Comparison of Three Methods of DNA Extraction in Endocervical Specimens for Chlamydia trachomatis Infection by Spectrophotometry, Agarose Gel, and PCR

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) attending Shahid 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 (phenol-chlorophorm, proteinase K, and boiling). DNA yield was evaluated by spectrophotometry, agarose gel, and PCR. The internal control was assayed by beta-globin primers (PCO4, GH20). The DNA cryptic plasmid 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 old and 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.

Prevalência de Chlamydia trachomathis em amostras endocervicais de mulheres em São Paulo e Santa Catarina pela PCR

“…O trabalho de Arráiz et al (1) apresentou prevalência de 13,7% para pacientes sintomáticas, 7,4% para assintomáticas e 10,4% no total, nos três casos com amostras coletadas por swab endocervical. Jenab et al (10) , com o mesmo tipo de amostra, mostraram prevalência de 21,25%. Patel et al (22) , em um trabalho realizado na Índia coletando-se, também, amostras do endocérvix por meio de swab, mostraram prevalência de 23%.…”

“…As amostras coletadas por meio de swab mostraram prevalência muito maior (75%) do que as coletadas por meio de raspado (53,44%). Todavia, o total de pacientes com amostras coletadas por swab é muito inferior ao total coletado com raspado, mesmo assim, isso é forte indicativo de que o swab, assim como nos trabalhos anteriores (1,10,22,23,25) , é mais eficiente do que o raspado na obtenção de material de melhor qualidade para o diagnóstico molecular, mesmo que possa parecer controverso.…”

“…Portanto, visto que é um agente com alta prevalência no mundo, faz-se necessário o emprego de metodologias de diagnóstico e monitoria adequada da CT (1) . Entre a vasta gama de metodologias, a mais utilizada é a identificação por métodos sorológicos (9,28) , no entanto, é fundamental o emprego de metodologias da Biologia Molecular na identificação, principalmente pelo material genético, do agente, entre elas destaca-se a PCR (2,6,8,10,12,22,25,27,(30)(31)(32) , por possuir eficácia e sensibilidade de 100%, enquanto o imunoensaioenzimático, por exemplo, possui sensibilidade de 58,8% e especificidade de 100% (14) .…”

Detection of Chlamydia trachomatis by Immunological and Genetic Methods in Female Sex Workers and the Local Female Population of Reproductive Age in Mymensingh, Bangladesh

“…This mixture was incubated at 1009 C for 15 min and then centrifuged at 10,000 rpm for 1 min. The supernatant was used as a DNA template, as described previously (16). PCR was used to amplify a 517-bp fragment from the common endogenous plasmid as described previously, using a pair of oligonucleotide primers, T1 and T2 (11).…”