Polymerase chain reaction-hybridization method using urease gene sequences for high-throughput Ureaplasma urealyticum and Ureaplasma parvum detection and differentiation. 2016

Chen Xu, and Nan Zhang, and Qianyu Huo, and Minghui Chen, and Rengfeng Wang, and Zhili Liu, and Xue Li, and Yunde Liu, and Huijing Bao
Fourth Central Hospital, Tianjin 300203, China.

In this article, we discuss the polymerase chain reaction (PCR)-hybridization assay that we developed for high-throughput simultaneous detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum using one set of primers and two specific DNA probes based on urease gene nucleotide sequence differences. First, U. urealyticum and U. parvum DNA samples were specifically amplified using one set of biotin-labeled primers. Furthermore, amine-modified DNA probes, which can specifically react with U. urealyticum or U. parvum DNA, were covalently immobilized to a DNA-BIND plate surface. The plate was then incubated with the PCR products to facilitate sequence-specific DNA binding. Horseradish peroxidase-streptavidin conjugation and a colorimetric assay were used. Based on the results, the PCR-hybridization assay we developed can specifically differentiate U. urealyticum and U. parvum with high sensitivity (95%) compared with cultivation (72.5%). Hence, this study demonstrates a new method for high-throughput simultaneous differentiation and detection of U. urealyticum and U. parvum with high sensitivity. Based on these observations, the PCR-hybridization assay developed in this study is ideal for detecting and discriminating U. urealyticum and U. parvum in clinical applications.

UI MeSH Term Description Entries
D009693 Nucleic Acid Hybridization Widely used technique which exploits the ability of complementary sequences in single-stranded DNAs or RNAs to pair with each other to form a double helix. Hybridization can take place between two complimentary DNA sequences, between a single-stranded DNA and a complementary RNA, or between two RNA sequences. The technique is used to detect and isolate specific sequences, measure homology, or define other characteristics of one or both strands. (Kendrew, Encyclopedia of Molecular Biology, 1994, p503) Genomic Hybridization,Acid Hybridization, Nucleic,Acid Hybridizations, Nucleic,Genomic Hybridizations,Hybridization, Genomic,Hybridization, Nucleic Acid,Hybridizations, Genomic,Hybridizations, Nucleic Acid,Nucleic Acid Hybridizations
D004269 DNA, Bacterial Deoxyribonucleic acid that makes up the genetic material of bacteria. Bacterial DNA
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D014509 Ureaplasma A genus of gram-negative, nonmotile bacteria which are common parasitic inhabitants of the urogenital tracts of humans, cattle, dogs, and monkeys. T-Mycoplasma,Ureaplasma parvum,Ureaplasma urealyticum biovar 1
D014510 Urease An enzyme that catalyzes the conversion of urea and water to carbon dioxide and ammonia. EC 3.5.1.5. Phytourease,Urea Amidohydrolase,Amidohydrolase, Urea
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D016990 Ureaplasma urealyticum A species of gram-negative bacteria found in the human genitourinary tract (UROGENITAL SYSTEM), oropharynx, and anal canal. Serovars 1, 3, 6, and 14 have been reclassed into a separate species UREAPLASMA parvum. Ureaplasma urealyticum biovar 2
D059014 High-Throughput Nucleotide Sequencing Techniques of nucleotide sequence analysis that increase the range, complexity, sensitivity, and accuracy of results by greatly increasing the scale of operations and thus the number of nucleotides, and the number of copies of each nucleotide sequenced. The sequencing may be done by analysis of the synthesis or ligation products, hybridization to preexisting sequences, etc. High-Throughput Sequencing,Illumina Sequencing,Ion Proton Sequencing,Ion Torrent Sequencing,Next-Generation Sequencing,Deep Sequencing,High-Throughput DNA Sequencing,High-Throughput RNA Sequencing,Massively-Parallel Sequencing,Pyrosequencing,DNA Sequencing, High-Throughput,High Throughput DNA Sequencing,High Throughput Nucleotide Sequencing,High Throughput RNA Sequencing,High Throughput Sequencing,Massively Parallel Sequencing,Next Generation Sequencing,Nucleotide Sequencing, High-Throughput,RNA Sequencing, High-Throughput,Sequencing, Deep,Sequencing, High-Throughput,Sequencing, High-Throughput DNA,Sequencing, High-Throughput Nucleotide,Sequencing, High-Throughput RNA,Sequencing, Illumina,Sequencing, Ion Proton,Sequencing, Ion Torrent,Sequencing, Massively-Parallel,Sequencing, Next-Generation

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