Comparison of DNA extraction from cervical cells collected in PreservCyt solution for the amplification of Chlamydia trachomatis.
OBJECTIVE
The aim of this study was to compare and evaluate three methods of DNA extraction for the amplification of Chlamydia trachomatis in uterine cervical samples collected in PreservCyt solution. ThinPrep is the trade name for the slide preparation.


METHODS
Thirty-eight samples collected in LCx buffer medium, which were identified as C. trachomatis infected by ligase chain reaction (LCR), were selected for this study. DNA from the PreservCyt samples was extracted by three methods: (i) QIAamp kit, (ii) boiling in Tris-EDTA buffer with Chelex purification, and (iii) Proteinase K digestion with Chelex purification. Sample DNA was tested for the presence of C. trachomatis by PCR using cryptic plasmid research (CTP) primers and major outer membrane protein research momp gene (MOMP) primers. Real-time (LightCycler) PCR for relative C. trachomatis quantification following DNA extraction was performed using primers (Hsp 60) for the 60 kDa heat-shock protein hsp60 gene.


RESULTS
Amplification using CTP primers was the most successful with each of the extraction protocols. Boiling in buffer was the least successful extraction method. QIAamp was the best extraction method, yielding the most positives with both the CTP and MOMP primers. Proteinase K-Chelex extraction gave similar sensitivity to QIAamp extraction with CTP primers but lower for MOMP primers.


CONCLUSIONS
The DNA extraction method must be carefully selected to ensure that larger PCR amplicons can be successfully produced by PCR and to ensure high sensitivity of detection of C. trachomatis. In this study it was found that the QIAamp extraction method followed by PCR with the CTP primers was the most successful for amplification of C. trachomatis DNA.
Keegan
H
H
Molecular Biomedical Research, School of Biological Sciences, Dublin Institute of Technology, Dublin, Ireland.
Boland
C
C
Malkin
A
A
Griffin
M
M
Ryan
F
F
Lambkin
H
H
D000942:Antigens, Bacterial / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D018834:Chaperonin 60 / Q000235:genetics; D002690:Chlamydia Infections / Q000175:diagnosis / Q000235:genetics / Q000382:microbiology*; D002692:Chlamydia trachomatis / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis*; D005260:Female; D005831:Genital Diseases, Female / Q000175:diagnosis / Q000235:genetics / Q000382:microbiology*; D006801:Humans; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D018834:Chaperonin 60; D004269:DNA, Bacterial