qPCR in Infectious Disease Testing

Extension Real-time polymerase chain reaction (qPCR) has become one of the most widespread in vitro diagnostic techniques for evaluating and managing infectious diseases. In this infographic, we explore the discovery of qPCR, what happens during qPCR and examples where qPCR is used in infectious disease testing. The of Real-time polymerase chain reaction (qPCR) has become one of the most widespread in vitro diagnostic techniques for evaluating and managing infectious diseases. In this infographic, we explore the discovery of qPCR, what happens during qPCR and examples where qPCR is used in infectious disease testing. On the back of a receipt in 1983, American biochemist Kary Mullis detailed an idea of reproducing the DNA replication process that takes place in cells in a laboratory tube. The first description of PCR using Taq polymerase was published in Science in 1985. Mullis’ discovery earned him the Nobel Prize for Chemistry in 1993, with PCR becoming one of the most important scientific advances of the 20th century. has come a PCR is used to amplify a specific DNA fragment of interest from a template DNA. There are now varying methods of PCR that can be used for sample analysis. These include: PCR Sometimes called end-point PCR, the products are evaluated at the end of the amplification process, typically by agarose gel electrophoresis. Thus, traditional PCR is semi-quantitative compared to other methods. Measurements are taken at the end of each amplification cycle using fluorescent reporters. Thus, the actual amplification process itself is tracked rather than just the final product making it fully quantitative. qPCR Rather than DNA the starting material in RT-qPCR is RNA. A reverse transcription step is incorporated to convert the RNA to complementary DNA (cDNA) before the normal qPCR amplification RT-qPCR Samples are diluted and partitioned to the extent that single template molecules can be amplified. The products are detected using fluorescent probes. dqPCR What is needed for a DNA (to be amplified) DNA primers PCR Buffer Deoxynucleotide triphosphates (dNTPs) DNA polymerase Flurescence probes or dye From a practical perspective, a qPCR experiment is relatively straightforward and can be completed in a few hours. In general, a qPCR experiment involves: AKA a PCR template or template DNA Primers are short pieces of single-stranded DNA (approximately 15–30 bases) which are required by DNA polymerases to indicate where they need to initiate amplification. Enables detection of target amplification. For example, an intercalating dye (e.g. SYBR® Green) displays weak fluorescence in its unbound form, and a strong fluorescent signal when bound to doublestranded DNA. All PCR reactions require a DNA polymerase that can work at high temperatures. DNA polymerases are enzymes that create DNA molecules by assembling nucleotides Taq polymerase is commonly used. Serve as the building blocks to synthesize the new strands of DNA and include the four basic DNA nucleotides (dATP, dCTP, dGTP, and dTTP). Ensures that optimal conditions are maintained throughout the PCR reaction DNA template DNA primers Buffer solution Action of SYBR Green I Dye 1. Dye in solution emits low florescence 2. Emission of the flurescence by binding dNTPs DNA polymerase denaturation Extension annealing Template DNA is heated to 95 °C for a few seconds. DNA strands separate due to hydrogen bonds breaking between them. The temperature is raised to optimal temperature (~72 °C) for DNA polymerase. DNA polymerase binds to one end of each primer and synthesizes new strands of DNA, complementary to the template DNA. The fluorescence produced by the probes/dye is detected by the fluorimeter and recorded by the computer. The reaction mixture is cooled for 30 seconds to 1 minute. Annealing temperatures are usually 50–65 °C. The optimal temperature depends on the primers’ length and sequence. 5’ 3’ 3’ 5’ DNA 94-95°C 5’ 3’ 5’ 3’ 5’ 3’ DNA Polymerase 72°C 3’ since 1983 of On the back of a receipt in 1983, American biochemist Kary Mullis detailed an idea of reproducing the DNA replication process that takes place in cells in a laboratory tube. The first description of PCR using Taq polymerase was published in Science in 1985. Mullis’ discovery earned him the Nobel Prize for Chemistry in 1993, with PCR becoming one of the most important scientific advances of the 20th century. has come a since 1983 PCR is used to amplify a specific DNA fragment of interest from a template DNA. There are now varying methods of PCR that can be used for sample analysis. These include: Sometimes called end-point PCR, the products are PCR evaluated at the end of the amplification process, typically by agarose gel electrophoresis. Thus, traditional PCR is semi-quantitative compared to other methods. Measurements are taken at the end of each qPCR amplification cycle using fluorescent reporters. Thus, the actual amplification process itself is tracked rather than just the final product making it fully quantitative. RT-qPCR Rather than DNA the starting material in RT-qPCR is RNA. A reverse transcription step is incorporated to convert the RNA to complementary DNA (cDNA) before the normal qPCR amplificatio dqPCR Samples are diluted and partitioned to the extent that single template molecules can be amplified. The products are detected using fluorescent probes. What is needed for a From a practical perspective, a qPCR experiment is relatively straightforward and can be completed in a few hours. In general, a qPCR experiment involves: 1 2 DNA (to be amplified) DNA polymerase AKA a PCR template or All PCR reactions require a DNA template DNA polymerase that can work at high temperatures. DNA polymerases are enzymes that create DNA molecules by assembling nucleotides Taq polymerase is commonly used. DNA template DNA polymerase 3 4 DNA primers Primers are short pieces Deoxynucleotide of single-stranded DNA (approximately 15–30 triphosphates bases) which are required by DNA polymerases to (dNTPs) indicate where they need to initiate amplification Serve as the building blocks to synthesize the new strands of DNA and include the four basic DNA nucleotides (dATP, dCTP, dGTP, and dTTP). DNA primers dNTPs 5 6 Flurescence probes PCR or dye Buffer Ensures that Enables detection of target amplification optimal For example, an intercalating dye (e.g. conditions are SYBR® Green) displays weak fluorescenc R® maintained in its unbound form, and a strong throughout the fluorescent signal when bound to double PCR reaction stranded DNA. Action of SYBR Green I Dye 1. Dye in solution emits 2. Emission of the Buffer solution low florescenc flurescence by binding Standard overview denaturation 1 Template DNA is heated to 95 °C for a few seconds. DNA strands separate due to hydrogen bonds breaking between them. 5’ 3’ 94-95°C DNA 3 2 3’ 5’ The The temperature reaction is raised to optimal mixture is cooled temperature (~72 °C) for DNA for 30 seconds to 1 minute. polymerase. DNA polymerase binds to one Annealing temperatures are usually end of each primer and synthesizes new 50–65 °C. The optimal temperature depends strands of DNA, complementary to the on the primers’ length and sequence. template DNA. The fluorescence produced by the probes/dye is detected by the fluorimete and recorded by the computer. 5’ 3’ 3’ 5’ 3’ 3’ 72°C 50-65°C F DNA Primer Polymerase F 3’ 5’ 3’ 3’ 5’ 3’ 5’ The is then By repeating the cycle 30 times, the double-stranded DNA molecules present at the beginning are converted into over 130 million new double-stranded molecules, each a copy of the region of the starting molecule delineated by the annealing sites of the two primers. AMPLIFICATION OF A SPECIFIC DNA SEQUENCE DNA template THE DNA DNA Buer REACTION primers dNTPs polymerase solution THERMAL 3’ 5’ CYCLER Denaturation 5’ 3’ 25-40 cycles 5’ 3’ Annealing 5’ 3’ 3’ 5’ 5’ 3’ Elongation 3’ 5’ 3’ 5’ Products Cycle 1 Cycle 2 Cycle 3 Cycle 4 Using to identify Selecting the appropriate diagnostic techniques for Infectious diseases are a major the rapid identification of global cause of morbidity pathogens is crucial for clinical and mortality, seriously disease diagnosis and public affecting public health and health management. socioeconomic stability. qPCR has a well-established place in both infectious disease diagnostics of humans, animals and plants and in the detection of genetic markers, such as mutations. qPCR can also be used on environmental samples to help epidemiologists monitor and predict the spread of disease. Quantitative data can help estimate pathogenic load, potentially informing treatment or management decisions. Mycobacterium tuberculosis One of the earliest recognized applications of PCR for clinical practice was M. tuberculosis detection. Standard testing before qPCR was introduced often consisted of culture-based experiments. These techniques resulted in inherent delays in test results which, given how important early detection is for infected patients, was a significant drawback for these standard testing techniques. Resultingly, qPCR has significantl improved the time from obtaining a sample to receiving test results can be generated. SARS-CoV-2 One of the most recent examples of qPCR (specificall RT-qPCR) uses in infectious disease testing, was throughout the COVID-19 pandemic. RT-qPCR was used to convert RNA from a SARS-CoV-2 sample to its cDNA sequence. The DNA would then be amplified by qPCR. COVID-19 diagnostic test using RT-qPCR Technique 1 2 Nasopharyngeal Collected swab specimen Cotton swab is inserted into Specimen is stored at nostril to absorb secretions. 2-8ºC for up to 72 hours or proceeded for RNA extraction. 3 RNA extraction Purified RNA is extracted from deactivated virus. Deactivated SARS-CoV-2 Purified DNA 4 5 RT-qPCR Test results Purified RNA is reverse Positive SARS-CoV-2 transcribed to cDNA an patients cross the amplified by qPCR. threshold line with 40.00 cycles (<40.00 Ct). Positive Threshold Reverse - Transcription Negative Cycles Researchers estimate that RT-qPCR can detect as little as ~1,000 copies of viral RNA per milliliter or 10 copies per analytical limit of detection. This means diagnosing someone as COVID-19 positive is possible even when their sample contains a very small amount of virus. Other tests – such as the rapid antigen test – were also developed throughout the COVID-19 pandemic to aid with mass testing. Below is a comparison between RT-qPCR and antigen testing for SARS-CoV-2: Antigen RT-qPCR test Tests for genetic material (RNA) Tests for pathogen proteins Less rapid test results produced Rapid test results produced (a few days) (within minutes) Less convenient Highly convenient Highly specifi Reduced specificit Very low viral load needed High viral load needed Low occurrence of false False-negative results are more negative results common (i.e. there can be too few virus proteins present in the sample to register positive on the test) Benefits of in testing The use of qPCR allows for rapid triage and the administration of early aggressive targeted therapies, appropriate isolation of contagious patients and diminished antibiotic resistance. Patients with unrecognized or difficult-to-diagnose infections can be identified and treated more quickly with qPCR. Inpatient stays may reduce alongside reduced iatrogenic events. To fully understand the benefits of qPCR, further research is needed on the societal benefits of the technology with attention to the relative costs of novel diagnostics concerning existing standards and how this may contribute to reducing healthcare costs. long way experiment? experiment repeated... Infectious Disease Infectious Disease discovery nnealing Fluorescence