Withdrawn Australian Society for Microbiology Annual Scientific Meeting 2013

A novel rapid multiplex PCR assay for the detection of 13 bacterial and viral causes of sexually transmitted infection (STI). (#233)

John R Melki 1 , Shoo Peng Siah 1 , Kiran Kaur 1 , jiny Nair 1 , William Rawlinson 2 , Doug Millar 1
  1. Genetic Signatures, Sydney, NSW, Australia
  2. Director of Virology, Prince of Wales Hospital, Randwick, NSW, Australia

Objective: It has been estimated that around 78-300 million new cases of genitourinary tract infections occur annually worldwide. Neiseria gonnorrhoeae and Chlamydia trachomatis are considered the most common causes of genital tract infections however other species such Mycoplasma, Trichomonas and Ureaplasma are also agents of disease.

Aims: Produce rapid multiplex real time PCR assays that could detect 13 causative agents of STI in less than 2 hours. The PCR panels were designed to correspond to clinical symptom groups thus the requesting physician could select the most appropriate panel for the presenting symptoms.

Methods: Pelleted urine samples or genital swabs were placed in sample buffer and heated at 95oC for 15 minutes. Sample purification can be completed using a number of platforms including the EZ1 or Qiasymphony (Qiagen), Kingfisher (ThermoFisher), Magnapure (Roche) or the Easymag (Biomerieux) extraction systems. Real time PCR analysis can be performed on the LC480 (Roche), 7500 Fast (Applied Biosystems), RotorgeneQ (Qiagen) and the Smartcycler II (Cepheid) instruments.

Results: Using genomic DNA from the target organisms of interest the sensitivity of all components of the multiplex assay was shown to be in the range of 1-10 copies. No cross reactivity was shown using a wide range of non-target bacteria and viruses. The assay detected all samples from a commercially available CT/NG validation panel (Zeptometrix, Buffalo). Testing previously characterised clinical samples with the multiplex PCR gave excellent concordance with conventional methods.

Conclusion: The panels were able to successfully detect the presence of specific agents responsible for STI from primary patient material in under 2 hours thus patients could be rapidly placed on the correct antibiotic regime. In addition, the tests require minimal hands on time and can be implemented in virtually any clinical laboratory that is equipped with a sample extraction platform and real-time instrument without further capital outlay for dedicated equipment.