ELECTROCHEMICAL DETECTION OF BACTERIAL AND/OR FUNGAL INFECTIONS
The present disclosure relates to methods and devices for amplifying a plurality of targets in a single PCR run while distinguishing between clinically relevant amplification and amplification from other sources such as from background contamination. The methods and devices further enable discrimination between gram-positive, gram-negative and fungal infections as wells as identify antimicrobial resistance genes. When applying the methods and devices of the invention, the species or genus of an infection(s), and genus of a fungal co-infection(s) or category of bacterial (gram-positive or negative) co-infection(s) are identified. Species identification of co-infections can also be achieved. Further, when applying the methods and devices of the invention, organisms which are likely to be contaminating organisms from a blood draw are identified.
1 . An in vitro method for the detection and/or identification of a hybridization complex comprising a human pathogen and/or genetic material thereof hybridized to a signal probe and a capture probe comprising: subjecting a sample comprising or suspected of comprising a human pathogen and/or genetic material thereof to a single multiplex polymerase chain reaction (PCR), wherein said method comprises amplification of PCR products under conditions appropriate for the substantial reduction in detection of contaminating pathogen and/or genetic material present in the sample and detecting the binding between the human pathogen and/or genetic material thereof and the signal probe and a capture probe.
2 . The method of claim 1 , wherein the human pathogen is gram-positive bacteria, gram-negative bacteria or fungal infection.
3 . The method of claim 1 , wherein the human pathogen is gram-positive bacteria, gram-negative bacteria, and fungal infection.
4 . The method of claim 1 , wherein the method can further distinguish between gram-positive bacteria species and can identify gram-negative bacteria and fungal infections but cannot distinguish gram-negative bacteria and fungal species if present in the sample.
5 . The method of claim 1 , wherein the method can further distinguish between gram-negative bacteria species and can identify gram-positive bacteria and fungal infections but cannot distinguish gram-positive bacteria and fungal species if present in the sample.
6 . The method of claim 1 , wherein the pathogen is a fungal infection and the method can distinguish between fungal species wherein the method can further identify gene determinants of antimicrobial resistance.
7 . The method of claim 1 , wherein the capture probe is bound to gold-plated electrodes and wherein the signal probe comprises at least one covalently attached ferrocene derivative.
8 . The method of claim 1 , wherein prior to subjecting the sample to a single multiplex PCR the sample is contacted with a compound which hydrolyzes nucleic acids.
9 . The method of claim 1 , wherein if four or more pathogens are identified in the sample, the sample is subject to further testing.
10 . The method of claim 1 , wherein prior to subjecting the sample to multiplex PCR it is loaded into any instrument bay at any time.
11 . The method of claim 1 , further comprising automatically generating an epidemiology report on a clinical instrument.
12 . The method of claim 1 , wherein the human pathogen identified is automatically reported to a hospital's laboratory information system (LIS).
13 . An in vitro method for the electrochemical detection and/or identification of a first human pathogen and/or genetic material thereof and a second human pathogen and/or genetic material thereof comprising subjecting a first portion of a sample comprising or suspected of comprising a first human pathogen and/or genetic material thereof to a first single multiplex polymerase chain reaction (PCR), wherein said method comprises amplification of PCR products under conditions appropriate for the substantial reduction in electrochemical detection of contaminating pathogen and/or genetic material present in the sample; subjecting a second portion of the sample comprising or suspected of comprising a second human pathogen and/or genetic material thereof to a second single multiplex PCR, wherein said method comprises amplification of PCR products under conditions appropriate for the substantial reduction in electrochemical detection of contaminating pathogen and/or genetic material present in the sample.
14 . The method of claim 13 , wherein the first human pathogen is gram-positive bacteria, gram-negative bacteria or fungal infection and wherein the second human pathogen is gram-positive bacteria, gram-negative bacteria or fungal infection.
15 . The method of claim 13 , wherein the first single multiplex PCR can further distinguish between gram-positive bacteria species and can identify gram-negative bacteria and fungal infections but cannot distinguish gram-negative bacteria and fungal species if present in the sample.
16 . The method of claim 13 , wherein the first single multiplex PCR can further distinguish between gram-negative bacteria species and can identify gram-positive bacteria and fungal infections but cannot distinguish gram-positive bacteria and fungal species if present in the sample.
17 . The method of claim 13 , wherein the first single multiplex PCR can further distinguish between fungal species.
18 . The method of claim 13 , wherein the method can further identify determinants of antimicrobial resistance.
19 . The method of claim 13 , wherein the second human pathogen is gram-positive bacteria, gram-negative bacteria or fungal infection and wherein the second human pathogen can be identified by its species.
20 . A microfluidic device for detecting a human pathogen and/or genetic material thereof comprising: a mixture of oligonucleotides and reagents for carrying out a single nucleic acid amplification reaction capable of distinguishing between contaminating pathogen and/or genetic material present in the sample and infectious pathogen and/or genetic material present in the sample and capable of distinguishing between gram-negative bacterial species and identifying by its genus a fungal co-infection or identifying by its type a gram-positive co-infection.