OBJECTIVE Molecular biology techniques such as polymerase chain reaction (PCR) and ligase chain reaction (LCR) are routinely used in research for detection ofC trachomatis DNA in synovial samples, and these methods are now in use in some clinical laboratories. This study aimed at determining the method best suited to molecular diagnosis ofC trachomatis by examining four standard DNA preparation methods using chlamydia spiked synovial tissue and chlamydia infected monocytes.

RESULTS In synovial tissue samples spiked with EB, and in monocytes persistently infected with the organism, preparation of template using the QIAmp tissue kit (method 1) and the hot phenol extraction technique (method 2) allowed sensitive detection of C trachomatis DNA. These methods also produced template from both sample types for LCR. DNA prepared by heat denaturation (method 3) allowed only low sensitivity chlamydia detection in LCR and did not work at all for PCR. Proteinase K digestion plus heat denaturation (method 4) gave template that did not allow amplification in either PCR or LCR assays.

CONCLUSIONS The sensitivity of detection for C trachomatisDNA in synovial tissue by PCR and LCR depends strongly on the method used for preparation of the amplification template. LCR targeting the multicopy chlamydial plasmid and two nested PCR assay systems targeting the single copy omp1 gene showed roughly equivalent sensitivity. Importantly, template preparation method and the specific PCR primer system used for screening must be optimised in relation to one another for highest sensitivity.