title: Optimised sample DNA preparation for detection of Chlamydia trachomatis in synovial tissue by polymerase chain reaction and ligase chain reaction.
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 degrees C phenol; (3) incubation at 97 degrees C; (4) proteinase K digestion at 97 degrees 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.
authors.affiliation: Division of Rheumatology, Medizinische Hochschule Hannover, Carl Neuberg Strasse 1, 30625 Hannover, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Freise
authors.forename: H C
authors.initials: HC
authors.lastname: Gérard
authors.forename: T
authors.initials: T
authors.lastname: Bunke
authors.forename: J A
authors.initials: JA
authors.lastname: Whittum-Hudson
authors.forename: H
authors.initials: H
authors.lastname: Zeidler
authors.forename: L
authors.initials: L
authors.lastname: Köhler
authors.forename: A P
authors.initials: AP
authors.lastname: Hudson
authors.forename: J G
authors.initials: JG
authors.lastname: Kuipers
mesh_terms: D002690:Chlamydia Infections / Q000175:diagnosis*; D002692:Chlamydia trachomatis / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000382:microbiology; D022521:Ligase Chain Reaction; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013583:Synovial Membrane / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial