Bacteriology
Application of an oligonucleotide array assay for rapid detecting and genotyping of Chlamydia trachomatis from urogenital specimens

https://doi.org/10.1016/j.diagmicrobio.2006.05.007Get rights and content

Abstract

An oligonucleotide array technology was established for rapidly detecting and genotyping Chlamydia trachomatis in urogenital infections. The VS1–VS2 region of the omp1 gene was used to design oligonucleotide probes. Eleven serovar-specific probes to serovars A, B, C, D, E, F, G, H, I, J, and K, and 3 group-specific probes to group B (B, Ba, D, E, L1, and L2), group C (A, C, H, I, J, K, and L3), and an intermediate group (F and G) were synthesized and spotted onto the nylon membrane. Two pairs of universal primers were designed for the nested polymerase chain reaction (PCR) amplification of the VS1–VS2 gene. Digoxigenin-labeled amplicons of the VS1–VS2 gene of C. trachomatis were hybridized to the membrane array. Hybridization signals were read by the nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate color development. The assay developed was tested with reference strains of C. trachomatis serovars and clinical samples. The sensitivity was evaluated for 57 samples previously found to be positive for C. trachomatis by using plasmid PCR, and 98.2% (56/57) concordance was obtained. Fourteen oligonucleotide probes were optimized by trying different reaction conditions, showing specific hybridization with the corresponding reference strains, but no cross-reactions with other urogenital microorganisms. Using this procedure, a total of 59 strains were detected from 56 chlamydial samples. Eight genotypes were found, and type D, E, F, and H were the most frequently observed types (77.9%). Three cases (5.4%) had multiple infections with serovars: 1.D/E, 2.D/F, and 3.F/K. To validate the reference strains and confirm the genotype identity as determined by the oligonucleotide array technology, we sequenced all reference strains and 10 selected specimens across variable sequence VS1 and VS2. No discrepancies were found between the array typing and the genotype identity confirmed by nucleotide sequencing of the PCR product. The findings from this study indicated that the oligonucleotide array is a simple, fast, and specific assay for directly detecting and genotyping C. trachomatis from clinical samples.

Introduction

Chlamydia trachomatis is considered to be one of the major causes of sexually transmitted diseases, and as such, it is a major public health problem worldwide (WHO, 1989). Chlamydial infections in the urogenital tract can cause urethritis, epididymitis, and prostatitis in men and cervicitis and pelvic inflammatory disease in women (Brunham et al., 1985), which, if untreated, may lead to serious complications, including ectopic pregnancy and tubal infertility. Usually, people with Chlamydia infection do not seek medical care, because more than 50% of men and 70% of women with genital infections are asymptomatic (Hay and Ghaem-Maghami, 1997). From a public health perspective, this leads to the persistence of an undetected C. trachomatis reservoir with significant perpetuation of transmission. Moreover, C. trachomatis is suggested to be a cofactor in transmission of human immunodeficiency virus (Fleming and Wasserheit, 1999).

C. trachomatis isolates occur as 15 distinct serovars. On the basis of serologic relation, these 15 serovars are divided into 3 serogroups: B (serovars B, Ba, D, E, L1, and L2), intermediate (serovars F and G), and C (serovars A, C, H, I, J, K, and L3) (Yuan et al., 1989). Serovars A, B, Ba, and C have been predominantly associated with endemic trachoma; D through K with infection in the urogenital tract; and L1, L2, and L3 with lymphogranuloma venereum.

The typing of C. trachomatis plays an important role in the epidemiologic and vaccination studies. The traditional immunotyping method requires culturing of clinical isolates and a large panel of monoclonal antibodies, which make it difficult to use in the clinical laboratory. Although nucleotide sequencing of the omp1 genes provides definite typing results (Van Duynhoven et al., 1998, Takourt et al., 2001), this method is laborious and not suitable for typing the isolates from a large number of clinical samples. Furthermore, ambiguous results were produced after analysis of a mixed sample because the serovars in the sample cannot be resolved unless the omp1 polymerase chain reaction (PCR) products were cloned and multiple clones were subsequently sequenced. The genotyping methods using restriction fragment length polymorphism (RFLP) analysis of the omp1 gene were shown to be more rapid and accurate in revealing C. trachomatis variants within a serovar as well as potential recombinants among serovars, and it is currently favored over the immunotyping method (Lan et al., 1993). However, the PCR-RFLP has proved to be a method with low sensitivity (49%) with a quite large (1.1 kb) fragment of omp1 gene required (Yang et al., 2000).

The single-copy omp1 gene consists of 5 regions of conserved sequence that alternate with 4 variable regions (VS1–VS4) (Stephens et al., 1987). The study on nucleotide and deduced amino acid sequences for the 4 variable domains of major outer membrane protein of the 15 C. trachomatis serovars indicated that the VS1 and VS2 are sufficiently different among all serovars to allow construction of serovar-specific synthetic oligonucleotides (Yuan et al., 1989). Recently, a line blot assay based on the VS2 was developed for the typing of C. trachomatis (Molano et al., 2004). However, because of the limited discrimination sequence used, some of the probes selected for C. trachomatis genotyping had only a single mismatch with related serovars (probe J to serovar K, probe I to serovar C), which might have resulted in nonspecific hybridization. In this study, we selected the VS1–VS2 of omp1 gene as the discrimination target. Digoxigenin (DIG)-linked enzyme-linked immunosorbent assay method was used for color development. An oligonucleotide array technology was developed. Using the assay, we rapidly detected and genotyped the reference and clinical strains by reverse dot blot (RDB) procedure.

Section snippets

Strains and clinical C. trachomatis samples

Eleven different reference serovars were used. The reference strains D, E, F, G, H, I, J, and K were kindly provided by Dr. Joke Spaargaren, Public Health Laboratory of the Municipal Health Service, Amsterdam, the Netherlands; serovar A, B, and C were provided by Dr. Peng Hui, Sun Yat-sen University, Guangzhou, China.

Fifty-seven consecutive C. trachomatis samples previously found to be positive for C. trachomatis by using plasmid PCR were collected from outpatients with nongonococcal urethritis

Amplification of VS1–VS2 region of the omp1 gene

All reference strains studied and 56 of 57 clinical samples previously found to be positive for C. trachomatis by using plasmid PCR assay were successfully amplified by the nested VS1–VS2 PCR. The primary PCR step amplified a 516-bp fragment, and the secondary PCR produced a 453-bp fragment of VS1–VS2 (Fig. 1).

Array and dotting of oligonucleotide probes

In our study, 14 oligonucleotide probes were designed and synthesized (Table 1) to match bacteria of different serovars /groups. Probes Gb, Gc, and Gi were group specific. Probes A to K

Discussion

Although DNA microarray technology has been widely used in gene expression monitoring, genotyping has emerged as another area of application in the last few years. Recent applications of DNA microarrays in genotyping include detection and genotyping of human papillomaviruses (Oh et al., 2004), detection of antibiotic resistance genes in Gram-positive bacteria (Perreten et al., 2005), toxin typing of Clostridium perfringens (Al-Khaldi et al., 2005), and serovar differentiation among mixed

Acknowledgments

The authors thank Dr. Bang-xing Hong and Yu-shan Hu for their technical assistance. We also thank Dr. Alan Walshe for proofreading the manuscript. This work was supported by the Guangdong Province Science Funds (no. 2005B34201011) and the Medical Science Funds of Guangdong Provincial Bureau of Public Health (no. A2005150).

References (25)

  • S.F. Al-Khaldi et al.

    Genotyping of Clostridium perfringens toxins using multiple oligonucleotide microarray hybridization

    Mol. Cell. Probes

    (2005)
  • R.C. Brunham et al.

    Chlamydia trachomatis: its role in tubal infertility

    J. Infect. Dis.

    (1985)
  • R.C. Brunham et al.

    The epidemiology of Chlamydia trachomatis within a sexually transmitted disease core group

    J. Infect. Dis.

    (1996)
  • T.Y. Choi et al.

    Evaluation of serotyping using monoclonal antibodies and PCR-RFLP for Chlamydia trachomatis serotype identification

    J. Korean Med. Sci.

    (2001)
  • D. Dean et al.

    Comparison of the major outer membrane protein variant sequence region of B/Ba isolation: a molecular epidemiologic approach to Chlamydia trachomatis infections

    J. Infect. Dis.

    (1992)
  • D. Dean et al.

    Major outer membrane protein variants of Chlamydia trachomatis are associated with severe distribution of urogenital. Chlamydia trachomatis strains in The Netherlands

    Genitourin. Med.

    (1995)
  • R. Ehricht et al.

    Optimized DNA microarray assay allows detection and genotyping of single PCR-amplifiable target copies

    Mol. Cell. Probes

    (2006)
  • D.T. Fleming et al.

    From epidemiological synergy to public health policy and practice: the contribution of other sexually transmitted diseases to sexual transmission of HIV infection

    Sex Transm. Infect.

    (1999)
  • R. Griffais et al.

    Detection of Chlamydia trachomatis

    Res. Microbiol.

    (1989)
  • P.E. Hay et al.

    Chlamydia and non-gonococcal urethritis

    Curr. Opin. Infect. Dis.

    (1997)
  • J. Lan et al.

    Direct detection and genotyping of Chlamydia trachomatis in cervical fragment length polymorphism analysis

    J. Clin. Microbiol.

    (1993)
  • J.B. Mahony et al.

    Comparison of plasmid- and chromosome-based polymerase chain reaction assays for detecting Chlamydia trachomatis nucleic acids

    J. Clin. Microbiol.

    (1993)
  • Cited by (26)

    • Epidemiology of transmissible diseases: Array hybridization and next generation sequencing as universal nucleic acid-mediated typing tools

      2018, Infection, Genetics and Evolution
      Citation Excerpt :

      This led to a huge number of papers describing the very efficient molecular detection of C. trachomatis and among these were also hybridization tests for further characterization of the organisms. Molecular serovar identification was described in the early 2000′s and the work by Zheng et al. (2007) nicely showed the feasibility of this approach. The array covered genetic diversity in the VS1-VS2 gene and allowed for sensitive detection and specific genotyping.

    • Novel multiplex real-time PCR system using the SNP technology for the simultaneous diagnosis of Chlamydia trachomatis, Ureaplasma parvum and Ureaplasma urealyticum and genetic typing of serovars of C. trachomatis and U. parvum in NGU

      2011, Molecular and Cellular Probes
      Citation Excerpt :

      The quality of the triplex real-time TaqMan-LNA PCR assays, with respect to PCR efficiency, limit of detection, and linear range, was similar to simplex real-time TaqMan-LNA PCR. The specific and easy differentiation of C. trachomatis, U. parvum and U. urealyticum will be of great value for the analysis of the biovars and pathogenicity of these microorganisms [13,14]. We also evaluated the efficacy of the triplex real-time TaqMan-LNA PCR assays for detection of the three pathogens in clinical specimens.

    • A high-resolution melting analysis for genotyping urogenital Chlamydia trachomatis

      2010, Diagnostic Microbiology and Infectious Disease
      Citation Excerpt :

      The potential of mistyping may occur from the alteration of the Tm and melting profiles. Previous studies have had limited epidemiologic data and very few mutations in the VS2 region (Lima et al., 2007; Lister et al., 2004; Zheng et al., 2007a). There were neither insertions nor deletions detected in our validation panel.

    View all citing articles on Scopus
    View full text