Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis , Neisseria gonorrhoeae , Mycoplasma hominis and Ureaplasma urealyticum

Abstract

Doc number: 433

Abstract

Background: Although sophisticated methodologies are available, the use of endpoint polymerase chain reaction (PCR) to detect 16S rDNA genes remains a good approach for estimating the incidence and prevalence of specific infections and for monitoring infections. Considering the importance of the early diagnosis of sexually transmitted infections (STIs), the development of a sensitive and affordable method for identifying pathogens in clinical samples is needed. Highly specific and efficient primers for a multiplex polymerase chain reaction (m-PCR) system were designed in silico to detect the 16S rDNA genes of four bacteria that cause genital infections, and the PCR method was developed.

Methods: The Genosensor Probe Designer (GPD) (version 1.0a) software was initially used to design highly specific and efficient primers for in-house m-PCR. Single-locus PCR reactions were performed and standardised, and then primers for each locus in turn were added individually in subsequent amplifications until m-PCR was achieved. Amplicons of the expected size were obtained from each of the four bacterial gene fragments. Finally, the analytical specificity and limits of detection were tested.

Results: Because they did not amplify any product from non-STI tested species, the primers were specific. The detection limits for the Chlamydia trachomatis , Neisseria gonorrhoeae , Mycoplasma hominis and Ureaplasma urealyticum primer sets were 5.12 × 10⁵ , 3.9 × 10³ , 61.19 × 10⁶ and 6.37 × 10⁵ copies of a DNA template, respectively.

Conclusions: The methodology designed and standardised here could be applied satisfactorily for the simultaneous or individual detection of Chlamydia trachomatis , Neisseria gonorrhoeae , Mycoplasma hominis and Ureaplasma urealyticum . This method is at least as efficient as other previously described methods; however, this method is more affordable for low-income countries.