Use of PCR and Reverse Line Blot Hybridization Assay for Rapid Simultaneous Detection and Serovar Identification of Chlamydia trachomatis

Abstract

The aim of this study was to develop and evaluate multiplex and nested PCR-reverse line blot (RLB) hybridization assays for detection and serovar identification of Chlamydia trachomatis. Two sets of primers targeting the VD2 region of the omp1 gene and one set targeting the cryptic plasmid were designed for use in multiplex (both targets) and nested PCR (omp1 only). For the RLB assay, labeled omp1 amplicons were hybridized to a membrane containing probes specific for 15 C. trachomatis serovars. The assays were used to test 429 clinical specimens, which had been previously tested for C. trachomatis using the COBAS AMPLICOR system. Specimens were tested without knowledge of the COBAS AMPLICOR result. Of 205 specimens that were positive by COBAS AMPLICOR, 201 (98%) were positive by multiplex PCR-RLB and 188 (92%) were also positive by omp1 nested PCR-RLB. In addition, three of 224 COBAS AMPLICOR-negative specimens were positive by omp1 nested PCR-RLB. One hundred sixty-six of 191 (87%) specimens in which C. trachomatis serovars were identified contained only one serovar and 25 (13%) contained two or three serovars. Serovars D, E, and F were found in 31 (16%), 83 (43%), and 51 (27%) specimens, respectively. Serovar E (41%) was the most commonly identified single serovar. Serovars J and K were found alone uncommonly (<2% each), but 18 of 25 (72%) specimens with multiple C. trachomatis serovars contained one or both (10 specimens) of these serovars. The nested (ompI) PCR-RLB is a specific and sensitive method for simultaneous detection and serovar identification of C. trachomatis, which can reliably identify mixed C. trachomatis serovars. It is suitable for use in epidemiological studies.

Development and Evaluation of anompAQuantitative Real-Time PCR Assay forChlamydia trachomatisSerovar Determination

“…The frequency of multiple serovars identified in this study (2.4%) is comparable to those in other C. trachomatis typing studies of between 1.5% and 5.4% (8,15,19,20,33). Interestingly, some studies have described multiple infections with frequencies as high as 8.7% and 13% (14,31) which principally involved use of PCR followed by reverse line blot procedures.…”

“…With this, a notable limitation of this qPCR assay was its marginally lower success rate in predicting C. trachomatis serovars compared to that of sequencing. Several of the published protocols for C. trachomatis serovar prediction incorporate an initial nested PCR followed by various methods of detection, including sequencing, qPCR, blotting, and microsphere suspension array, to improve assay sensitivity (7,8,9,14,31,33). Therefore, as a step to potentially enhancing the current assay, it is possible that a nested PCR precedes the primary consensus/group-specific PCR.…”

Comparison of three genotyping methods to identify Chlamydia trachomatis genotypes in positive men and women

“…Multiple infections will cause sequencing difficulties 217 leading to a non interpretable Omp1 sequence. In other studies 4-12% of the Ct infections 218 contained multiple serovars (11,16), making the Ct-DT-RHA more suitable for serovar 219 distribution studies and future Ct vaccine studies compared to Omp1 sequencing. All LGV 220 strains detected by Omp1 sequencing and the pmpH PCR were recognized by the Ct-DT-221 RHA, indicating that the Ct-DT assay can also be used for LGV serovar detection.…”

“…Because LGV Ct 75 infections require longer antibiotic treatment, it is highly recommended to differentiate them 76 from non-LGV serovars (9). 77 A number of reverse line blot assays were developed making genotyping faster and less 78 laborious, compared to sequencing (10)(11)(12). The Ct-Detection genoTyping (DT) assay 79 consists of a Ct amplification step (PCR), a Ct Detection step (DNA Enzyme Immuno Assay; 80 DEIA) and a Ct genotyping step (Reverse Hybridization Assay; RHA).…”

Use of PCR and Reverse Line Blot Hybridization Macroarray Based on 16S-23S rRNA Gene Internal Transcribed Spacer Sequences for Rapid Identification of 34 Mycobacterium Species

“…The RLB assay was performed by using a system which has previously been described in detail (28,31). Briefly, the slots in a Miniblotter 45 apparatus (Immunetics) were filled with 150 l of optimized concentrations of probe solution.…”