PCR assays can be both qualitative and quantitative, depending on the method used. Qualitative PCR, often referred to as conventional PCR, detects the presence or absence of a specific DNA sequence. In contrast, quantitative PCR (qPCR or real-time PCR) measures the amount of DNA, providing information on the quantity of the target sequence in a sample. Thus, PCR can serve both purposes based on the specific assay design.
When sampling for pathogens, it's essential to test for specific microorganisms such as bacteria, viruses, and fungi that can cause disease. Common targets include pathogens like Salmonella, E. coli, Listeria, and various viruses depending on the source (e.g., food, water, or clinical samples). The testing process typically involves using culture methods, molecular techniques like PCR, or immunological assays to detect and quantify these pathogens effectively. Additionally, assessing the sample's environmental conditions and potential contamination sources is crucial for accurate results.
To quantify a virus in a sample, techniques such as quantitative PCR (qPCR) can be employed, which measures the amount of viral genetic material present. Another common method is plaque assay, where viral particles are diluted and added to a cell culture, and the number of plaques formed indicates viral concentration. Additionally, techniques like ELISA can measure viral proteins, providing another means of quantification. Each method has its own sensitivity and specificity, depending on the virus and sample type.
Its a technique adopted during DNA sequencing in which while joining the contigs we find whether there is any sequence present in between the contigs or not.If there is a sequence inbetween they re sequence the ends of the DNA.If not PCR is used to close the gaps.
Certainly rt-PCR is qualitative and can also theoretically be quantitative. Anneal the RNA to get a 1:1 RNA to DNA copy, then proceed with quantitative PCR.
In qualitative PCR specific DNA fragment is detected while in quantitative PCR our target DNA sequence not only is detected but its amount is determined (after reaction we can calculate the amount of DNA we had in our sample)
: Differentiate between quantitative and real time PCR.
Quantitative PCR Technology is used in biochemistry, in particular molecular biology. The PCR stands for polymerase chain reaction and is used to "amplify" pieces of DNA to make millions of copies of a particular DNA strand.
Molecular assays are laboratory techniques that detect and analyze the genetic material (DNA or RNA) of organisms. These assays are used to identify specific genes, mutations, or pathogens, and are widely used in research, diagnostics, and pharmaceutical development. Examples of molecular assays include polymerase chain reaction (PCR), next-generation sequencing (NGS), and hybridization assays.
Real-time PCR, also known as quantitative PCR (qPCR), has been around since the mid-1990s. It gained popularity for its ability to monitor the amplification of DNA during the PCR process in real time, providing quantitative data on DNA or RNA targets.
Quantitative real-time PCR for Hepatitis B Virus (HBV) measures the amount of viral DNA in a blood sample. This test is used to monitor the levels of HBV in patients undergoing treatment and to assess disease progression and response to therapy. It helps healthcare providers determine the stage of infection and make treatment decisions.
PCR is a biotechnological method to amplify your gene (DNA) of your interest. It produce millions of your DNA fragments hence used in cloning. There are variants of this method using the same thermocycling principle such as touch down PCR, gradient PCR, RFLP, multiplex PCR, Q PCR, RT PCR and so on.
PCR allows amplification of DNA for a specific gene, after too many cycles of PCR the result will reach saturation, basically meaning all of the DNA has been amplified. Conventional PCR will basically tell you whether or not a gene is expressed in your sample. This can be done semi-quantitavely if the PCR is performed for a low number of cycles, ie it will tell you whether one sample expresses more of your gene of interest than another sample. The results are seen by separating the PCR products by agarose gel/ethidium bromide electrophoresis. Real-time PCR will record exactly what cycle of PCR a detectable level of amplified product became detectable, giving a far more accurately quantifiable estimation of gene expression.
You would employ reverse transcription PCR (RT-PCR) to amplify a desired gene from an RNA virus like human immunodeficiency virus (HIV) because RT-PCR can convert the RNA into complementary DNA (cDNA) before amplification, making it suitable for RNA viruses. To monitor disease progression and therapy in HIV, you would measure viral load by quantifying the amount of viral RNA in the blood using techniques like quantitative PCR or real-time RT-PCR. Additionally, you could monitor immune function by measuring CD4 cell counts to assess the impact of antiretroviral therapy and disease progression.
There are several advantages of using real-time PCR over other methods. Real-time PCR assays are thousand folds more sensitive than RNase protection assays or dot blot hybridization. It allows you to quite precisely calculate and compare of the amount of template in each cycle, instead of determining the amount of product at the end of the reaction. Quantitative RT-PCR is commonly used for clinical applications. For example, you could use this method to quantify the amount of HIV RNA particles per ml of blood plasma in a patient who is undergoing treatment with antiviral drugs to see if it's working or not. The main problem with real-time PCR is that it requires specialised thermal cyclers (PCR machine) with fluorescence monitors and its reagents are quite expensive.
TaqMan Gene Expression Assays consist of a pair of unlabeled PCR primers and a TaqMan probe with a FAM or VIC dye label on the 5' end, and minor groove binder (MGB) nonfluorescent quencher (NFQ) on the 3' end.