Comparison of DNA extraction from cervical cells collected in PreservCyt solution for the amplification of Chlamydia trachomatis.
نویسندگان
چکیده
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.
منابع مشابه
PreservCyt transport medium used for the ThinPrep Pap test is a suitable medium for detection of Chlamydia trachomatis by the COBAS Amplicor CT/NG test: results of a preliminary study and future implications.
The commercial COBAS Amplicor CT/NG test (Roche Diagnostic Systems, Meylan, France) is a sensitive and specific method for detection of Chlamydia trachomatis infections. This test currently consists of using a nucleic acid amplification method to detect C. trachomatis in first-void urine specimens and in endocervical swabs collected in 2-sucrose-phosphate (2SP) transport medium. We conducted a ...
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ورودعنوان ژورنال:
- Cytopathology : official journal of the British Society for Clinical Cytology
دوره 16 2 شماره
صفحات -
تاریخ انتشار 2005