Monday, November 25, 2013

PCR: 30 Years Young and Still Going Strong

The polymerase chain reaction (PCR) is a molecular biology method that exponentially amplifies very small quantities of DNA (e.g., one or a few copies). The result is millions of copies of a specific DNA sequence that can be used to detect the presence of a specific microorganism or pathogen. The method was direst developed 30 years ago (1983) by Kary Mullis. I came across a nice review detailing the advancements of PCR over the past 30 years in Genetic Engineering & Biotechnology News. Below is an excerpt from this article.

Novel techniques, automated machines, sophisticated synthetic and bioprospected polymerases, and developments in symbiotic sequencing technologies have transformed PCR into a multifaceted, adaptable molecular tool.

Such advancements, however, have tethered PCR to expensive laboratories, requiring ever more specialized equipment and technicians. Looking to the future, improvements in PCR technologies will simplify and speed up the process, reduce costs, and enable miniaturization of PCR devices, taking diagnostics out of the lab and placing it firmly at the point of care (POC).

PCR is increasingly used to interrogate the genomes of humans, animals, bacteria, and viruses in different ways. Though the process has long been routinely used for detection of infectious diseases such as malaria and flu, quantitative PCR (qPCR) and digital PCR (dPCR) have significantly increased the sensitivity and precision of such tests.

The ability to identify single nucleotide polymorphisms (SNPs) or regions of DNA associated with infectious disease drug resistance is crucial to effectively treating diseases, and thereby preventing further spread within the population, or overprescribing therapeutics that will have no benefit.

The development of dPCR has seen advances in the precision and sensitivity of PCR for absolute quantification of nucleic acid targets, key capabilities for the detection and treatment of food-borne pathogens such as salmonella and E. coli, as well as rare mutations such as those seen in cancerous cells.

Neoplastic cells have been successfully detected in the sputum, urine, and stool of patients with lung, bladder, and colon cancer respectively, and circulating tumor cells have been shown to be a useful biomarker for metastatic breast cancer.

In addition, the development of dPCR as a noninvasive prenatal testing (NIPT) diagnostic using maternal blood is a promising focus of current research.

High-Quality Diagnostics

Though PCR is now used for a multitude of diagnostic applications, from infectious diseases to NIPT, these tests are costly, and it can take weeks to return results to the patient. As technology moves forward, and more complex automated devices allow for real-time results with higher specificity and resolution, affordability and accessibility are expected to improve. But such gains have yet to materialize.

As the cost of standard polymerases falls, new, high-priced PCR machines, which require costly reagents and polymerases, are incrementally introduced to maintain the market share of nextgen technologies.

Rather than allowing more accessibility, these new PCR machines require increasingly well-stocked labs and well-trained staff. The infrastructure and skill levels required prohibit the use of such tests outside the developed world. Reversing this trend requires new thinking and disruptive, game-changing technology.

Recent advances in microfluidics now allow for POC PCR. This technology will make PCR-based diagnostic testing accessible in various settings worldwide, especially in remote, underserved communities where the detection of drug resistance markers for infectious diseases could revolutionize healthcare provision.

Microfluidics and PCR

Microfluidic techniques have developed from the advent of dPCR, which relies upon the ability to generate small-reaction volumes in an accurate and repeatable manner. Single-molecule isolation technologies have shown promise of late, with Quanterix utilizing single-molecule array technology to produce an extremely sensitive assay.

Innovative researchers have been applying microfluidics techniques to PCR, with the recent production of thermal cyclers utilizing microwells, capillaries, and microdroplet formation. Companies such as RainDance Technologies and QuantaLife provide extremely high levels of sensitivity with their microdroplet PCR devices. Further microfluidic advancement is pivotal to future advances in amplification techniques and nucleic acid detection.

Microfluidics technology increases the speed of PCR by orders of magnitude. Speed is only theoretically limited by the processivity of the polymerase and the length of the region to be amplified.

In reality, the limiting factor is often the time it takes to cycle the reaction mixture through the reaction temperatures, so-called ramping. Ramping in conventional PCR machines is time- and energy-intensive due to the thermal mass of the block and is not readily made portable.

Improvements in buffer systems and engineered enzymes have increased the speed of standard benchtop thermal cyclers. Typically, however, these methods still take over 90 minutes, with 40 minutes being considered fast. This is far too long to be practical in a clinical POC setting.

Very few commercially available devices are capable of truly rapid PCR, none of which can be considered portable. BJS Technologies, though it doesn’t utilize true microfluidics technology, has developed the xxpress benchtop microwell thermal cycler. This device is capable of 40 cycles in just 10 minutes because it has a ramp rate of 10°C per second.

Microfluidic PCR systems fall into two categories—microwell reactors and continuous flow. Microwell thermal cyclers are essentially miniaturized versions of traditional PCR machines, reducing the input and chamber volumes such that the time it takes to heat and cool the reaction mix is likewise reduced.

Roche’s LightCycler holds PCR mix within capillaries, thereby increasing the surface-to-volume ratio and reducing the cycle time. Despite a runtime of approximately 30 minutes, the LightCycler technology has been commercially successful.

Continuous flow devices, on the other hand, require heating zones of different temperatures, over or between which the reaction mixture is passed in microfluidic channels. This lab-on-chip approach greatly reduces energy consumption, as once heat has been added to the system it only has to be maintained and not ramped. Provided there is good thermal contact between the flowing reaction mixture and the heaters, extremely rapid PCR can be achieved.

With microfluidic channels, surface area is increased and the time to reach thermal equilibrium at any given temperature zone becomes fractions of seconds. This equates to rapid ramping. An additional benefit of continuous flow devices is that they utilize disposable plastic microfluidics cassettes in lieu of large batch processing machines, lowering the cost per test and eliminating the possibility of cross-contamination between samples.

The increased surface-to-volume ratio in microfluidic PCR is not without its challenges. Polymerase can become denatured on the walls of microfluidic channels, and primer concentration often has to be increased for the same reasons. Inhibition can be overcome by careful choice of materials and utilizing surface chemistry to increase hydrophobicity.

Alternatively, active or passive passivation layers can be used to decrease the high adsorption rate of reagents by the materials. Pumping mechanisms and channel dimensions also require careful consideration because of their effect on laminar flow and dispersion. Research teams worldwide are optimizing continuous flow PCR, with advances in materials, heating and cooling systems, and connectors, all required to provide more efficient integrated systems.

Continuous flow PCR was born out of capillary electrophoresis in the early 1990s. It was pioneered by Andreas Manz, who has been central in the early development of microchip devices for chemical and biological analysis. However, during this time it was not commercialized.

More recently, academic labs, such as that of Niel Crews, have further developed the early iterations of continuous flow PCR, although the majority of these devices have only been used in the research setting. Thermal Gradient has produced a commercially available continuous flow PCR device capable of sub-10 minute runtime by pumping PCR reaction mix through a sandwich of two or three heating zones.

The single-use devices are intended for integration into POC devices. While rapid PCR would save time and money for researchers, it could also save lives if put into the hands of healthcare workers as part of a POC diagnostic test.

Continuous flow PCR is ideal for POC applications with amplification of DNA possible in just a few minutes, microfluidic PCR opens up the possibility for rapid diagnostic testing. When combined with automated sample preparation and DNA detection technologies, such as QuantuMDx’s Q-POC device, the development of which also benefits from microfluidics engineering, a portable, fully integrated POC diagnostic device could extend MDx to resource-limited settings.

Continuous flow PCR, with low power, sample size, and reagent volume requirements, is ideal for inclusion in a handheld device, which will be produced at much lower cost than traditional PCR machines due to the lack of robotics. Per-test costs also will be lower due to the much smaller volumes of reagents required.

Moreover, due to the speed of of amplification the speed of molecular analysis will be improved, and with assay times in the 10-minute range, this makes it relevant to in-field or in-clinic MDx testing.

The resulting sample-to-result device will not only be less costly than traditional PCR machines but significantly easier to operate, negating the need for well-stocked laboratories and highly skilled technicians. Such a technology leap could provide resource-scarce settings with access to the high-quality PCR-based diagnostic testing that is available in the developed world.

Providing diagnostic results in minutes rather than days, weeks, or months could revolutionize healthcare worldwide by surmounting many of the obstacles associated with traditional healthcare provision models in the developing world.

Lack of reliable electricity supply, clean water, transportation (particularly cold chain transportation for reagents), and highly skilled technicians are all hurdles that could be overcome by a simple POC test. Such a test would make complex diagnostics affordable and accessible, and could improve outcomes through early detection and treatment of disease.

Advancements in speed, accuracy, and cost control will open up developed and developing healthcare economies to the advantages of state-of-the-art PCR and related diagnostic technologies, including reduced test costs as healthcare costs skyrocket, reduced waiting times as hospitals overflow, and improved accuracy for priority diagnostics.

This will aid in diagnosis and treatment of patients worldwide, whether for a drug-resistant infectious disease, cancer, or a genetic condition. The development of such cutting-edge diagnostics is dependent on the creation of novel rapid microfluidic PCR methods, and the resulting technologies are now set to change the basis of genetic diagnosis.

Source: Genetic Engineering & Biotechnology News

Thursday, November 21, 2013

Rapid Detection of Superbugs

A new lab test that detects antibiotic resistance genes quickly could help doctors choose the right drugs to knock out superbugs.

Patients affected by a bacterial infection can usually be treated with an antibiotic. But sometimes a resistant bacterial strain is causing the infection. In a hospital setting, doctors ideally want to know if they are dealing with such bacteria and which drugs they should choose. But if the doctor runs a test it can take days to get a result. Now, a European project is paving the way for much more rapid tests using DNA biochips. The aim is to rapidly screen disease-causing bacteria using a microarray to spot which resistant genes are present in bacteria.

The scientists, as part of the Antiresdev project, developed an array that could test for 116 antibiotic resistant genes from one class of bacteria, and 90 resistant genes from the other class of bacteria. The "arrays" contain pits with a DNA probe that lights up if a specific gene is present.

It is important to search for many resistance genes because bacteria have a habit of exchanging bits of DNA that make for useful anti-drug defences, making the bacteria resistant or even untreatable. This is bad news for patients infected with a superbug. "A lot of these resistance genes are on mobile elements. They can transfer all sorts of different resistant genes if they are put under the right pressures," explains project scientist Peter Mullany, who is also a molecular microbiologist at University College London, UK. He adds: "There are lots of resistance genes too, with at least 30 for tetracycline [an antibiotic] alone."

Quick detection technologies could therefore provide a diagnostic without delay to help direct the therapeutic choice. "The chip arrays are rapid and will tell us if any of the resistance genes on the array are present in a particular environment. If resistance genes are present, which allow the bacteria to resist a clinically important antibiotic, clinicians may choose to use an alternative antibiotic to which no resistance genes are present," Mullany tells

The team also discovered a new genetic fragment that gives bacteria an ability to resist an antibiotic called minocycline and an antiseptic for wounds called cetrimide bromide. Identifying the fragment's presence could allow doctors to take steps preventing its spread to harmful bacteria.

Until now, the DNA biochips were used to investigate how various antibiotics influenced the kinds of resistant bacteria that were present. It also helped determine which persisted at various sites in the human body – mouth, skin and nose. This gave insight into how antibiotics affect 'friendly' microorganisms naturally present in our bodies. "There are a lot of unknowns in this area, as the majority of microorganisms can't be cultured," says Mullany. "But if you can get rapid knowledge about which antibiotics genes are present, you have a better chance that treatment will be successful."

Experts welcome this development. Microarray allows the simultaneous detection of a large number of genes in the bacterial cell in one test. "The result which is obtained is a complex pattern of spots or dots, representing the individual resistance genes, which is read by machine and tells us which genes are present," explains Chris Teale, head of antimicrobial resistance at the Animal Health and Veterinary Laboratories Agency in the UK, "This technique is a great benefit when screening isolates for resistance because so many different genes can be screened at the same time."

The threat of antibiotic resistant pathogens is global, notes Richard Goering professor of medical microbiology and immunology at Creighton University, Omaha, Nebraska, USA, whose research deals with antibiotic resistance genes in Methicillin-resistant Staphylococcus aureus (MRSA). "The issue in dealing with these is two-fold, diagnosis and treatment. There are long standing worldwide problems with slowdown in pharma research and development for new drugs but still the bottom line is that the sooner the diagnosis is made the better the potential therapeutic outcome," Goering explains.

Quick detection is therefore a positive step. "So, genomics and the ability to rapidly sequence bacterial chromosomes holds great promise in identifying bacterial genes associated with accurate detection of both the bug and what is it susceptible to treatment," Goering tells
However, more work lies ahead. "More such tests are needed especially for use in resource-poor regions where morbidity and mortality associated with infectious disease are always high," he adds, concluding that just because a gene is present, does not mean it is actively expressed, and therefore that molecular techniques will need to address this issue too.


Monday, November 18, 2013

Rapid Testing to Diagnose Influenza Leads to More Appropriate Care in the Emergency Department

When patients in the emergency department (ED) are diagnosed with influenza by means of a rapid test, they get fewer unnecessary antibiotics, are prescribed antiviral medications more frequently, and have fewer additional lab tests compared to patients diagnosed with influenza without testing, according to a new study. Published online in the Journal of the Pediatrics Infectious Diseases Society, the findings suggest that diagnosing influenza with a rapid diagnostic test leads to more appropriate, specific, and efficient care.

In the study, researchers used data from the National Hospital Ambulatory Medical Care Survey, a nationally representative sample of ED visits in the U.S. They identified children and adults across three influenza seasons (2007-2009) who were diagnosed with influenza in the ED. They looked at how the patients were diagnosed—either with the use of a rapid influenza test or without it—and the subsequent care they received.

Among patients diagnosed with influenza without rapid testing, 23 percent of the ED visits included a prescription for antibiotics, which are not effective in treating influenza, a viral infection. However, for patients who were diagnosed by rapid testing, only 11 percent of ED visits resulted in the patient getting antibiotics. Additional laboratory tests, including chest X-rays, blood tests, and urinalysis, were also ordered less frequently for patients whose influenza illness was diagnosed with a rapid test.

Notably, prescriptions for antiviral drugs, which can be effective in treating influenza when used early and appropriately, were more frequent (56 percent of ED visits) among patients diagnosed with influenza using a rapid test, compared to antiviral use among influenza patients diagnosed without testing (19 percent of ED visits).

"When results of influenza tests are available to physicians at the 'point of care,' they use this information to provide more appropriate patient management," said lead study author Anne J. Blaschke, MD, PhD, of the University of Utah School of Medicine. "While other studies have shown that physicians can accurately diagnose influenza without testing, our results suggest that using an influenza test increases diagnostic certainty and leads to the physician providing more specific and appropriate care."

The study suggests a significant impact from rapid influenza testing on physician decision making, patient care, and use of health care resources, the authors wrote, despite the limited sensitivity of currently available rapid tests, which miss a number of true cases of influenza. The development of more accurate and faster tests for influenza available at the bedside could further improve care for patients with influenza or other respiratory illness, they noted.

The researchers' findings build on previous studies by others, focused primarily on children, that found that rapid influenza testing can influence patient care in specific settings. This latest study breaks new ground, Dr. Blaschke said, by using nationwide data and by demonstrating that the findings apply to both adults and children, and across different practice types.

Source: Pediatric Infectious Diseases Society

Purdue U. Device Speeds Concentration Step in Food-Pathogen Detection

Researchers have developed a system that concentrates foodborne salmonella and other pathogens faster than conventional methods by using hollow thread-like fibers that filter out the cells, representing a potential new tool for speedier detection.

The machine, called a continuous cell concentration device, could make it possible to routinely analyze food or water samples to screen for pathogens within a single work shift at food processing plants.

"This approach begins to address the critical need for the food industry for detecting food pathogens within six hours or less," said Michael Ladisch, a distinguished professor of agricultural and biological engineering at Purdue University. "Ideally, you want to detect foodborne pathogens in one work shift, from start to finish, which means extracting the sample, concentrating the cells and detection."

A report from the Centers for Disease Control and Prevention (CDC) indicates a lack of recent progress in reducing foodborne infections and highlights the need for improved prevention. Although many foodborne illnesses have declined in the past 15 years, the number of laboratory-confirmed salmonella cases did not change significantly in 2012 compared with 2006 to 2008.

The first step in detecting foodborne pathogens is concentrating the number of cells in test samples. The new system enables researchers to carry out the concentration step within one hour, compared to a day for the standard method now in commercial use, said Ladisch, also a professor of biomedical engineering and director of Purdue's Laboratory of Renewable Resources Engineering (LORRE)

Findings are detailed in a research paper to appear in November in the journal Applied and Environmental Microbiology.

The paper was authored by doctoral student Xuan Li; LORRE research scientist Eduardo Ximenes; postdoctoral research associate Mary Anne Roshni Amalaradjou; undergraduate student Hunter B. Vibbert; senior research engineer Kirk Foster; engineering resources manager Jim Jones; microbiologist Xingya Liu; Arun K. Bhunia, a professor of food microbiology; and Ladisch.

Findings showed the system was able to concentrate inoculated salmonella by 500 to 1,000 times the original concentration in test samples. This level of concentration is required for accurate detection. Another finding showed the system recovered 70 percent of the living pathogen cells in samples, Ladisch said.

"This is important because if you filter microorganisms and kill them in the process that's self-defeating," he said. "The goal is to find out how many living microorganisms are present."

The machine was used to concentrate cells in a sample of chicken meat. The sample is first broken down into the consistency of a milkshake and chemically pretreated to prevent the filtering membranes from clogging. The fluid is then passed through 12 hollow-fiber filters about 300 microns in diameter that are contained in a tube about the size of a cocktail straw. The filtering process continues until pathogens if present are concentrated enough to be detected.

The technique, developed by researchers from Purdue's colleges of Engineering and Agriculture, could be performed during food processing or vegetable washing before the products are shipped.

The U.S. Department of Agriculture will test the system, which is not yet ready for commercialization.

One feature that could make the machine practical for commercial application is that it can be quickly cleaned between uses. The tubes are flushed with sodium hydroxide and alcohol.

Purdue has filed a patent application for the concept.

The research is funded by the U.S. Department of Agriculture, Purdue's Agricultural Research Programs and Center for Food Safety Engineering, and the Department of Agricultural and Biological Engineering.

Above Picture: Purdue University doctoral students, from left, Xuan Li and Seockmo Ku operate a new system that concentrates foodborne salmonella and other pathogens faster than conventional methods, representing a potential new tool for speedier detection. The research is led by Michael Ladisch, center, a distinguished professor of agricultural and biological engineering. (Purdue University photo/Steven Yang). Click on picture to enlarge.


Rapid Sample Processing for Detection of Food-Borne Pathogens via Cross-Flow Microfiltration  

Xuan Li,a,b Eduardo Ximenes,a,b Mary Anne Roshni Amalaradjou,d Hunter B. Vibbert,a,e* Kirk Foster,c Jim Jones,c Xingya Liu,a,b, Arun K. Bhunia,d,f Michael R. Ladisch a,b,c

Laboratory of Renewable Resources Engineering,a Department of Agricultural and Biological Engineering,b Weldon School of Biomedical Engineering,c Department of Food Science,d Department of Chemistry,e Department of Comparative Pathobiology,f  Purdue University

This paper reports an approach to enable rapid concentration and recovery of bacterial cells from aqueous chicken homogenates as a preanalytical step of detection. This approach includes biochemical pretreatment and prefiltration of food samples and development of an automated cell concentration instrument based on cross-flow microfiltration. A polysulfone hollow-fiber membrane module having a nominal pore size of 0.2 um constitutes the core of the cell concentration instrument. The aqueous chicken homogenate samples were circulated within the cross-flow system achieving 500- to 1,000-fold concentration of inoculated Salmonella enterica serovar Enteritidis and naturally occurring microbiota with 70% recovery of viable cells as determined by plate counting and quantitative PCR (qPCR) within 35 to 45 min. These steps enabled 10 CFU/ml microorganisms in chicken homogenates or 102 CFU/g chicken to be quantified. Cleaning and sterilizing the instrument and membrane module by stepwise hydraulic and chemical cleaning (sodium hydroxide and ethanol) enabled reuse of the membrane 15 times before replacement. This approach begins to address the critical need for the food industry for detecting food pathogens within 6 h or less.

Source: Purdue University