Comparison of rRNA-based and DNA-based nucleic acid amplifications for detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Ureaplasma urealyticum in urogenital swabs

A total of 123 urogenital swabs were collected from 123 outpatients with suspected genital infections in our hospital, which consisted of 64 male and 59 female with a median age of 29 years old (range: 19–63). Swabs were placed into 2 ml of sterile normal saline and the tubes were vortexed. For each sample, the two NAATs were performed at the same time. RNase-free tubes and tips were used in the SAT assay.

This study was exempted from a requirement for a statement of ethical approval from ethical committee of the affiliated hospital of academy of military medical sciences.

CT, NG, and UU qPCR kits (Liferiver Biotech) were used in the DNA-based NAAT. 1 ml of each sample was centrifuged at 13000 g for 5 min at room temperature. The supernatant was discarded and the pellet was washed twice with normal saline. After the second wash, the pellet was resuspended in 100 μl extraction buffer and heated at 100 °C for 10 min. After another centrifugation at 13000 g for 5 min, the supernatants were collected and used as DNA extracts.

The reaction mixtures were prepared as described by the manufacturer’s instructions. The amplifications were performed on a Roche LightCycler® 480 system under the following conditions: 94 °C 2 min, 40 cycles of 93 °C 15 s and 60 °C 60 s. If the qPCR assay was positive, the amplified segments were subjected to cloning and sequencing to confirm whether they were true or not. Both negative and positive controls were included in each run.

Briefly, 16S rRNA was isolated from samples by a capture oligomer via target capture by magnetic microparticles. The target rRNA was then reverse transcribed to generate cDNA fragments using Moloney murine leukemia virus reverse transcriptase and multiple RNA copies (100–1000) were produced from each cDNA copy by the T7 RNA polymerase. Afterwards, these RNA copies were transcribed into cDNA again and bound to fluorescence-labeled specific probes, which were labeled with 6-carboxyfluorescein (FAM) phosphoramidite at the 5′ end and with 4-[4-(dimethylamino) phenylazo] benzoic acid N-succinimidyl ester (DABCYL) at the 3′ end [10]. The SAT assay was performed according to the manufacturer’s instructions (Shanghai Rendu Biotechnology Co, Ltd). 400 μl of samples and 100 μl RNA nucleic acid extraction buffer were mixed in a 1.5 ml microcentrifuge tube and heated at 60 °C for 5 min. After incubation at room temperature for 10 min, RNA extraction was performed by magnetic beads. 30 μl of RNA extracts and 40 μl of amplification detection buffer were mixed as the final reaction mixture. After incubation at 60 °C for 10 min, the reaction mixture was immediately placed at 42 °C for 5 min. 10 μl of pre-heated (42 °C) enzyme reagent was then added. The tubes were immediately placed on a Roche LightCycler® 480 system and subjected to amplification with the following conditions: 40 cycles of 42 °C for 1 min. Both negative and positive controls were included in each run.

To evaluate the reproducibility of SAT, high (original) and low (100-fold diluted) concentrations of the positive controls in the kits (CT: 10⁴ and 10² copies/μl; NG: 10⁴ and 10² copies/μl; UU: 2 × 10⁵ and 2 × 10³ cfu/ml) were tested, respectively. Each control sample was tested five times.

After 19 of the 33 CT-positive, 19 of the 32 NG-positive, 19 of the 29 UU-positive, and 29 pathogen-negative samples were tested by SAT, 100% agreements between the SAT assay and the qPCR assay were observed in samples with a concentration more than 1 × 10³ copies/ml, as shown in Table 1. Specifically, the sensitivity and specificity were 100% (19/19) and 100% (10/10) for CT, 100% (19/19) and 100% (10/10) for NG, and 100% (19/19) and 100% (10/10) for UU, respectively. No discrepancy was found. In addition, SAT exhibited a good reproducibility. Specifically, the coefficient of variations (CV) of CT, NG, and UU were 1.91, 2.94, and 1.25% for high concentration of positive controls, and 3.14, 0.28, and 2.28% for low concentration of positive controls, respectively.

Because the minimum quantification limits of CT, NG, and UU qPCR kits (Liferiver Biotech) were 1 × 10³ copies/ml, samples with concentrations less than the limit could not be accurately quantified or might be detected as negative. Thus, samples with concentrations less than 1 × 10³ copies/ml would be ideal to evaluate the sensitivities. However, due to less availability of this kind of samples, especially NG samples, diluted samples were used here. The remaining 14 of the 33 CT-positive, 13 of the 32 NG-positive, and 10 of the 29 UU-positive were diluted to 1 × 10² copies/ml. Then, nucleic acids (DNA and RNA) of these diluted samples were simultaneously isolated and tested using both of the SAT assay and the qPCR assay.

Among the 14 CT samples, 28.57% (4/14) was positive in the qPCR assay, while 57.14% (8/14) was positive in the SAT assay (Table 2). Among the 13 NG samples, none was positive in the qPCR assay, while 46.15% (6/13) was positive in the SAT assay (Table 2). Among the 10 UU samples, none was positive in the qPCR assay, while 80% (8/10) was positive in the SAT assay (Table 2). These results indicated that the SAT assay had higher sensitivities than the qPCR assay in the detection of CT, NG, and UU.