OBJECTIVES: A method based on a nested polymerase chain reaction (PCR) was developed to detect and to type Chlamydia trachomatis from low titre samples by amplifying a large portion of the major outer membrane protein gene. The sensitivity of this procedure was evaluated in urogenital clinical samples in comparison with culture. SPECIMENS: A series of 787 urogenital specimens, including 37 (4.7%) positive by culture, together with 227 other samples that had been found to yield less than 25 chlamydial inclusions in culture were tested. METHODS: Samples were pelleted, resuspended in 1 mM NaOH, heated and amplified without further purification. After 40 cycles of PCR, 1 microliters of product was amplified by a further 30 cycles of PCR using a second set of primers nested within the initial pair. Positives were detected by agarose gel electrophoresis and confirmed by repeating the PCR analyses and determining the serovar of both amplified samples by restriction fragment length polymorphism. RESULTS: Nested PCR allowed detection of 96% and culture 77% of positives with only three samples repeatedly positive by PCR but considered false positives because a different serovar was identified in the two amplifications. Of culture-positive samples with less than 11 chlamydia inclusion-forming-units 97% could be detected by nested PCR and most still gave a positive signal when diluted hundred fold. CONCLUSIONS: Nested PCR provided the basis for a very sensitive C trachomatis detection and typing strategy. Repetition and typing positive samples facilitated detection of false-positive PCR specimens resulting from contamination of the PCR process or any reagent except the original sample.
OBJECTIVES: A method based on a nested polymerase chain reaction (PCR) was developed to detect and to type Chlamydia trachomatis from low titre samples by amplifying a large portion of the major outer membrane protein gene. The sensitivity of this procedure was evaluated in urogenital clinical samples in comparison with culture. SPECIMENS: A series of 787 urogenital specimens, including 37 (4.7%) positive by culture, together with 227 other samples that had been found to yield less than 25 chlamydial inclusions in culture were tested. METHODS: Samples were pelleted, resuspended in 1 mM NaOH, heated and amplified without further purification. After 40 cycles of PCR, 1 microliters of product was amplified by a further 30 cycles of PCR using a second set of primers nested within the initial pair. Positives were detected by agarose gel electrophoresis and confirmed by repeating the PCR analyses and determining the serovar of both amplified samples by restriction fragment length polymorphism. RESULTS: Nested PCR allowed detection of 96% and culture 77% of positives with only three samples repeatedly positive by PCR but considered false positives because a different serovar was identified in the two amplifications. Of culture-positive samples with less than 11 chlamydia inclusion-forming-units 97% could be detected by nested PCR and most still gave a positive signal when diluted hundred fold. CONCLUSIONS: Nested PCR provided the basis for a very sensitive C trachomatis detection and typing strategy. Repetition and typing positive samples facilitated detection of false-positive PCR specimens resulting from contamination of the PCR process or any reagent except the original sample.
Authors: J Moncada; J Schachter; G Bolan; J Engelman; L Howard; I Mushahwar; G Ridgway; G Mumtaz; W Stamm; A Clark Journal: J Clin Microbiol Date: 1990-08 Impact factor: 5.948