Simultaneous strand displacement amplification and fluorescence polarization detection of Chlamydia trachomatis DNA.

Strand displacement amplification (SDA) is an isothermal DNA amplification technology that uses a restriction enzyme and polymerase. We have developed a target-specific method which allows simultaneous SDA and detection in a homogeneous format. This is accomplished by including a detector oligodeoxynucleotide labeled with 5-(4,6-dichlorotriazin-2-yl)amino fluorescein in the SDA reaction. Fluorescence polarization is used to monitor hybridization of the detector probe to the amplification product as it rises in concentration during SDA. We have demonstrated real-time SDA detection for the cryptic plasmid of Chlamydia trachomatis with high sensitivity in only 30 min.

[1]  C. Spargo,et al.  Detection of M. tuberculosis DNA using thermophilic strand displacement amplification. , 1996, Molecular and cellular probes.

[2]  D. Blakeslee,et al.  Immunofluorescence using dichlorotriazinylaminofluorescein (DTAF). I. Preparation and fractionation of labelled IgG. , 1976, Journal of immunological methods.

[3]  W. Stamm.  Diagnosis of Chlamydia trachomatis genitourinary infections , 1988, Annals of internal medicine.

[4]  B. Dwyer,et al.  Comparative evaluation of detection assays for Chlamydia trachomatis , 1991, Journal of clinical microbiology.

[5]  J. Clark,et al.  DNA synthesis on discontinuous templates by DNA polymerase I of Escherichia coli. , 1991, Gene.

[6]  G. Walker,et al.  Hybridization of fluorescein-labeled DNA oligomers detected by fluorescence anisotropy with protein binding enhancement. , 1995, Analytical chemistry.

[7]  H. Handsfield,et al.  Use of sequential enzyme immunoassay and direct fluorescent antibody tests for detection of Chlamydia trachomatis infections in women , 1990, Journal of clinical microbiology.

[8]  Helen H. Lee,et al.  Diagnosis of Chlamydia trachomatis genitourinary infection in women by ligase chain reaction assay of urine , 1995, The Lancet.

[9]  M. Ward,et al.  Analysis of the entire nucleotide sequence of the cryptic plasmid of Chlamydia trachomatis serovar L1. Evidence for involvement in DNA replication. , 1988, Nucleic acids research.

[10]  Thomas J. White,et al.  PCR protocols: a guide to methods and applications. , 1990 .

[11]  A. Murakami,et al.  Fluorescent-labeled oligonucleotide probes: detection of hybrid formation in solution by fluorescence polarization spectroscopy. , 1991, Nucleic acids research.

[12]  G. Walker,et al.  Strand displacement amplification--an isothermal, in vitro DNA amplification technique. , 1992, Nucleic acids research.

[13]  J. Burczak,et al.  Detection of Chlamydia trachomatis in urine specimens from women by ligase chain reaction , 1995, Journal of clinical microbiology.

[14]  Helen H. Lee,et al.  Diagnosis of Chlamydia trachomatis infections in men and women by testing first-void urine by ligase chain reaction , 1994, Journal of clinical microbiology.

[15]  H. Rousseau,et al.  Comparison of three techniques for detection of Chlamydia trachomatis in endocervical specimens from asymptomatic women , 1988, Journal of clinical microbiology.

[16]  M. Domeika,et al.  Diagnosis of genital Chlamydia trachomatis infections in asymptomatic males by testing urine by PCR , 1994, Journal of clinical microbiology.

[17]  G. Walker.  Empirical aspects of strand displacement amplification. , 1993, PCR methods and applications.

[18]  G. Walker,et al.  DNA detection by strand displacement amplification and fluorescence polarization with signal enhancement using a DNA binding protein. , 1996, Nucleic acids research.

[19]  G. Walker,et al.  Strand displacement amplification (SDA) and transient-state fluorescence polarization detection of Mycobacterium tuberculosis DNA. , 1996, Clinical chemistry.

[20]  C. Spargo,et al.  Chemiluminescent detection of strand displacement amplified DNA from species comprising the Mycobacterium tuberculosis complex. , 1993, Molecular and cellular probes.

[21]  M. Loeffelholz,et al.  Diagnosis of Chlamydia trachomatis urethritis in men by polymerase chain reaction assay of first-catch urine , 1993, Journal of clinical microbiology.

[22]  J B Findlay,et al.  Automated closed-vessel system for in vitro diagnostics based on polymerase chain reaction. , 1993, Clinical chemistry.

[23]  G. Walker,et al.  Detection of Mycobacterium tuberculosis DNA with thermophilic strand displacement amplification and fluorescence polarization. , 1996, Clinical chemistry.

[24]  S. T. Isaacs,et al.  Post-PCR sterilization: a method to control carryover contamination for the polymerase chain reaction , 1991, Nucleic Acids Res..