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Annals of the Rheumatic Diseases logoLink to Annals of the Rheumatic Diseases
. 2001 Feb;60(2):140–145. doi: 10.1136/ard.60.2.140

Optimised sample DNA preparation for detection of Chlamydia trachomatis in synovial tissue by polymerase chain reaction and ligase chain reaction

J Freise 1, H Gerard 1, T Bunke 1, J Whittum-Hudson 1, H Zeidler 1, L Kohler 1, A Hudson 1, J Kuipers 1
PMCID: PMC1753467  PMID: 11156547

Abstract

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.
METHODS—Synovial 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.
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 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.



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Figure 1  .

Figure 1  

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Representative polymerase chain reaction sensitivities in detection of C trachomatis in synovial tissue samples spiked with elementary bodies (EB) using template DNA prepared by the QIAmp tissue kit (method 1) and homogenisation in hot phenol (method 2), with set 1 and set 2 primers. (A) DNA preparation method 1 with primer set 1; (B) DNA preparation method 2 with primer set 1; (C) DNA preparation method 1 with primer set 2; (D) DNA preparation method 2 with primer set 2. Assays were performed as given in "Methods"; amplification product size in base pairs (bp) is given at the right. In each panel lanes C1 and C2 used template DNA from synovial tissue of the patient with rheumatoid arthritis without added EB, and primer sets 1 and 2, respectively. The positive (+) control and negative (−) control lanes contained pure EB DNA and water only, respectively, as amplification template.

Figure 2  .

Figure 2  

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Representative polymerase chain reaction (PCR) sensitivities in detection of C trachomatis using chlamydia infected monocytes in synovial tissue samples and template DNA prepared by the QIAmp tissue kit (method 1) as well as homogenisation in hot phenol (method 2), with set 1 and set 2 primers. (A) DNA preparation method 1 with primer set 1; (B) DNA preparation method 2 with primer set 1; (C) DNA preparation method 1 with primer set 2; (D) DNA preparation method 2 with primer set 2. Assays were performed as given in "Methods"; amplification product size in base pairs (bp) is given at right. In each panel lanes C1 and C2 PCR was done using the set 1 and set 2 primers, respectively, and template DNA from synovial tissue of the patient with rheumatoid arthritis without added EB. The positive (+) control and negative (−) control lanes contained pure EB DNA and water only, respectively, as amplification template.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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