A Infectious diseases account for about 3 million deaths per year. The advent of molecular techniques has led to an enormous improvement in their diagnosis, both in terms of sensitivity and specificity and in terms of the speed with which a clinically useful result can be obtained. Digital PCR, or 3rd generation PCR, is based on a series of technical modifications that result in more sensitive techniques, more resistant to the action of inhibitors and capable of direct quantification, without the need for standard curves.This review presents the main applications that have been developed for the diagnosis of viral, bacterial and parasitic infections, and the potential prospects for the clinical use of this technology.

The viral nervous necrosis virus (VNNV) is the causative agent of an important disease affecting fish species cultured worldwide. Early and accurate diagnosis is at present the most effective control and prevention tool, and the molecular techniques have been strongly introduced and accepted by official organizations. Among those, real time quantitative polymerase chain reaction (rt-qPCR) is nowadays displacing other molecular techniques. However, another PCR-based technology, the droplet digital PCR (ddPCR), is on the increase. It has many advantages over qPCR, such as higher sensitivity and more reliability of the quantification. Therefore, we decided to design and validate a protocol for diagnosis and quantification of SJ and RG type VNNV, using reverse transcription-ddPCR (RT-ddPCR). We obtained an extremely low limit of detection, 10 to 100-folds lower than with RT-qPCR. Quantification by RT-ddPCR, with a dynamic range of 6.8 – 6.8 x 104 (SJ type) or 1.04 x 101 – 1.04 x 105 (RG) cps/rctn, was more reliable than with RT-qPCR. The procedure was tested and validated in field samples, providing the high clinical sensitivity and negative predictive values. In conclusion, we propose this method to substitute RT-qPCR protocols because it exceeds the expectations of qPCR in the diagnosis and quantification of VNNV.