Multiplex pcr assay for chlamydia-like bacteria detection

Introduction: Waddlia chondrophila and Parachlamydia acanthamoebae are well-known and best-studied representatives of Сhlamydia-related bacteria carrying a potential zoonotic threat. These bacteria are associated with miscarriage, ectopic pregnancy, diseases of the respiratory system in both humans and animals. Despite the importance of these Сhlamydia-like organisms for human medicine along with veterinary medicine, studies on their prevalence in Ukraine were not conducted due to the lack of available tests. The aim of our work was to create relatively cheap and easy method for detection Waddlia chondrophila and Parachlamydia acanthamoebae.

Materials and methods: GenBank database was used to find nucleotide sequences of the 16S rRNA gene of bacteria Chlamydiales' order. Alignment was performed using the MEGA7 software, in order to detect the presence of polymorphic hybridization sites specifically attributed to Waddlia chondrophila and Parachlamydia acanthamoebae. PrimerBLAST software was used to design oligonucleotide primers, to evaluate the critical parameters of the primer, in particular, the melting temperature, difference between melting temperatures for the primer pairs, the GC content, the self-complementarity, etc.

Results and conclusions: The amplification of control DNA of Parachlamydia acanthamoebae and Waddlia chrondophila in single PCR using the corresponding primers and subsequent gel electrophoresis of PCR products determined the size of the amplified DNA fragments 88 b.p. and 123 b.p, respectively; the fragments were in line with the expected sizes.The analytical specificity test was performed by amplifying the control DNA of 15 species of the order Chlamydiales.