NOW OFFERING NEXT GENERATION DNA SEQUENCING
Molecular Engineering
Polymerase Chain Reaction (PCR) molecular assays are powerful tools used in molecular biology to amplify specific DNA sequences, making them easier to analyze. The creation of PCR assays involves several key steps:
- Target Identification: The first step is identifying the specific DNA sequence of interest, which could be a pathogen’s genetic material or a gene associated with a particular condition.
- Primer Design: Primers are short sequences of nucleotides that are complementary to the target DNA sequence. Two primers are designed for each assay—one for each strand of the DNA. The design must ensure specificity to avoid amplifying non-target sequences. Tools and software programs can assist in designing effective primers by analyzing potential secondary structures and ensuring they bind only to the intended target.
- Reagent Preparation: A PCR reaction requires several components:
- Template DNA: This is the sample containing the target sequence.
- DNA Polymerase: An enzyme that synthesizes new DNA strands.
- Deoxynucleotide Triphosphates (dNTPs): The building blocks for new DNA strands.
- Buffer Solution: Provides optimal conditions for the reaction, including pH and ionic strength.
- Primers: As previously mentioned, these initiate the amplification process.
- Thermal Cycling: The PCR process involves repeated cycles of heating and cooling:
- Denaturation (94-98°C): The double-stranded DNA melts open to single strands.
- Annealing (50-65°C): Primers bind to their complementary sequences on the single-stranded DNA.
- Extension (72°C): DNA polymerase extends the primers, synthesizing new strands of DNA.
- Optimization: Each PCR assay may require optimization of various parameters such as temperature, time, and concentrations of reagents to achieve maximum efficiency and specificity.
- Validation: After development, assays must be validated through rigorous testing against known standards and controls to ensure accuracy, reproducibility, and reliability.
Use in Diagnostics
PCR molecular assays have become indispensable in diagnostics due to their ability to detect minute quantities of nucleic acids from pathogens or genetic mutations associated with diseases:
- Pathogen Detection: PCR is widely used for detecting infectious agents like bacteria, viruses, and fungi in clinical samples (e.g., blood, urine, swabs). For example, RT-PCR (Reverse Transcription PCR) is employed for RNA viruses like SARS-CoV-2.
- Genetic Testing: These assays can identify genetic mutations linked to hereditary diseases or cancer predisposition by amplifying specific regions of genes known to harbor mutations.
- Forensic Analysis: In forensic science, PCR assists in amplifying trace amounts of DNA from crime scenes for identification purposes.
- Transplant Compatibility Testing: PCR can determine compatibility between organ donors and recipients by analyzing specific genetic markers.
- Monitoring Treatment Efficacy: In some cases, PCR is used to monitor viral load in patients undergoing treatment for infections like HIV or hepatitis C.
Sensitivity of PCR Assays
One of the most significant advantages of PCR is its high sensitivity:
- Detection Limitations: Standard PCR can detect as little as one copy of target DNA within a sample due to its exponential amplification nature; this makes it highly sensitive compared to traditional culture methods or antigen tests.
- Quantitative PCR (qPCR): This variant allows quantification of nucleic acids in real-time during amplification cycles using fluorescent dyes or probes, providing not just presence/absence data but also information on viral load or gene expression levels.
- Limitations & Challenges:
- While highly sensitive, false positives can occur due to contamination or non-specific amplification if proper controls are not implemented.
- Sensitivity may vary based on sample quality and handling; thus, stringent protocols must be followed during sample collection and processing.
In summary, PCR molecular assays are meticulously designed through a series of steps involving target identification, primer design, reagent preparation, thermal cycling optimization, and validation processes. Their application spans various diagnostic fields due to their high sensitivity and specificity in detecting pathogens and genetic abnormalities.
Menu / Price List
Molecular PCR Assays
With Direct Support from AxisBio LLC.
MOLECULAR 24T RESPIRATORY
28
Adenovirus, SARS-CoV-2 (COVID-19), Coronavirus (not COVID-19), Human metapneumovirus, Human rhinovirus/enterovirus, Multiple types of influenza A and B, Parainfluenza viruses 1–4, Respiratory syncytial virus A and B, Chlamydophila pneumoniae, Mycoplasma pneumoniae
MOLECULAR UTI
28
Gram Positive Bacteria: Staphylococcus saprophyticus, Streptococcus agalactiae, Enterococcus faecalis/faecium
Gram Negative Bacteria: Acinetobacter baumannii, Klebsiella pneumoniae, Morganella morganii, Pseudomonas aeruginosa, Providencia stuartii, Proteus spp., Escherichia coli, Klebsiella oxytoca, Klebsiella aerogenes, Enterobacter cloacae complex, Citrobacter freundii complex
Fungi: Candida albicans
MOLECULAR STI
30
Virus: HSV-1, HSV-2
Gram Positive Bacteria: Gardnerella vaginalis, Neisseria gonorrhoeae, Treponema pallidum, Ureaplasma parvum/urealyticum
Gram Negative Bacteria: Mycoplasma genitalium, Mycoplasma hominis, Chlamydia trachomatis
Parasite: Trichomonas vaginalis Add a description about this item
PLEASE CONTACT FOR FULL MENU