Extended report
Optimised sample DNA preparation for detection of
Chlamydia trachomatis in synovial tissue by
polymerase chain reaction and ligase chain reaction
J Freisea, H C Gérardb, T Bunkea, J A Whittum-Hudsonc, H Zeidlera, L Köhlera, A P Hudsonb, J G Kuipersa
a Division of
Rheumatology, Medizinische Hochschule Hannover, Carl Neuberg Strasse 1, 30625 Hannover, Germany, b Department of Immunology and Microbiology, Wayne
State University School of Medicine, 540 East Canfield Avenue, Detroit
MI 48201 USA, c Department of Internal Medicine, Wayne State
University School of Medicine
Correspondence to: Dr Kuipers Kuipers.Jens{at}mh-hannover.de
Accepted for publication 7 June 2000
OBJECTIVE
Molecular
biology techniques such as polymerase chain reaction (PCR) and ligase
chain reaction (LCR) are routinely used in research for detection of
C 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 of
C trachomatis by examining four standard DNA
preparation methods using chlamydia spiked synovial tissue and
chlamydia infected monocytes.
METHODSSynovial
tissue from a chlamydia negative patient with rheumatoid arthritis was
spiked with defined numbers of C trachomatis elementary bodies (EB). Purified human peripheral monocytes from normal
donors were infected with the organism at a multiplicity of infection
1:1 in vitro and harvested after four days. DNA was prepared from all
samples by four methods: (1) QIAmp tissue kit; (2) homogenisation in
65°C phenol; (3) incubation at 97°C; (4) proteinase K digestion at
97°C. DNA from methods 1 and 2 was subjected to PCR using two
different primer sets, each targeting the C
trachomatis omp1 gene. LCR was done
on DNA prepared by each method.
RESULTSIn 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.
CONCLUSIONSThe
sensitivity of detection for C trachomatis
DNA 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.
© 2001 by Annals of the Rheumatic Diseases
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