BacteriologyApplication of an oligonucleotide array assay for rapid detecting and genotyping of Chlamydia trachomatis from urogenital specimens
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).
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