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Nucleic acid-based testing in point-of-care settings

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Also listed as: NABT in point-of-care settings
Related terms
Background
Methods
Research
Implications
Limitations
Safety
Future research
Author information
Bibliography

Related Terms
  • Alternate site testing, ancillary testing, bedside testing, biological weapon, decentralized testing, DNA microarray, electronic health records, forensic marker, genetic marker, home health care, home healthcare, lab-on-a-chip, LOC, MEMS, microelectromechanical systems, microfluidic chip, molecular diagnostics, NABT, nanotechnology, near patient testing, near-patient testing, nucleic acid-based technology, nucleic acid-based testing, pathogens, PCR, POCT, point-of-care testing, polymerase chain reaction, telemedicine.

Background
  • Point-of-care testing (POCT): Point-of-care testing (POCT) are tests that are designed to be used at or near the site where the patient is located. POCT may not require a permanent dedicated space and may be performed outside the physical facilities of clinical laboratories. POCT is also called near-patient testing, bedside testing, ancillary testing, and decentralized testing.
  • POCT includes simple self-care tests that patients can perform by themselves at home, tests that are performed in the doctor's office, and tests that are performed by personnel in, for example, an intensive care unit, an emergency room, a ward, or in a battlefield area. POCT may be in the form of transportable, portable, or handheld instruments (e.g., a blood glucose meter) and test kits (e.g., an HIV salivary assay).
  • The major benefits of POCT are that it is convenient, cost-effective, and provides timely results. In turn, this aids in early diagnosis and prompt decisions regarding treatment for the disease or condition. It also helps to monitor the disease, thereby helping to reduce complications.
  • Nucleic acid-based testing (NABT): Nucleic acids are deoxyribose nucleic acids (DNA) and ribonucleic acids (RNA). DNA is a long, thread-like (double-helix) molecule made up of large numbers of nucleotides. Nucleotides are the building blocks of DNA and are made of nitrogen bases, sugars, and phosphate. Nitrogen bases are of two types: purines, such as adenine (A) and guanine (G), and pyrimidines, such as cytosine (C) and thymine (T). Long strands of nucleotides form nucleic acids. The sequence of bases in DNA serves as the carrier of genetic information in the form of genes. RNA is a nucleic acid that helps in protein synthesis, which is important for the growth and maintenance of the body. RNA is formed under the direction of DNA, and both help to form amino acids, which are the building blocks of protein.
  • Genes provide instructions for all cells in the body and are passed from parents to their children as genetic (inherited) characteristics. Genes, which are located inside cells, control the development and function of an organism by instructing cells to make new molecules (usually proteins). A genetic trait refers to certain characteristics (e.g., physical characteristics, such as height or eye color) brought about by the expression of a gene or many genes.
  • NABT is an analysis technique that is based on the detection, amplification (production of multiple copies), and/or analysis of DNA and RNA. NABT involves the identification and characterization of disease-causing microorganisms (pathogens) and genetic markers. Genetic markers refer to a gene or DNA sequence that produces a recognizable trait and can be used in family and population studies. A genetic marker is associated with a certain disease and can be detected (e.g., in blood) to determine whether an individual is at risk of developing that disease. NABT has widespread applications, such as disease detection and diagnosis, evaluation of the degree or severity of a disease caused by pathogens (i.e., its virulence), drug resistance, etc. Drug resistance in NABT refers to decreased reactivity of an organism to the anti-infective action of a drug as a result of changes in the genetic makeup of an organism, such as gene mutations. Mutations are changes or alterations of a sequence within a gene of an organism that results in the creation of a new character or trait not found in the parent (original) type.
  • The advantage of NABT techniques in comparison to conventional disease detection tools is its high specificity and sensitivity, which requires very little sample (e.g., blood), its rapid analysis, and its application in both qualitative (identifying) and quantitative (counting) analysis assays. Specificity refers to the quality of the test's detection and identification of specific organisms or genetic markers. Sensitivity indicates the probability of a test to correctly identify or detect the pathogen or a marker present in a very small quantity.
  • Nucleic acid-based testing in a point-of-care setting: The integration of NABT used at point-of-care settings has potential in the identification of new pathogens and diseases. NABT may also help identify genetic markers that indicate a potential risk for diseases or adverse drug reactions. In addition, NABT may also help identify pathogens used in global bioterrorism. Some examples of NABT at POCT settings include the handheld advanced nucleic acid analyzer (HANAA) and the LiatT analyzer (by IQuum).

Methods
  • Nucleic acids are deoxyribose nucleic acids (DNA) and ribonucleic acids (RNA). DNA is a long, thread-like (double-helix) molecule made up of large numbers of nucleotides. Nucleotides are building blocks of DNA and are made of nitrogen bases, sugars, and phosphate. Nitrogen bases are of two types: purines, such as adenine (A) and guanine (G), and pyrimidines, such as cytosine (C) and thymine (T). Long strands of nucleotides form nucleic acids. The sequence of bases in DNA serves as the carrier of genetic (hereditary) information in the form of genes. RNA is a nucleic acid that helps in protein synthesis, which is important for the growth and maintenance of the body. RNA is formed under the direction of DNA, and both help to form amino acids, which are the building blocks of protein.
  • Nucleic acid-based tests (NABT) are DNA/RNA-based detection, identification, amplification (multiplication), and analysis techniques chiefly used for disease detection and diagnosis. Point-of-care-testing (POCT), or bedside testing, are tests that may be used at or near the site where the patient is located. These further assist in starting treatment at the earliest time possible and in preventing complications related to the infection or disease. Several devices have been developed using a combination of NABT in a POCT setting, and they are described below.
  • Sample preparation: The conventional method of sample preparation involves cell separation or isolation (e.g., separating white blood cells from blood), breakage of cells (cell lysis), and nucleic acid extraction and isolation followed by amplification (multiplication). These processes and functionalities have been miniaturized to microchip level for nucleic acid-based devices in POCT. The cell's isolation has been simplified by the use of filters based on the cell size, dielectrophoresis (DEP), and paramagnetic particles. DEP involves separation of cells such as bacteria, cancer cells, etc., based on their physical structure, electrical charge, and many other factors. Paramagnetic particles involve separation of cells by coating magnetic beads with antibodies against specific antigens (agent that triggers an immune response) on the target cells (e.g., E. Coli bacteria).
  • Cell lysis involves use of chemical, mechanical, thermal (temperature), and electrical methods. This process is followed by the isolation of nucleic acids by use of chemicals and amplification.
  • Handheld advanced nucleic acid analyzer (HANAA): A HANAA is a handheld device based on nucleic acid analysis that was developed by the Lawrence Livermore National Laboratory in 1999. HANAAs aid in easy, direct testing of blood and urine samples in the field (on-site locations) and was employed by United Nations inspectors in Iraq during the search for biological weapons in 2003.
  • A HANAA is about the size of a brick and weighs less than one kilogram (2.2 pounds). The analyzer is highly sensitive, as it can detect 200 organisms per milliliter. The handheld system can test four samples at one time and provide results in less than 30 minutes, thereby helping to detect the cause of the infection or disease quickly, as compared to routine laboratory tests that take hours to days to provide results. The process of using a HANAA for the identification or detection of a disease-causing organism (pathogen) is described below.
  • Sample preparation: The process involves the preparation of the sample to be tested by placing the sample in a liquid reagent (a solution containing chemical substances) and adding chemicals to make the sample ready for measurement. Sample preparation is done to isolate the target cells by separating their nucleic acids (DNA or RNA). It also helps to remove interfering elements that may mask the analysis of the component of interest (e.g., bacteria or viruses).
  • Addition of DNA probe: A DNA probe that is attached or tagged with a fluorescent dye is introduced into the sample to facilitate easy detection. Then the sample is inserted into a heater chamber where thermal cycling (heating and cooling) of the sample's DNA takes place. The DNA probes are single-stranded DNA sequences with base pairs complementary to the target DNA segment. A complementary strand is a nucleic acid sequence that can form a double-stranded structure by matching base pairs (adenine (A) to thymine (T) and guanine (G) to cytosine (C)). For example, a complementary strand for G-T-A-C is C-A-T-G. The operator must have an idea of the substance involved and then decide the kind of pathogen that needs to be tested because each DNA probe is designed to target a specific organism (e.g., anthrax or plague). If the organism is present in the sample, the probe attaches to its DNA and amplifies using the polymerase chain reaction (PCR) process.
  • Making several copies of a component of interest: Amplification of the sample's DNA is done to help analyze the sample, thereby helping to detect the pathogen. Hence, the PCR process is used to amplify the sample's DNA, which involves repeated heating and cooling. Every time the DNA is heated, the two strands of DNA unwind and come apart. As the sample cools down, the DNA makes a copy of itself, in the presence of DNA polymerase, thus doubling the amount of DNA after every cycle. DNA polymerase is an enzyme that makes new DNA strands using preexisting DNA strands as a template, thereby assisting in DNA replication. The handheld analyzer measures the sample's fluorescence to detect the presence or absence of the targeted organism. A HANAA can detect as few as 10 individual bacteria in one-hundredth of a milliliter in less than 30 minutes.
  • Nucleic acid sequence-based amplification (NASBA) analyzer: Handheld NASBA sensors may be used to detect or monitor microbial populations in the field or in clinical settings, thus facilitating diagnoses. A sensor is a device designed to respond to physical features (in the form of a stimulus), such as temperature, light, magnetism, or movement, and to transfer (transmit) resulting signals (in the form of impulses) for interpretation and recording. NASBA is an isothermal (constant temperature)-based method of RNA amplification that uses enzymes (such as reverse transcriptase and RNA polymerase) at a fixed temperature to amplify the target RNA.
  • The reverse transcriptase enzyme aids in the formation of single-stranded DNA from single-stranded RNA used as a template. The RNA polymerase enzyme aids in the formation of single-stranded RNA from a single-stranded RNA or DNA. In this system, probes tagged with fluorescent dye are added, and if the organism is present in the sample, the probe attaches to the target RNA. The handheld detection system can perform real-time analysis of samples and obtain the results within minutes.
  • IQuum's LiatT Analyzer: The LiatT analyzer is useful for first responders or healthcare workers to conduct nucleic acid tests at the patient's bedside or in the field, aiding in the detection of viral or bacterial DNA or RNA. The LiatT system automates all nucleic acid testing processes, including sample preparation and amplification, using both PCR and reverse transcription-polymerase chain reaction (RT-PCR) and real-time detection in one portable device. Results are provided in 30-60 minutes. Reverse transcription is a process of making DNA under the direction of RNA, i.e., it is the opposite of transcription wherein synthesis of RNA takes place under the direction of DNA. RT-PCR is also used for real-time PCR. A wide variety of samples, such as whole blood, plasma, and urine, can be used in the analyzer, providing flexibility to the operator in choosing the sample.
  • DNA microarray: A DNA microarray, also called A DNA microchip or a "lab-on-a-chip" (LOC), is an analytical device that combines several laboratory functions on a single chip that is only a few millimeters to square centimeters in size. A microarray is a collection of miniaturized tests arranged on a surface that permits many tests to be performed at the same time in order to achieve higher throughput. The commonly used solid or physical support materials for the microarray chips include, glass slides, silicon, microwells or nanowells, nitrocellulose membranes, and microbeads. A nitrocellulose membrane is a thin layer formed by a mixture of pulpy or cotton-like polymers (large chains of molecules) of cellulose (a form of glucose), nitric acid, and sulfuric acids. Microbeads are very small beads of glass or silicon that can be used instead of a flat glass or silicon surface. These microbeads are coated with bait or target molecules for easy detection.
  • The basis of DNA microchips are the tiny fixed nucleic acid spots that act as bait or target molecules, which may be detected by allowing them to react with probes tagged (attached) with a signal-generating molecules (e.g., fluorescent tags). A probe is a molecule complementary to the target molecule on the microarray chip to which it can bind. The tagging molecule generates signals; the signal intensity of each spot is proportional to the quantity of applied tagged molecules bound to the bait molecule, thereby helping in detection and measurement (both qualitative and quantitative analysis) of DNA or RNA from the sample of interest. The spot pattern image is captured, analyzed, and compared with biological information with the help of a computer.
  • LOC devices involve the use of extremely small fluid volumes (<1 picoliter, or one trillionth of a liter) and are a subset of microelectromechanical systems (MEMS devices). MEMS are also known as micro total analysis systems (µTAS). MEMS is the technology of the very minute and is used to perform biochemical analysis by integrating laboratory processes.
  • Microfluidic chip: Microfluidic chips are also LOC devices that perform operations on small bodies of fluids and/or on particles suspended in a fluid. Microfluidic instruments are capable of performing amplification (the production of multiple copies of DNA or RNA) and analysis on a single sample. A microfluidic instrument may be used in the field by extracting or separating a sample, preferably a nucleic acid, from a fluid-based sample (either water-based or biological) loaded on a glass chip. The combination of portability, low cost, and high performance helps in the detection of disease-causing organisms and may also have the potential to make healthcare more accessible.
  • Microfluidic chips are made of glass, polymers (long chains of molecules), and fused silica called quartz. This technology uses a network of channels and wells that are etched onto the chips to build minilabs. The microfluidic chip contains one or more sample deposit areas, measuring devices, mixing chambers, fluid channels (to move the mixtures around), and reaction chambers that are temperature-controlled. The microchannels are manufactured to different sizes, enabling the fluid to flow from one location to another. Capillary forces and electrophoresis are used to pump the fluids from one location on the chip to another. Capillary force is the ability of a substance to draw another substance into it and electrophoresis is a technique that uses electrical current to separate and analyze proteins and DNA by electrical charge. The LabChip® 90 system (by Caliper Life Sciences, Massachusetts) is an example of microfluidic chip technology.
  • Advantages: Some of the advantages of using the lab-on-a-chip platforms include simplified handling, fast and accurate results, reduced consumption of the sample and the reagents (chemical solutions) with minimal waste production, and low production cost and instrument size. Since only small amounts of reagents are required, it helps reduce cost. Non-skilled people may also perform the complex tests. Also, some reaction products may be harmful to humans; lab-on-a-chip devices reduce human interaction, thereby decreasing the harmful effects of the reagent products. When LOCs are produced in large quantities, it may be cost-effective. They are fabricated using nanotechnology, which is the field of research that deals with the engineering and creation of things from materials that are smaller than 100 nanometers (one billionth of a meter) in size, especially single atoms or molecules.

Research
  • General: Nucleic acids are deoxyribose nucleic acids (DNA) and ribonucleic acids (RNA). Nucleic acid-based testing (NABT) is an analysis technique that is based on detection, amplification (production of multiple copies), and/or analysis of DNA and RNA.
  • Handheld fluorometric instrument: Researchers have developed a heated fluorometric instrument for performing real-time isothermal (constant temperature) nucleic acid amplification and detection. A digital fluorometer measures the fluorescence (illumination) of a specimen in an automatic manner. The instrument includes a detection chamber, an LED (light-emitting diode) light source, and a photo-detector (a light-sensing device). The instrument enables an inexpensive, compact approach to in-the-field screening of disease-causing organisms.
  • Electronic health records and telemedicine: NABT in point-of-care testing (POCT) has being linked with laboratory information system and electronic medical records. POCT means tests that are designed to be used at or near the site where the patient is located. Electronic medical records involve gathering, recording, and communicating structured andcoded data into an understandable electronic format. Results should be available immediately through electronic medical records and may be shared instantaneously with all members of the medical team through telecommunication links, which is crucial in improving healthcare. This may help in goal-directed therapy and thereby reduce morbidity (the incidence of a particular disease or disorder in a population) as well as mortality (death due to a particular disease).

Implications
  • General: Nucleic acids are deoxyribose nucleic acids (DNA) and ribonucleic acids (RNA). DNA is a long, thread-like (double-helix) molecule made up of large numbers of nucleotides. Nucleotides are the building blocks of DNA and are made of nitrogen bases, sugars, and phosphate. Nitrogen bases are of two types: purines, such as adenine (A) and guanine (G), and pyrimidines, such as cytosine (C) and thymine (T). Long strands of nucleotides form nucleic acids. The sequence of bases in DNA serves as the carrier of genetic (hereditary) information in the form of genes. RNA is a nucleic acid that aids in protein synthesis, which is important for the growth and maintenance of the body. RNA is formed under the direction of DNA, and both help to form amino acids, which are the building blocks of protein. Genes provide instructions for all cells in the body and are passed from parents to their children as genetic characteristics.
  • Nucleic acid-based testing (NABT) is an analysis technique that is based on detection, amplification (production of multiple copies) and analysis of DNA and RNA. Point-of-care testing (POCT) refers to tests that are used at or near the site where the patient is located.
  • Detection and Identification of pathogenic organisms: NABT at the point of care (POC) help detect and identify disease-causing organisms. For example, a handheld nucleic acid based analyzer has been used to detect Yersinia pestis, the causative agent of plague, which is a fatal bacterial infection that spreads by droplets in the air. Another example where a handheld device based on NABT has been used is the detection and identification of the virus causing severe acute respiratory syndrome (SARS). SARS is severe and fatal lung infection that spreads rapidly and extensively when exposed in a particular area or population (known as an epidemic outbreak). Hence, NABT-based POCT devices have found extensive application in identifying these pathogenic organisms, facilitating the initiation of quick treatment for affected individuals and helping to control the epidemic outbreak.
  • Genetic markers: A genetic marker is a specific sequence of a DNA or RNA that may be altered (mutated) and associated with a specific disease or disorder. NABT in POCT has been used to identify some genetic markers, which may help in the detection and/or diagnosis of a disease, monitoring the course of a disease, or indicating the disease stage. Some examples include mutations (alterations in the sequence of a gene) used as genetic markers, e.g., those associated with increased susceptibility to breast, intestinal, and skin cancers. These genetic markers can be analyzed and quantitated (counted) from very minimal tissue material or tissue extracts, thereby creating new possibilities for monitoring cancer treatment and therapy.
  • Detection of biological weapons: Handheld nucleic acid devices have been used successfully to identify disease-causing organisms such as Bacillus anthracis Ames, a bacterium that causes inhalational anthrax. Inhalation anthrax is a form of anthrax in humans resulting from inhalation of (breathing in) anthrax bacteria, which initially presents with flu-like symptoms. This is followed by the involvement of the lungs and brain, which is most often fatal. Individuals may be exposed to anthrax when it is used as a biological weapon. According to the U.S. Centers for Disease Control and Prevention (CDC), anthrax spores were deliberately spread in powder-containing envelopes through the U.S. postal system in 2001, causing widespread panic. Hence, handheld nucleic acid analyzers have the potential to detect a pathogen in the case of bioterrorism attack, thereby facilitating immediate treatment.
  • Identification of plant pathogens: Portable nucleic acid analysis devices may be used to detect certain pathogens in plants, e.g., Erwinia herbicola, a bacterium growing on many plants. Certain strains of Erwinia are known to cause galls, or tumor-like structures, in plants such as gypsophila. Hence, early identification of pathogens using nucleic acid analyzers may help prevent the disease from affecting the plants and spreading to other plants.
  • Agriculture: The handheld analyzer helps in the detection of bacterial pathogens in water, air, or treated manure. It facilitates the investigation of the colonization of pathogens by dissemination (being spread) in manure, thereby helping to prevent the infection of plants. The analyzer also has the capacity to detect evolved organisms that may have become resistant to some pesticides used to treat the plants. As a result, the handheld analyzer may assist in selecting a different treatment or control method for such organisms.
  • Identification of pathogens affecting the environment: The handheld nucleic acid sequence-based analyzer has been used for the field detection and quantification (counting) of Karenia brevis, a microscopic, single-celled organism (algae) that may cause ''algal bloom'' in coastal waters. Algal bloom is commonly known as ''red tide,'' in which algae accumulates rapidly and forms dense visible patches near the water's surface, leading to discoloration of the water. This phenomenon causes a lot of problems for coastal communities and leads to fish and mammal deaths. Also, tourism and fisheries are affected by the accumulation of algae. Handheld NABT analyzers have been a valuable tool for monitoring Karenia brevis in field environments, thereby ensuring better harvests of fishes for human consumption.
  • Forensic marker: NABT analysis helps in the definitive identification of human remains. This test may assist in criminal cases because it may provide evidence about the identity of crime victims and crime perpetrators who leave any biological material, such as hair shafts, at crime scenes.

Limitations
  • Nucleic acids are deoxyribose nucleic acids (DNA) and ribonucleic acids (RNA). Nucleic acid-based testing (NABT) is an analysis technique that is based on the detection, amplification (production of multiple copies), and/or analysis of DNA and RNA. Handheld nucleic acid analyzers are NABT devices that have limited target identification because the analyzer provides little information about the presence of multiple bacterial species within a genus or whether the bacteria are in their growth phase or not. Hence, the system may not be able to indicate much about the replication of the bacteria, and thereby may not help determine the infective nature (virulence) of the disease or infection.
  • The operator of the handheld nucleic acid analyzers must have an idea of the disease-causing organism (pathogen) involved and then decide the kind of pathogens that need to be tested because each DNA probe is designed to target a specific organism. Hence, the drawback is the need for a skilled operator; it would be trial and error to try to find the pathogen if the operator does not have an idea about the possible cause of infection. This may be limiting if a very small amount of sample is available.
  • Point-of-care testing (POCT) equipment should be linked to the laboratory information system and constantly monitored for performance and quality, so as to give reliable and accurate results.

Safety




Future research
  • Detection of Aspergillus: Researchers are conducting studies to diagnose invasive aspergillosis, a fungal infection caused by the Aspergillus fungus, using a nucleic acid-based testing (NABT) device. This fungal infection is difficult to detect despite advances made in imaging and laboratory tests. Further research is required to verify the accuracy of the NABT analyzer in the identification of the fungus. NABT is an analysis technique based on the detection, amplification (production of multiple copies), and/or analysis of DNA and RNA.

Author information
  • This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).

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The information in this monograph is intended for informational purposes only, and is meant to help users better understand health concerns. Information is based on review of scientific research data, historical practice patterns, and clinical experience. This information should not be interpreted as specific medical advice. Users should consult with a qualified healthcare provider for specific questions regarding therapies, diagnosis and/or health conditions, prior to making therapeutic decisions.

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