Comparison of analytical sensitivity of DNA-based and RNA-based nucleic acid amplification tests for reproductive tract infection pathogens: implications for clinical applications

Abstract

ABSTRACT Currently, DNA-based nucleic acid amplification tests (NAATs) and RNA-based NAATs are employed to detect reproductive tract infection (RTI) pathogens including Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Ureaplasma urealyticum (UU). Although evaluations of DNA-based NAATs have already existed, the comparison of the two methods is scarce. Thus, we compared the limits of detection (LODs) of DNA-based and RNA-based NAATs on the same experimental conditions. Inactivated culture supernatants of CT, NG, and UU with determined pathogen DNA and RNA load were used to evaluate LODs of seven DNA kits and one RNA kit. The LODs of the seven DNA kits for CT, NG, and UU ranged between 38–1,480, 94–20,011, and 132–2,011 copies/mL, respectively. As for RNA kits, they could detect samples at RNA concentrations of 3,116, 2,509, and 2,896 copies/mL, respectively. The RNA concentrations of CT, NG, and UU were 40, 885, and 42 times that of corresponding pathogen DNA concentrations in the employed supernatants, so RNA kits could detect pathogen DNA concentrations as low as 78 copies/mL, 3 copies/mL, and 69 copies/mL, respectively, but the level of pathogen load that the RNA tests could detect was primarily dependent on the infectious phase and transcriptional level of RNA. Thus, a schematic of bacterial dynamics during the period of reproductive tract infections was provided, which suggests that in terms of the analytical sensitivity of pathogen detection, RNA tests are more suitable for detecting active infection and recovery phase, while DNA tests are more suitable for detection in the early stage of infection. IMPORTANCE Reproductive tract infections have considerable effects on the health of humans. CT, NG , and UU are common pathogens. Although evaluation of DNA-based tests has already existed, the comparison between DNA-based and RNA-based tests is rare. Therefore, this study compared the limits of detection of the two tests on the same experimental conditions. Results suggested that most DNA-based NAATs could detect CT, NG, and UU at DNA concentrations lower than 1,000 copies/mL, while RNA-based NAATs could detect bacteria at RNA concentrations around 3,000 copies/mL. Considering the copy number of RNA per bacterium is dynamic through the growth cycle, further comparison is combined with a schematic of bacterial dynamics. Results suggested that in terms of the analytical sensitivity of pathogen detection, RNA tests are more suitable for detecting active infection and recovery phase, while DNA tests are more suitable for detection in the early stage of infection.

Keywords

• reproductive tract infection pathogens
• DNA-based NAAT
• RNA-based NAAT
• analytical sensitivity