Abstract:
The present invention relates to a Polymerase Chain Reaction and High Resolution Melt genetic identification system, and, more specifically, to a tactical and portable Polymerase Chain Reaction and High Resolution Melt genetic analysis and identification system that is configured to determine and communicate analysis and identification results and a tiered confidence/alert level related to the analysis and identification results.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a Polymerase Chain Reaction and High Resolution Melt genetic identification system, and, more specifically, to a tactical and portable Polymerase Chain Reaction and High Resolution Melt genetic analysis and identification system that is configured to determine and communicate analysis and identification results and a tiered confidence/alert level related to the analysis and identification results. 
     2. Description of the Related Art 
     Polymerase Chain Reaction (“PCR”) is a ubiquitous molecular biology tool used in thousands of different applications. In brief, this molecular biology tool is used to produce (“amplify”) a sufficient number (sometimes millions to billions) of copies of a particular DNA sequence so that the sequence can adequately be used in these applications. Essentially, PCR makes a sample of DNA that is large enough so that the sample can be appropriately analyzed. As just one example of a typical application for PCR amplification, it is a common detection and/or identification method and tool used, for example, in clinical applications, scientific investigations, and in biological warfare agent (“BWA”) detection systems. 
     High Resolution Melt (“HRM”) is another molecular biology tool. This tool is used to detect a variety of differences (e.g., mutations, other types of genetic sequence differences) in samples of double stranded DNA, which is based on the detected melting temperature/behavior of the double stranded DNA sequence(s) at issue. HRM is performed post PCR amplification, which is performed to obtain a sufficient number of copies of the DNA sequence(s) of interest. 
     An end user would like the ability to perform genetic PCR and HRM genetic identity testing outside of the lab environment, i.e., in the field. This is a need because most commercially available PCR and PCR/HRM instruments are designed to be operated in a controlled and generally clean laboratory environment. In the field, however, it is expected that there will be wider temperature and humidity fluctuations, there may be no electrical supply, and other ambient environmental conditions may be outside the normal operating environment of these systems. 
     Accordingly, there is a continued need for a portable PCR/HMR genetic analysis and identification system. 
     BRIEF SUMMARY OF THE INVENTION 
     It is therefore a principal object and advantage of the present invention to provide a tactical and portable PCR and HMR genetic analysis and identification system that is configured to determine and communicate analysis and identification results and a tiered confidence/alert level related to the analysis and identification results. 
     Another object and advantage of the present invention is to provide a tactical and portable PCR and HMR genetic analysis and identification system that simple and is easy to use, is an all inclusive analysis, results, and reporting/communication system, provides accurate geo-location mapping, can run on most power devices including vehicle power, concealable, minimum size, weight, and power, has GPS capability, mechanically isolated for shock and vibration, easily transportable and deployable around the world (e.g., maximum 2 person carry and set-up), does not require a “technical” person to operate, and has a reliability and probability detection rating of up to 95.0%. 
     Another object and advantage of the present invention is to provide a tactical and portable PCR and HMR genetic analysis and identification system that is intended to be primarily portable and tactical in nature. This is accomplished, in part, by combining and modifying existing commercial-based technologies and combining them with custom hardware and software into a complete system. 
     In accordance with the foregoing objects and advantages, a tactical and portable PCR and HMR genetic analysis and identification system is provided comprising one or more of the following: a PCR/HRM module encompassed within a ruggedized enclosure, wherein the PCR/HRM module is structured and configured to perform PCR amplification and an HRM analysis on unknown DNA samples in the field; a computer processor encompassed within the ruggedized enclosure, interconnected to the PCR/HRM module, and programmed to compare the results of the PCR amplification and an HRM analysis on at least one unknown DNA sample by the PCR/HRM module to known control DNA samples, assign a probability of a match to one of the known control DNA samples, and to assign a confidence level to the DNA sample based on the probability of a match to the known control DNA; a communication module interconnected to the computer processor and configured to wirelessly transmit data from the computer processor and the PCR/HRM module to a computer device; wherein the computer processor is programmed to assign a confidence level as part of a tiered confidence level routine comprising a plurality of confidence levels, wherein a highest confidence level indicates a high probability of a match to one of the known control DNA samples and a lowest confidence level indicates a low probability of a match to one of the known control DNA samples. 
     Current commercial PCR/HRM systems are not designed to survive and operate in an environment outside of a controlled indoor laboratory environment. In addition, these machines are designed for standalone operation and have many features unnecessary for “field use” scenario, such as, LED&#39;s, push buttons, and the actual enclosures and packaging. These items can be removed and only the “critical to function” items can be used in the portable and tactical design, represented by the PCR/HRM module in  FIG. 2 , described below. “Field use” can be defined as outside of the normal laboratory environment and in a naturally occurring environment caused by terrestrial weather and an un-natural environment caused by dropping, road shock &amp; vibration and any other human incident. 
     The details of one or more embodiments are described below and in the accompanying drawings. Other objects and advantages of the present invention will in part be obvious, and in part appear hereinafter. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) 
       The present invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which: 
         FIG. 1  is a schematic/flow chart representation of a tactical and portable PCR and HMR genetic analysis and identification system according to an embodiment of the present invention; 
         FIG. 2  is a schematic representation of a tactical and portable PCR and HMR genetic analysis and identification system  200 , according to an embodiment of the present invention; and 
         FIG. 3  is a schematic representation of results from a tactical and portable PCR and HMR genetic analysis and identification system being viewed and analyzed on various devices with GUIs, according to an embodiment of the present invention. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Advantages of the invention are illustrated by the Examples set forth herein. However, the particular conditions and details are to be interpreted to apply broadly in the art and should not be construed to unduly restrict or limit the invention in any way. 
     As discussed further below, the tactical and portable PCR and HMR genetic analysis and identification system according to an embodiment of the present invention can include a PCR/HRM device, a power source, minimal liquid handling equipment, and kits for DNA extraction &amp; purification and a well plate (e.g., a pre-made 96 well plate or 386 well plate). The plate can contain reagents necessary for PCR and HRM, where some plate wells are filled with reagents for the unknown DNA samples, and other wells contain control DNAs and reagents for comparison to the unknowns during the PCR/HRM run. 
     Once the sample or samples of concern are identified, a small portion is subjected to DNA extraction and purification using the commercial kits. The DNA is then distributed to a few wells the 96 well plate in a manner common for PCR. The plate, once filled with reagents, is placed into the PCR/HRM device. 
     The PCR/HRM device is turned on and a pre-determined PCR/HRM routine can be run on the device that subjects the sample to the PCR and subsequent HRM in a manner that is diagnostic for all known target samples. DNAs that are amplified during PCR and that have the appropriate HRM melt profiles in comparison to control DNAs are binned into three match groups: High probability of match to an known standard (&gt;95%), moderate probability of a match to a known standard (&gt;90; ≦94.9%), or low probability of a match to a known standard (&lt;90.0%) Upon successful matching of the unknown DNA to a standard (here termed “genome identification”), the software of an embodiment of the present invention will provide the end-user with a tiered alert level. For example, a three (3) tier system could be established where a level of confidence or highest probability of a successful analysis and result would categorize a sample at a Level 1, 2 or 3, as set forth in Table 1 below. 
     
       
         
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Levels 
                 Description 
               
               
                   
               
             
             
               
                 1 
                 The end user is provided with a result identifying the sample as a  
               
               
                   
                 probable match to a known DNA type and having a low risk 
               
               
                   
                 of either a false negative or false positive. 
               
               
                   
                 This response is one of clear and probable target, based on a  
               
               
                   
                 greater than 95% similarity to standard target DNAs. A Level 
               
               
                   
                 1 result would provide the end-user with a specific genetic type 
               
               
                   
                 and a regional mapping of the source&#39;s geographical location 
               
               
                   
                 and/or point of origin via the software GUI. 
               
               
                 2 
                 The end user is provided with a result identifying the sample as 
               
               
                   
                 a less close match to a known DNA and would be considered 
               
               
                   
                 to have a medium risk of a false negative or false positive. 
               
               
                   
                 The sample may require additional analysis. A Level 2 result  
               
               
                   
                 would provide the end-user with, for example, a less confident 
               
               
                   
                 assignment of geospatial origin (owing to perhaps a overlapping 
               
               
                   
                 matches to several standard target DNA. This lack of certainty  
               
               
                   
                 will also result in a more expansive regional mapping of the 
               
               
                   
                 source material&#39;s geographical location and/or more likelihood  
               
               
                   
                 for more than one point of origin via the software GUI. A Level 
               
               
                   
                 2 result could require additional tactical and portable PCR and  
               
               
                   
                 HMR genetic analysis and identification system analysis, but 
               
               
                   
                 would be most benefitted by further home station post analysis. 
               
               
                 3 
                 The end user is provided with a result identifying the sample as  
               
               
                   
                 having a low probability of match to the target DNA standards, 
               
               
                   
                 and therefore presents a high risk of a false positive or false  
               
               
                   
                 negative, and the sample is not considered reliable for geolocation 
               
               
                   
                 source assignments; the sample requires post analysis. A Level  
               
               
                   
                 3 result would require home station (not in-field) post analysis. 
               
               
                   
               
             
          
         
       
     
     Referring now to the drawings, wherein like reference numerals refer to like parts throughout, there is seen in  FIG. 1  a schematic/flow chart representation of a tactical and portable PCR and HMR genetic analysis and identification system  10 , according to an embodiment of the present invention. A power system  15 , a PCR/HRM system  20 , and a communication portion of the system  25  are shown. The communication portion  25  of the system can transmit results data wirelessly at  65 . The wireless transmission can be accomplished through any wireless protocol/technology, including, but not limited to, ZigBee standards-based protocol, Bluetooth technology, and/or Wi-Fi technology. The wireless transmission can be over a network which can be any suitable wired or wireless network capable of transmitting communication, including but not limited to a telephone network, Internet, Intranet, local area network, Ethernet, online communication, offline communications, wireless communications and/or similar communications means. Further, this data can be encrypted as needed based on the sensitivity of the data, the location of the obtainment of the field sample, and or the identification of the collector, for example. Field DNA sample  45  is inputted at  50  (and outputted at  52  to the communication portion of the system  25 ) and can be analyzed in the field by the tactical and portable PCR and HMR genetic analysis and identification system  10  or remotely by the Post Analysis/Home Station  60 . 
     As shown in the Communication of Tactical Analysis Results  66 , if a Level 1 is indicated (low risk/clear/probable target), no further analysis of the field DNA sample  45  is required. If a Level 2 is indicated, (medium risk) more analysis of the field DNA sample  45  may be required. Arrow  30  shows follow-up analysis of the field DNA sample  45  by the tactical and portable PCR and HMR genetic analysis and identification system  100 . Arrow  35  shows further analysis of the field DNA sample  45  by a remote PCR/HMR system  60 . If a Level 3 is indicated, (high risk), post analysis of the field DNA sample  45  is required. Arrow  40  shows further analysis of the field DNA sample  45  by a remote PCR/HMR system  60 . 
     As shown in  FIG. 2 , a tactical and portable PCR and HMR genetic analysis and identification system  200 , according to an embodiment of the present invention is shown. The PCR/HRM module  20  can be designed and packaged in a ruggedized enclosure  205  (to protect from dropping, shaking, weather etc.) along with a battery power module  15 ″, power supply PCB  15 ′, single board computer  210  and a communications PCB  25 ′. The communication portion  25  of the system can transmit results data wirelessly at  65 , and a DNA sample going in  50  and out  52  is also shown. 
     The ruggedized enclosure  205  can be custom designed to accommodate mounting and interconnection of the hardware items shown in  FIG. 2 . The material used for the design can be lightweight alloy or composite materials, such as aluminum or carbon fiber. These materials are known for excellent strength to weight ratio, their ability to dissipate heat efficiently and can be easily machined or formed into many different configurations. 
     The battery power module  15 ″ is where DC batteries are placed to provide power to the system in a remote environment or away from shore power. The batteries supply power to the power supply PCB  15 ′ that takes the incoming battery power or shore power and converts to useful power for the single board computer  210 , communications PCB  25 ′ and the PCR/HRM module  20 . 
     The single board computer  210  provides the functionality to the system to control the PCR/HRM module  20 , communications PCB  25 ′ and to post-process the results of PCR/HRM DNA sample. Within the single board computer  210  is a bioforensics software module/tool/program code that enables classification and probability assignment results of the DNA samples. These classification and probability assignments can be filtered through a single board computer  210  database (the database can be internal or external where the computer  210  is wirelessly or in wired connection with the database (not shown)) in such a way as to provide confidence/alert level reporting (as discussed herein) to the user in a way that will report a high confidence of identification, a medium confidence of identification or a low confidence of identification. Those results are provided to the communications PCB  25 ′. The communications PCB  25 ′ will take the results  315  and transfer that information to a host of computer devices e.g., desktop, laptop  300 , smart phone  310 , cell phone, computer tablet, and/or other portable computer like device where the results are displayed on an easy to read GUI  305  for an end user to assess and evaluate. See  FIG. 3 . 
     As will be appreciated by one skilled in the art, aspects of the present invention may be embodied as a system, method or computer program product. Accordingly, aspects of the present invention may take the form of an entirely hardware embodiment, an entirely software embodiment or an embodiment combining software and hardware aspects that may all generally be referred to herein as a “circuit,” “module” or “system.” Furthermore, aspects of the present invention may take the form of a computer program product embodied in one or more computer readable medium(s) having computer readable program code embodied thereon. 
     Any combination of one or more computer readable medium(s) may be utilized. The computer readable medium may be a computer readable signal medium or a computer readable storage medium. A computer readable storage medium may be, for example, but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, or device, or any suitable combination of the foregoing. More specific examples (a non-exhaustive list) of the computer readable storage medium would include the following: an electrical connection having one or more wires, a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), an optical fiber, a portable compact disc read-only memory (CD-ROM), an optical storage device, a magnetic storage device, or any suitable combination of the foregoing. In the context of this document, a computer readable storage medium may be any tangible medium that can contain, or store a program for use by or in connection with an instruction performance system, apparatus, or device. 
     The program code may perform entirely on the user&#39;s computer, partly on the user&#39;s computer, as a stand-alone software package, partly on the user&#39;s computer and partly on a remote computer or entirely on the remote computer or server. In the latter scenario, the remote computer may be connected to the user&#39;s computer through any type of network, including a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider). 
     Although the present invention has been described in connection with a preferred embodiment, it should be understood that modifications, alterations, and additions can be made to the invention without departing from the scope of the invention as defined by the claims.