To achieve this we have developed a protocol for onbead padlock probing and rolling circle amplification. This technique is analogous to digital PCR, in which fluorescence-labelled amplicon clusters deriving from individual templates are enumerated, but our approach does not require compartmentalization in water-inoil emulsion or Theaflavin massively parallel microwell plates. The total reaction time of the protocol is 30 min with retained LOD. This study furthermore presents stream-lining of the padlock probe and PLA protocols, to present a protocol that comprise several identical reaction mixtures and that should lend itself to automation. To summarize, the protocols and instrumentation presented herein represents a substantial improvement from previous protocols and instrumentation, and pose a unique and promising opportunity for quick and sensitive detection of pathogens Furthermore, we herein for the first time apply PLA with a digital readout for an analyte in solution. The bio-monitoring approach presented here is based on the homogeneous amplified single-molecule detection technique described by 2-Thiouracil Jarvius et al. A protocol for molecular probing and amplification which is quick and amenable to automate has been developed and a dedicated prototype instrument has been constructed. In this study the focus has been bio-security applications, however, the system is generic and could be useful for several applications, including water and food quality testing, and high-precision digital analysis for research and diagnostic applications. The time of the molecular probing and amplification procedures have been reduced from about six hours to 30 min with retained sensitivity. The number of processing steps has also been reduced from six to four. The analysis of RCPs was previously performed using a confocal microscope as fluorescence detector and a separated pump for driving the microfluidic system. We have developed a dedicated detection instrument which is rapid, and with an at least tenfold increased sensitivity. Furthermore, incorporation of multiple lasers and detectors in the instrument enabled, for example, simultaneous screening of PA and EC. The approach developed in this study enables digital detection of both DNA and protein in the same analysis platform. This versatile feature enables multiple detection strategies for challenging agents, such as spores, where the DNA can be difficult to address directly. We applied the assays for detection of EC and PA on a DNA-level with a LOD of approximately 30 bacteria. For the genetic analysis the specificity was further demonstrated by discriminating between the two bacterial species based on a single-nucleotide difference between the target DNA sequences of EC and PA.