowak ◽

...

Keyword(s):

Dna Sequences ◽

Puccinia Triticina ◽

Blumeria Graminis ◽

Pyrenophora Tritici Repentis ◽

Field Samples ◽

Expected Length ◽

Pcr Assays ◽

Primer Sets ◽

Wheat Leaves ◽

Species Specific

We developed new PCR assays that target beta-tubulin (TUB2) and 14 alpha-demethylase (CYP51) genes and used them for the species-specific detection of Blumeria graminis f. sp. tritici (Bgt). Based on fungi DNA sequences available in the NCBI (National Center for Biotechnology Information) GenBank database we developed simplex and duplex PCR assays. The specificities of the primer sets were evaluated using environmental samples of wheat leaves collected during the 2015/2016 growing season across Poland. Primer sets LidBg17/18 and LidBg21/22 strongly amplified fragments of the expected length for all 67 tested samples. Primer specificity was confirmed using field samples of Zymoseptoria tritici, Puccinia triticina (syn. P. recondita f. sp. tritici), P. striiformis f. sp. tritici, and Pyrenophora tritici-repentis.

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A 16S rDNA-based nested PCR protocol to detect Campylobacter gracilis in oral infections

Pesquisa Odontológica Brasileira ◽

10.1590/s1517-74912003000200008 ◽

2003 ◽

Vol 17 (2) ◽

pp. 142-146 ◽

Cited By ~ 9

Author(s):

José Freitas Siqueira Júnior ◽

Isabela das Neves Rôças

Keyword(s):

16S Rdna ◽

High Sensitivity ◽

High Specificity ◽

Cross Reactivity ◽

Nested Polymerase Chain Reaction ◽

Oral Infections ◽

Root Canals ◽

Infected Root ◽

Pcr Products ◽

Species Specific

The aim of this study was to describe a 16S rDNA-based nested polymerase chain reaction (nPCR) assay to investigate the occurrence of Campylobacter gracilis in oral infections. Samples were collected from ten infected root canals, ten cases of acute periradicular abscesses and eight cases of adult marginal periodontitis. DNA extracted from the samples was initially amplified using universal 16S rDNA primers. A second round of amplification used the first PCR products to detect C. gracilis using oligonucleotide primers designed from species-specific 16S rDNA signature sequences. The nPCR assay used in this study showed a detection limit of 10 C. gracilis cells and no cross-reactivity was observed with nontarget bacteria. C. gracilis was detected in the three types of oral infections investigated - 4/10 infected root canals; 2/10 acute periradicular abscesses; and 1/8 subgingival specimens from adult periodontitis. The method proposed in this study showed both high sensitivity and high specificity to directly detect C. gracilis in samples from root canal infections, abscesses, and subgingival plaque. Our findings confirmed that C. gracilis may be a member of the microbiota associated with distinct oral infections, and its specific role in such diseases requires further clarification.

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A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands

Frontiers in Immunology ◽

10.3389/fimmu.2021.817604 ◽

2022 ◽

Vol 12 ◽

Author(s):

Katharina Radakovics ◽

Claire Battin ◽

Judith Leitner ◽

Sabine Geiselhart ◽

Wolfgang Paster ◽

...

Keyword(s):

Limit Of Detection ◽

Ectopic Expression ◽

High Sensitivity ◽

High Specificity ◽

Reporter System ◽

Specificity And Sensitivity ◽

Highly Sensitive ◽

Definition Of ◽

The Individual ◽

Allergen Extracts

Toll-like receptors (TLRs) are primary pattern recognition receptors (PRRs), which recognize conserved microbial components. They play important roles in innate immunity but also in the initiation of adaptive immune responses. Impurities containing TLR ligands are a frequent problem in research but also for the production of therapeutics since TLR ligands can exert strong immunomodulatory properties even in minute amounts. Consequently, there is a need for sensitive tools to detect TLR ligands with high sensitivity and specificity. Here we describe the development of a platform based on a highly sensitive NF-κB::eGFP reporter Jurkat JE6-1 T cell line for the detection of TLR ligands. Ectopic expression of TLRs and their coreceptors and CRISPR/Cas9-mediated deletion of endogenously expressed TLRs was deployed to generate reporter cell lines selectively expressing functional human TLR2/1, TLR2/6, TLR4 or TLR5 complexes. Using well-defined agonists for the respective TLR complexes we could demonstrate high specificity and sensitivity of the individual reporter lines. The limit of detection for LPS was below 1 pg/mL and ligands for TLR2/1 (Pam3CSK4), TLR2/6 (Fsl-1) and TLR5 (flagellin) were detected at concentrations as low as 1.0 ng/mL, 0.2 ng/mL and 10 pg/mL, respectively. We showed that the JE6-1 TLR reporter cells have the utility to characterize different commercially available TLR ligands as well as more complex samples like bacterially expressed proteins or allergen extracts. Impurities in preparations of microbial compounds as well as the lack of specificity of detection systems can lead to erroneous results and currently there is no consensus regarding the involvement of TLRs in the recognition of several molecules with proposed immunostimulatory functions. This reporter system represents a highly suitable tool for the definition of structural requirements for agonists of distinct TLR complexes.

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Loop-Mediated Isothermal Amplification for Detection ofStaphylococcus aureusin Dairy Cow Suffering from Mastitis

Journal of Biomedicine and Biotechnology ◽

10.1155/2012/435982 ◽

2012 ◽

Vol 2012 ◽

pp. 1-5 ◽

Cited By ~ 9

Author(s):

Zhang Tie ◽

Wang Chunguang ◽

Wei Xiaoyuan ◽

Zhao Xinghua ◽

Zhong Xiuhui

Keyword(s):

Staphylococcus Aureus ◽

High Sensitivity ◽

High Specificity ◽

Isothermal Amplification ◽

Lamp Method ◽

Specific Primers ◽

Loop Mediated Isothermal Amplification ◽

Reaction Tube ◽

Specificity And Sensitivity ◽

Detectable Concentration

To develop a rapid detection method ofStaphylococcus aureususing loop-mediated isothermal amplification (LAMP), four specific primers were designed according to six distinct sequences of thenucgene. In addition, the specificity and sensitivity of LAMP were verified and compared with those of PCR. Results showed that the LAMP reaction was completed within 45 min at 62.5°C, and ladder bands were appeared in LAMP products analyzed by gel electrophoresis. After adding 1x SYBR Green l, the positive reaction tube showed green color and the negative reaction tube remained orange, indicating that the LAMP has high specificity. The minimal detectable concentration of LAMP was1×102 CFU/mL and that of PCR was1×104 CFU/mL, indicating that the LAMP was 100 times more sensitive than the PCR. The LAMP method for detection ofStaphylococcus aureushas many advantages, such as simple operation, high sensitivity, high specificity, and rapid analysis. Therefore, this method is more suitable for the rapid on-site detection ofStaphylococcus aureus.

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A Preliminary Evaluation of a Prototype Western Blot Confirmatory Test Kit for Syphilis

International Journal of STD & AIDS ◽

10.1177/095646249400500606 ◽

1994 ◽

Vol 5 (6) ◽

pp. 409-414 ◽

Cited By ~ 4

Author(s):

H Young ◽

P J Walker ◽

D Merry ◽

A Mifsud

Keyword(s):

Western Blot ◽

False Positive ◽

High Sensitivity ◽

High Specificity ◽

Confirmatory Test ◽

Preliminary Evaluation ◽

Serological Tests ◽

Western Blot Test ◽

Specificity And Sensitivity ◽

Test Kit

A prototype Western blot kit was evaluated as a confirmatory test for syphilis using 131 sera characterized by other serological tests for syphilis. There were 114 treponemal sera (including 94 cases of early syphilis, 83 of which were untreated) and 17 non-treponemal problem sera (11 gave false positive reactions on screening with the TmpA recombinant antigen enzyme immunoassay (EIA), 3 gave false positive fluorescent treponemal antibody absorbed (FTA-abs) tests, and 3 false positive Captia Syphilis G EIA results). Based on the manufacturer's criteria of reactivity in multiple bands for designating a positive result the Western blot test gave a sensitivity of 99.1% (113/114) and a specificity of 88.2% (15/17) when indeterminate reactions were scored positive and 98.2% (112/114) and 100% (17/17) when indeterminate reactions were scored negative. Sensitivity was high in both treated and untreated infection. Corresponding sensitivities for the TPHA and FTA-abs when equivocal reactions were scored negative were 97.5% (111/114) and 99.1% (113/114). The high sensitivity of the FTA-abs in this study is probably due to the large number of untreated primary infections. Our results with the Western blot, confirm earlier studies using ‘in-house’ test systems and, support a role for a commercial Western blot test in the confirmatory diagnosis of syphilis. Further studies are required to confirm the high specificity and sensitivity of the kit in a larger series including a wider variety of non-treponemal cases as well as patients with untreated and treated infection.

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Development of Candida-Specific Real-Time PCR Assays for the Detection and Identification of Eight Medically Important Candida Species

Microbiology Insights ◽

10.4137/mbi.s38517 ◽

2016 ◽

Vol 9 ◽

pp. MBI.S38517 ◽

Cited By ~ 15

Author(s):

Jing Zhang ◽

Guo-Chiuan Hung ◽

Kenjiro Nagamine ◽

Bingjie Li ◽

Shien Tsai ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Candida Species ◽

Rapid Detection ◽

Corneal Transplantation ◽

High Sensitivity ◽

Turnaround Time ◽

Pcr Assays ◽

Primer Sets ◽

Pcr Primer

Culture-based identification methods have been the gold standard for the diagnosis of fungal infection. Currently, molecular technologies such as real-time PCR assays with short turnaround time can provide desirable alternatives for the rapid detection of Candida microbes. However, most of the published PCR primer sets are not Candida specific and likely to amplify DNA from common environmental contaminants, such as Aspergillus microbes. In this study, we designed pan- Candida primer sets based on the ribosomal DNA-coding regions conserved within Candida but distinct from those of Aspergillus and Penicillium. We demonstrate that the final two selected pan- Candida primer sets would not amplify Aspergillus DNA and could be used to differentiate eight medically important Candida pathogens in real-time PCR assays based on their melting profiles, with a sensitivity of detection as low as 10 fg of Candida genomic DNA. Moreover, we further evaluated and selected species-specific primer sets covering Candida albicans, Candida glabrata, Candida tropicalis, and Candida dubliniensis and show that they had high sensitivity and specificity. These real-time PCR primer sets could potentially be assembled into a single PCR array for the rapid detection of Candida species in various clinical settings, such as corneal transplantation.

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

10.21203/rs.3.rs-357601/v1 ◽

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.

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