Association Of Taqman Pcr And Hrm Technologies For Clinical Diagnosis Of Infections And For Rapid Sterility Testing For Pharmaceuticals, Biotechnology Products And Devices

Abstract

Purpose: : Bacterial and fungal cultures ("gold standard") of corneal, aqueous and vitreous samples can be biased if growth requirements are not optimal. Moreover, the confirmation of culture negativity requires 10 days or more. The goal of the present work was to develop a new strategy to detect, semi quantify and automatically characterize microbial DNA sequence variations by associating real-time Taqman PCR and High Resolution Melting(HRM).

Methods: : DNA was extracted from 100 µl using the MagNA Pure. Two in-house kit prototypes were prepared; the first kit consists of 4 tubes for Bacteria (real-time PCR) and the second of 2 tubes for Fungi (HRM). Viability was assessed by introducing samples into 2 tubes containing culture media, the first incubated at 4°C and the second at 37°C for 4 h before DNA extraction

Results: : The equivalent of 0.1 CFU/µl or less of Bacteria and Fungi were detected in all the tested samples; negativity for both could be confirmed in less than 2.30 hours after DNA extraction with inexpensive in-house prepared kits. Rates of positivity compared with culture for clinical samples were 10-fold higher, with a specificity of 100 %. The HRM differentiated filamentous fungi from yeasts, and in addition, identified simultaneously yeasts associated with pathology. The signals produced by residual microbial DNA were separated from those produced by viable organisms.This new molecular strategy, that can be easily adapted to routine molecular equipment: a) detects and quantifies all Bacteria; b) identifies most of the Genera implicated in the contamination of pharmaceuticals and in human pathology; c) improves true positive results with no increase in false positives or negatives; d) produces reproducible signature of Fungi with no need for probes (radioactive or non radioactive) or post amplification procedures.

Conclusions: : The first test associating real-time TaqMan PCR and HRM for rapid diagnosis of infections and for sterility testing shows higher sensitivity than culture and dramatically reduces the time for the detection of viable microorganisms. Larger studies are necessary to confirm its usefulness for clinical diagnosis and therapeutic follow up, and for sterility testing of pharmaceuticals, cell therapy derivatives, food, cosmetics, fluids, tissues, environmental samples and medical devices.

Keywords: bacterial disease • fungal disease • clinical laboratory testing