Liat? analyzer
Related Terms
Algal bloom, bedside diagnostics, biodetectors, biological weapon, BioSeeq? device, bioterrorism detection, deoxyribonucleic acid, DNA, DNA probe, Escherichia coli, fluorometric instrument, HANAA, handheld advanced nucleic acid analyzer, Karenia brevis, lab-on-a-chip technology, LiatT analyzer, microfluidic instrument, NASBA analyzer, nucleic acid sequence-based amplification, nucleic acids, pathogens, PCR, polymerase chain reaction, Razor? system, red tide, reverse transcription, ribonucleic acid, RNA, thermocycler, Verigene?, Yersinia pestis.
Background
General: Genes are often called the building blocks of life. They are located inside cells and control an organism's development and functions by instructing cells to make new molecules (usually proteins). Proteins are organic compounds required for the growth and maintenance of the body. They are composed of amino acids, the sequence of which is defined by a gene.
DNA (deoxyribonucleic acid) is a long threadlike molecule made up of large numbers of nucleotides. The sequence of bases in DNA serves as the carrier of genetic, or hereditary, information. Nucleotides are molecules composed of a nitrogen-containing base, a five-carbon sugar, and one or more phosphate groups. Long strands of nucleotides form nucleic acids. RNA (ribonucleic acid) is a nucleic acid that is essential in protein synthesis. RNA is formed under the direction of DNA, and both nucleic acids are involved in forming amino acids.
Handheld nucleic acid analyzer: A handheld nucleic acid analyzer is a real-time polymerase chain reaction-based system used to detect disease-causing organisms. It can be used in settings such as the patient's bedside or in the field, such as a clinic or mobile medical unit, and it provides results within 30-60 minutes. Polymerase chain reaction (PCR) is an enzymatic method used to copy the two DNA strands of a particular gene sequence many times for laboratory study.
The nucleic acid analyzer was initially designed for emergency responders, such as firefighters and police, who are the first to arrive at sites where bioterrorism may have occurred. A bioterrorism attack is the deliberate release of viruses, bacteria, or other germs used to cause widespread illness or death in people, animals, or plants.
Since PCR was first used in the late 1980s, investigators have tried to expand the potential of this technology to that of on-site, "bedside" diagnostics, in which clinical decisions can be determined in real time for quick results, without the need to transport samples to a remote testing facility.
The requirement for a thermocycler, a large specialized piece of equipment used in PCR, was a limiting factor for use in certain areas, such as field testing and point-of-care diagnosis. A number of U.S. government-sponsored projects have succeeded in reducing the size of PCR equipment to a handheld format, thereby making it possible to determine the cause of infection or disease at the patient's bedside. This allows treatment to begin earlier and may prevent complications related to the infection or disease.
Some of the commercially available systems of handheld nucleic acid analyzer are the Razor? system from Idaho Technology Inc, Utah; the BioSeeq? device from Smiths Detection, Maryland; and the Verigene? system from Nanosphere, Illinois.
Methods
Handheld nucleic acid analyzers are based on polymerase chain reaction (PCR), an enzymatic method used to copy the two DNA or RNA strands of a particular gene sequence for laboratory study. Types of handheld nucleic acid analyzers include handheld the advanced nucleic acid analyzer (HANAA), the nucleic acid sequence-based amplification (NASBA) analyzer, and the LiatT analyzer.
Handheld advanced nucleic acid analyzer (HANAA):
General: HANAA was developed by the Lawrence Livermore National Laboratory in 1999. HANAA is the first handheld device that allows easy testing of blood and urine samples in the field, and it was used by the United Nations inspectors in Iraq during the search for biological weapons in 2003. HANAA is about the size of a brick, weighs less than one kilogram (2.2 pounds), and can be held in one hand. The analyzer is highly sensitive and can detect 200 organisms per milliliter of sample. 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 compared with routine laboratory tests that can take hours or days to produce results. Below are some of the steps involved for identifying or detecting disease-causing organisms using the handheld analyzer.
Sample preparation: Sample preparation involves placing the sample in a liquid buffer and adding chemicals to make it ready for measurement. A buffer is a chemical used to maintain the hydrogen-ion concentration (pH) by neutralizing within limits both acids and bases. Sample preparation is done to isolate the target cells by separating their nucleic acids. It also helps to remove compounds that might mask the analysis of the component of interest (e.g., bacteria or a virus).
Addition of a DNA probe: The next step is to attach a DNA probe with a fluorescent dye to the sample. DNA probes are single-stranded DNA sequence base pairs that are 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) with thymine (T), and guanine (G) with cytosine (C). For example, the complementary strand for G-T-A-C is C-A-T-G. The researcher performing this step must decide what kind of disease-causing organism is being tested because each DNA probe is designed to target a specific organism, such as anthrax or plague. If the organism is present in the sample, the probe attaches to its DNA and makes multiple copies using the PCR process. The probes are provided along with the kit.
Amplifying the component of interest: There may not be enough DNA extracted from the sample of interest (e.g. blood) for further use in several genetic analysis techniques. The PCR process used to make multiple copies of the sample's DNA involves repeated heating and cooling of the sample. Every time the DNA is heated, the two strands of DNA unwind and come apart. As the sample cools down, DNA makes a copy of itself. Therefore, after each heating-cooling cycle, the amount of DNA is doubled. The handheld analyzer measures the sample's fluorescence (glow) to detect the presence or absence of the targeted organism. 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 for detection or monitoring of microbial populations in the field or in clinical settings, thus facilitating diagnosis of an infection or disease. A sensor is a device designed to respond to physical stimuli such as temperature, light, magnetism, or movement, and to transmit resulting impulses for interpretation, recording, movement, or operating control. NASBA uses a stable temperature to make multiple copies of RNA. 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 rapidly perform analysis of samples and obtain results within minutes.
IQuum's LiatT analyzer:
The LiatT analyzer detects the DNA or RNA of viruses and bacteria and is useful for first responders or healthcare workers to conduct nucleic acid tests at a patient's bedside or in the field. All nucleic acid testing processes, including sample preparation, amplification using both PCR and reverse transcription-polymerase chain reaction (RT-PCR), and real-time detection are automatic in one portable device in the LiatT system. Results are available within 30-60 minutes. Reverse transcription is a process of DNA synthesis under the direction of RNA, and is the opposite of transcription in which RNA synthesis takes place under the direction of DNA. A wide variety of samples, such as whole blood, plasma (the liquid component of blood in which blood cells are suspended), and urine, can be used in the analyzer, which provides flexibility to the operator in choosing the sample.
Research
General: Research initiatives have made several commercial handheld instruments available to researchers, clinicians, and epidemiologists, people who study factors that influence the health of populations toward developing strategies to prevent illness. The handheld instruments are capable of rapidly performing polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR). Scientists are conducting studies to develop devices that detect target nucleic acids without the need for amplification, or the production of multiple copies. PCR is an efficient and sensitive laboratory technique to amplify a specific sequence of DNA. Reverse transcription is a process of DNA synthesis under the direction of RNA and is the opposite of transcription in which RNA synthesis takes place under the direction of DNA. Several studies are being conducted to improve the existing handheld nucleic acid analyzers.
Handheld fluorometric instrument: Researchers have developed a heated fluorometric instrument for performing real-time nucleic acid amplification and detection at stable temperatures (without the need for heating and cooling). The instrument includes a detection chamber, LED (light-emitting diode) light source, and photo-detector. A digital fluorometer automatically measures the fluorescence of a specimen. Several fluorescent dyes and probes have been designed for genetic tracing and identification of disease-causing organisms. The instrument enables an inexpensive, compact approach to onsite or bedside screening of disease-causing organisms.
Implications
Detection of biological weapons: The handheld advanced nucleic acid analyzer (HANAA) has been used successfully for the identification of disease-causing organisms such as Bacillus anthracis Ames, a bacterium that lives in a spore and causes anthrax by inhalation. According to the 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.
The handheld nucleic acid analyzer has the potential to detect a pathogen used in a bioterrorism attack, making it possible to deliver treatment immediately and avoid complications related to the infection. A bioterrorism attack is the deliberate release of viruses, bacteria, or other germs used to cause widespread illness or death in people, animals, or plants.
Identification of plant pathogens: HANAA may also be used to detect disease-causing organisms such as Erwinia herbicola, a bacterium that grows on many plants. Certain strains of Erwinia are known to cause galls, tumor-like structures, in plants such as gypsophila, a flowering plant (Gypsophila paniculata L.), and affect its capacity to produce flowers. Identification of pathogens using HANAA may help prevent the disease from affecting these plants.
Agriculture: The handheld analyzer aids in the detection of bacterial pathogens in water, air, or treated manure. Pathogens may spread in manure used as fertilizer, which may cause infection in humans who consume the plant or its produce. The handheld analyzer may help analyze the sample to check for multiplication of the pathogen and thereby prevent infection in plants and humans.
There was an outbreak of Salmonella Saintpaul infections in the United States beginning in April 2008. Most people became ill by May or June, after having eaten jalapeno peppers. Early evidence suggested that contamination of the produce at the farm or during processing may have been the cause of infection. Hence, additional measures such as the use of handheld analyzers are required to enhance food safety and prevent infections or illnesses from the consumption of raw produce.
The analyzer also has the potential to detect genetically modified organisms, foods that are altered genetically to enhance positive qualities or downplay negative ones, and that may have become resistant to some of the pesticides used to treat the plants. This allows researchers and clinicians to select different treatments for such organisms.
Identification of disease-causing organisms that affect the environment: The handheld nucleic acid sequence-based amplification (NASBA) analyzer has been used to detect and count the number of Karenia brevis, a microscopic single-celled organism that may cause "algal bloom," commonly known as red tide, in coastal waters. In algal bloom, the algae accumulate rapidly and form dense visible patches near the water surface, leading to the discoloration of the water. This phenomenon causes problems for affected coastal communities, including death of fish and mammals and negative effects on tourism and fisheries. The NASBA analyzer has been a valuable tool for monitoring Karenia brevis in field environments, thereby ensuring the safety of shellfish harvested for human consumption.
Identification of pathogenic organisms: The handheld nucleic acid analyzer can detect organisms such as Escherichia coli, a type of bacteria commonly found in the intestine of warm-blooded animals. Some strains of the bacteria may cause food poisoning in humans. The handheld analyzer has also helped detect Yersinia pestis, the agent that causes plague, a deadly bacterial infection that spreads by droplets in air. Hence, the analyzer assists in identifying disease-causing organisms and making possible quick and appropriate treatment.
Limitations
The handheld nucleic acid analyzers can identify only limited targets because the analyzer provides little information about the presence of multiple bacterial species within a genus or whether the bacteria are in a growth phase. The system may not be able to indicate whether bacteria are multiplying and therefore, may not provide information about the ability of the infection or disease to be transmitted to others.
The researcher using a handheld nucleic acid analyzer must have an idea of the disease-causing organism he or she is looking for prior to using the device because each DNA probe is designed to target a specific organism. If nothing is known about the disease-causing organism prior to testing, it would be an inefficient trial and error process.
Future research
Microfluidic system (lab-on a chip technology): Several studies are being conducted to improve the portable handheld nucleic acid analyzer. Miniaturization, or creating smaller versions of biodetectors in a single, integrated, "lab-on-a-chip" system, offers great potential for environmental monitoring. These options provide improved accuracy, lower power consumption, easy disposability, and automation features, and require only small quantities of a sample.
Scientists have presented an inexpensive, portable, and integrated instrument that is capable of producing and analyzing multiple copies of DNA or RNA on a single sample. The combination of portability, cost-effectiveness, and high performance makes for easy detection of disease-causing organisms and also has the potential to make healthcare more accessible.
Detection of Aspergillus: Researchers are conducting studies using the NASBA (nucleic acid sequence-based amplification) analyzer to diagnose invasive aspergillosis, a fungal infection caused by Aspergillus fungus. This fungal infection normally affects those with weakened immune systems and is difficult to detect despite advances made in imaging and laboratory tests. Further research is required to verify the accuracy of the NASBA analyzer in identifying the fungus.
Author information
This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).
Bibliography
Casper ET, Patterson SS, Bhanushali P, et al. A handheld NASBA analyzer for the field detection and quantification of Karenia brevis. Harmful Algae. 2007 Jan;6(1):112-8.
Higgins JA, Nasarabadi S, Karns JS, et al. A handheld real time thermal cycler for bacterial pathogen detection. Biosens Bioelectron. 2003 Aug 15;18(9):1115-23.
IQuum, Inc. .
Kaigala GV, Hoang VN, Stickel A, et al. An inexpensive and portable microchip-based platform for integrated RT-PCR and capillary electrophoresis. Analyst. 2008 Mar;133(3):331-8.
Lawrence Livermore National Laboratory. .
Natural Standard: The Authority on Integrative Medicine. .
Nizan-Koren R, Manulis S, Mor H, et al. The regulatory cascade that activates the Hrp regulon in Erwinia herbicola pv. gypsophilae. Mol Plant Microbe Interact. 2003 Mar;16(3):249-60.
Perlin DS, Zhao Y. Molecular diagnostic platforms for detecting Aspergillus. Med Mycol. 2008 Jun 12:1-10.
Smith MC, Steimle G, Ivanov S, et al. An integrated portable hand-held analyser for real-time isothermal nucleic acid amplification. Anal Chim Acta. 2007 Aug 29;598(2):286-94.
Schummer M, Ng W, Nelson PS, et al. Inexpensive handheld device for the construction of high-density nucleic acid arrays. Biotechniques. 1997 Dec;23(6):1087-92.
United States Department of Agriculture. .
University of South Florida. College of Marine Science. .