Using a Deoxyribozyme Ligase and Rolling Circle Amplification To Detect a Non-nucleic Acid Analyte, ATP

An allosteric ribozyme (aptazyme) has been used to transduce the binding of a small organic analyte (ATP) into the ligation of a circular template for rolling circle amplification (RCA). An ATP-activated deoxyribozyme ligase was immobilized on a glass slide and, upon addition of ATP, catalyzed the ligation of a circular padlock probe. The ligated products could be directly amplified and visualized via RCA. The coupled reaction exhibited could detect as little as 1 μM of ATP and could discriminate against structurally similar nucleotides such as GTP, CTP, and UTP. Cooperative ATP activation of the deoxyribozyme was faithfully mimicked by RCA, yielding an amplified “switch” that was responsive to ATP concentration

Exclusivity of the porcine and chicken material detection method.

Exclusivity of the porcine and chicken material detection method.</p

Summary of collaborative trial results for the POD test.

Summary of collaborative trial results for the POD test.</p

Inclusivity of the porcine and chicken material detection method.

Inclusivity of the porcine and chicken material detection method.</p

Characterization and Phylogenetic Analysis of Allergenic Tryp_alpha_amyl Protein Family in Plants

Most known allergenic proteins in rice (Oryza sativa) seed belong to the Tryp_alpha_amyl family (PF00234), but the sequence characterization and the evolution of the allergenic Tryp_alpha_amyl family members in plants have not been fully investigated. In this study, two specific motifs were found besides the common alpha-amylase inhibitors (AAI) domain from the allergenic Tryp_alpha_amyl family members in rice seeds (trRSAs). To understand the evolution and functional importance of the Tryp_alpha_amy1 family and the specific motifs for the allergenic one, a BLAST search identified 75 homologous proteins of trRSAs (trHAs) from 22 plant species including main crops such as rice, maize (Zea mays), wheat (Triticum aestivum), and sorghum (Sorghum bicolor) from all available sequences in the public databases. Statistical analysis showed that the allergenicity of trHAs is closely associated with these two motifs with high number of cysteine residues (<i>p</i> value = 0.00026), and the trHAs with and without the two motifs were clustered into separate clades, respectively. Furthermore, significant difference was observed on the secondary and tertiary structures of allergenic and nonallergenic trHAs. In addition, expression analysis showed that trHA-encoding genes of purple false brome (Brachypodium distachyon), barrel medic (Medicago truncatula), rice, and sorghum are dominantly expressed in seeds. This work provides insight into the understanding of the properties of allergens in the Tryp_alpha_amyl family and is helpful for allergy therapy

Protocol and results.

Protocol and results of inter-laboratory collaborative validation trial for species specific real-time PCR assays of porcine and chicken-derived material in meat products. (DOCX)</p

Characterization and Phylogenetic Analysis of Allergenic Tryp_alpha_amyl Protein Family in Plants

Most known allergenic proteins in rice (Oryza sativa) seed belong to the Tryp_alpha_amyl family (PF00234), but the sequence characterization and the evolution of the allergenic Tryp_alpha_amyl family members in plants have not been fully investigated. In this study, two specific motifs were found besides the common alpha-amylase inhibitors (AAI) domain from the allergenic Tryp_alpha_amyl family members in rice seeds (trRSAs). To understand the evolution and functional importance of the Tryp_alpha_amy1 family and the specific motifs for the allergenic one, a BLAST search identified 75 homologous proteins of trRSAs (trHAs) from 22 plant species including main crops such as rice, maize (Zea mays), wheat (Triticum aestivum), and sorghum (Sorghum bicolor) from all available sequences in the public databases. Statistical analysis showed that the allergenicity of trHAs is closely associated with these two motifs with high number of cysteine residues (p value = 0.00026), and the trHAs with and without the two motifs were clustered into separate clades, respectively. Furthermore, significant difference was observed on the secondary and tertiary structures of allergenic and nonallergenic trHAs. In addition, expression analysis showed that trHA-encoding genes of purple false brome (Brachypodium distachyon), barrel medic (Medicago truncatula), rice, and sorghum are dominantly expressed in seeds. This work provides insight into the understanding of the properties of allergens in the Tryp_alpha_amyl family and is helpful for allergy therapy

Summary of false-positive and false-negative rates.

Summary of false-positive and false-negative rates.</p

Quantity of the reference genes in three sets of assays as compared with the Jimai 19 calibrator (Q = E^ΔCt).

<p>Boxplot shows median, interquartile and range.</p

DNA sequence of oligonucleotides used in this study.

DNA sequence of oligonucleotides used in this study.</p