SENSITIVE DETECTION AND TYPING OF CHLAMYDIA-TRACHOMATIS USING NESTED POLYMERASE CHAIN-REACTION

Objectives-A method based on a nested polymerase chain reaction (PCR) was developed to detect and to type Chlamydia trachomatis from low tit re samples by amplifying a large portion of the major outer membrane p rotein gene. The sensitivity of this procedure was evaluated in urogen ital clinical samples in comparison with culture. Specimens-A series o f 787 urogenital specimens, including 37 (4.7%) positive by culture, t ogether 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 furth er purification. After 40 cycles of PCR, 1 mul of product was amplifie d by a further 30 cycles of PCR using a second set of primers nested w ithin the initial pair. Positives were detected by agarose gel electro phoresis and confirmed by repeating the PCR analyses and determining t he serovar of both amplified samples by restriction fragment length po lymorphism. 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 P CR and most still gave a positive signal when diluted hundred fold. Co nclusions-Nested PCR provided the basis for a very sensitive C trachom atis detection and typing strategy. Repetition and typing positive sam ples facilitated detection of false-positive PCR specimens resulting f rom contamination of the PCR process or any reagent except the origina l sample.