title: Polymerase chain reaction-hybridization method using urease gene sequences for high-throughput Ureaplasma urealyticum and Ureaplasma parvum detection and differentiation.
abstract: 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.
authors: Fourth Central Hospital, Tianjin 300203, China.
authors: Chen
authors: C
authors: Xu
authors: School of Laboratory Science, Tianjin Medical University, Tianjin 300203, China.
authors: Nan
authors: N
authors: Zhang
authors: School of Laboratory Science, Tianjin Medical University, Tianjin 300203, China.
authors: Qianyu
authors: Q
authors: Huo
authors: School of Laboratory Science, Tianjin Medical University, Tianjin 300203, China.
authors: Minghui
authors: M
authors: Chen
authors: School of Laboratory Science, Tianjin Medical University, Tianjin 300203, China.
authors: Rengfeng
authors: R
authors: Wang
authors: School of Laboratory Science, Tianjin Medical University, Tianjin 300203, China.
authors: Zhili
authors: Z
authors: Liu
authors: School of Laboratory Science, Tianjin Medical University, Tianjin 300203, China.
authors: Xue
authors: X
authors: Li
authors: School of Laboratory Science, Tianjin Medical University, Tianjin 300203, China.
authors: Yunde
authors: Y
authors: Liu
authors: School of Laboratory Science, Tianjin Medical University, Tianjin 300203, China. Electronic address: kris_10713@126.com.
authors: Huijing
authors: H
authors: Bao
mesh_terms: D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014509:Ureaplasma / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification*; D016990:Ureaplasma urealyticum / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification*; D014510:Urease / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D014510:Urease
keywords: Differentiation; Ureaplasma parvum; Ureaplasma urealyticum; Urease gene