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Arch. Immunol. Ther. Exp. (2010) 58:227–234 DOI 10.1007/s00005-010-0076-z

ORIGINAL ARTICLE

Comparison of Three Methods of DNA Extraction in Endocervical Specimens for Chlamydia trachomatis Infection by Spectrophotometry, Agarose Gel, and PCR
Anahita Jenab • Rasoul Roghanian Naser Golbang • Pouran Golbang • Leili Chamani-Tabriz
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Received: 9 April 2009 / Accepted: 4 February 2010 /Published online: 17 April 2010 Ó L. Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland 2010

Abstract Chlamydia trachomatis is the major cause of sexually transmitted disease in the world. The aim of this study was to determine the best method of DNA extraction for detecting C. trachomatis by polymerase chain reaction (PCR) in sexually active women (n = 80) attendingShahid Beheshti Hospital in Isfahan, Iran. Endocervical swabs were collected from 80 women, 22 of whom were asymptomatic and 58 symptomatic. Three different DNA extraction methods were used in this study (phenolchlorophorm, proteinase K, and boiling). DNA yield was evaluated by spectrophotometry, agarose gel, and PCR. The internal control was assayed by b-globin primers (PCO4, GH20). The DNA crypticplasmid was selected as the target for C. trachomatis and samples were examined by PCR using specific KL1 and KL2 primers. It was shown that DNA extraction by boiling was the most sensitive with the highest yield of DNA. Of the 80 samples, 17 (21.25%) showed positivity for C. trachomatis by PCR. The highest rate of C. trachomatis infection was found in the group aged between 35 and 45 years oldand those who used withdrawal or an intrauterine device as methods of contraception. It was demonstrated that DNA extraction by boiling was the least expensive and a very rapid method

that gave the highest DNA yield. The infection rate in the sexually active women, including symptomatic and asymptomatic, was 21.25%, with a presumably high prevalence compared with other studies done in this field.Keywords Chlamydia trachomatis Á Endocervical smears Á DNA extraction Á PCR Á Agarose gel Á Spectrophotometry Abbreviations IUD Intrauterine device PID Pelvic inflammatory disease

Introduction Chlamydia trachomatis (serotypes D-K, Da, Ia) is the most common cause of bacterial sexually transmitted infections, although up to 80% of infected women and 50% of infected men may be asymptomatic(Nisyrios 2006). When symptoms do occur, usually 1–3 weeks following exposure, they are followed by dysuria, abdominal pain, and abnormal vaginal discharge (Fallah et al. 2005; Manavi 2006; Nisyrios 2006). In women, untreated infection or chronic (persistent) infection of C. trachomatis can spread into the uterus or fallopian tubes and cause pelvic inflammatory disease. Pelvic inflammatory disease cancause permanent damage to the fallopian tubes, uterus, and surrounding tissues. The damage can lead to chronic pelvic pain, infertility, potentially fatal ectopic pregnancy, and premature delivery. Infants born to pregnant mothers with genital Chlamydia infection are also often at risk of developing conjunctivitis and pneumonia (Lehmann et al. 1999; Manavi 2006). Since most C. trachomatisinfections

A. Jenab Á R. Roghanian (&) Á N. Golbang Section of Microbiology, Department of Biology, Faculty of Science, University of Isfahan, Isfahan, Iran e-mail: rasoul_roghanian@yahoo.co.uk P. Golbang Shahid Beheshti Hospital, Isfahan, Iran L. Chamani-Tabriz Department of Reproductive Infections, Reproductive Biotechnology Research Center, Avesina Research Institute, Tehran, Iran

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Arch.Immunol. Ther. Exp. (2010) 58:227–234

are asymptomatic, early diagnosis is essential for the timely treatment of C. trachomatis-infected women to prevent the development of sequelae and the transmission of C. trachomatis infection to susceptible individuals. Polymerase chain reaction (PCR) has been established as the standard assay for the rapid diagnosis of genital C. trachomatis infection...