We have all been hearing a lot about RT-PCR, rRT-PCR and RT-qPCR lately, and for good reason. Real-Time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) is the technique used in by the Center for Disease Control (CDC) to test for COVID-19. Real-time RT-PCR, or quantitative RT-PCR (RT-qPCR)*, is a specialized PCR technique that visualizes amplification of the target sequesnce as it happens (in real time) and allows you to measure the amount of starting target material in your reaction. You can read more about the basics of this technique, and watch a webinar here. For more about RT-PCR for COVID-19 testing, read this blog.
Both qPCR and RT-qPCR are powerful tools for scientist to have at their disposal. These fundamental techniques are used to study biological process in a wide range of areas. Over the decades, Promega has supported researchers with RT-qPCR and qPCR reagents and systems to study everything from from diseases like COVID-19 and cancer to viruses in elephants and the circadian rhythm of krill.
Over the last few months we have published several blogs about qPCR—from basic pointers on avoiding contamination in these sensitive reactions to a collection of tips for successful qPCR. Today we look in depth at a paper that describes the design and and optimization of a qPCR assay, and in keeping with the season of winter in the Northern hemisphere, it is only fitting that the assay tests for the abundance and identity of ice-nucleating bacteria.
Ice-nucleating bacteria are gram-negative bacteria that occur in the environment and are able to “catalyze” the formation ice crystals at warmer temperatures because of the expression of specific, ice-nucleating proteins on their outer membrane. Ice-nucleating bacteria are found in abundance on crop plants, especially grains, and are estimated to cause one-billion dollars in crop damage from frost in the United States alone.
In addition to their abundance on crop plants, ice-nucleating bacteria are also found on natural vegetation and have been isolated from soil, snow, hail, cloud water, in the air above crops under dry conditions and during rain fall. They have even been isolated from soil, seedlings and snow in remote locations in Antarctica. For the bacteria, ice nucleation may be a method to promote dissemination through rain and snow.
Although ice-nucleating bacteria have been isolated from clouds, ice and rain, little is known about their true contribution to precipitation or other events such as glaciation. Are such bacteria the only source of warm-temperature (above temperatures at which ice crystals form without a catalyst) ice nucleation? Can they trigger precipitation directly? What are the factors that trigger their release from vegetation into the atmosphere? Can we determine their abundance and variety in the environment? Continue reading “Do you want to build a snowman? Developing and optimizing a qPCR assay to detect ice-nucleating activity”
In areas of the world where the electricity is intermittent, resources are limited and transporting bulky equipment and reagents that are sensitive to temperature fluctuations is difficult, diagnosis of viruses like dengue can be challenging. If you could reduce or eliminate the need for electricity dependent equipment for diagnostic assays without sacrificing sensitivity or specificity, it would be a boon to field workers. An article published in PLOS ONE describes how researchers developed a multiplex isothermal amplification method that could assess a potential dengue infection with a visual real-time or endpoint detection in a single tube. Continue reading “A Potential Single-Tube Multiplex Assay for Detecting Dengue Virus in the Field”
The polymerase chain reaction (PCR) has revolutionized modern biology as a quick and easy way to generate amazing amounts of genomic data. However, when PCR doesn’t work, it can be frustrating. At these times, PCR and reverse transcription PCR (RT-PCR) inhibitors seem to be everywhere: They lie dormant in your starting material and can co-purify with the template of interest, and they can be introduced during sample handling or reaction setup. The effects of these inhibitors can range from partial inhibition and underestimation of the target nucleic acid amount to complete amplification failure. What is a scientist to do?