Polymerase chain reaction (PCR) is a molecular technique used to simultaneously detect a specific DNA sequence in a sample. DNA from samples is extracted then undergoes repeated cycles of heating and cooling to allow for separation of the DNA into single strand, annealing of oligonucleotide primers to target sequences and amplification of the target via DNA polymerase. This results in exponential amplification of the target DNA sequence allowing for detection of small amounts of original sample DNA. Quantitative polymerase chain reaction (qPCR) also called real-time PCR (RT-PCR), is a modified PCR technique and is used to simultaneously detect and determine the actual copy number (amount) of a specific DNA sequence in a sample relative to a standard. Baseline: The background fluorescence signal detected by the instrument

Efficiency of PCR (Slope): Ideally the efficiency (E) of a PCR should be 100%, meaning that for each cycle the amount of product doubles (E=2). This efficiency is calculated from the slope of the standard curve. For an efficiency of 100%, the slope is -3.32. Good reactions should have an efficiency between 90% and 110%, which corresponds to a slope between -3.58 and -3.10. Absolute Quantitation: Cycle Threshold (Ct) is defined as the number of cycles required for the fluorescent signal to cross the threshold. The level is set to be above the baseline but sufficiently low to be within the exponential growth region of an amplification curve to ensure all reagents are still in excess. The Ct value of each qPCR reaction depends on the initial template amount (copy number) of the target sequence, and it is inversely proportional to the log of this copy number. The Ct values of serially diluted standards of known concentrations can be plotted to generate a standard curve, which produces a linear relationship between Ct and initial amounts of total RNA or cDNA, allowing for the determination of the concentration of unknowns based on their Ct values. Syndromic Panel Testing

New advances in molecular techniques have led to the ability to take a syndromic approach to diagnostics in which a single test is capable of detecting all the microorganisms most commonly responsible for an infection. PCR has become a major method for these multiplex tests. These assays are highly sensitive and faster than non-molecular methods. Common panels include: -Respiratory Virus Panel -Meningitis -Gastrointestinal infections (GI) -Sepsis

CT/GC Test A major molecular test preformed in clinical labs which uses PCR to detect the presence of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) DNA in a single test. PCR enables quicker results as compared to non-molecular methods. These tests are qualitative and some new systems are even able to test additionally for Trichomonas vaginalis.

Resistance Gene Markers PCR can also be used to detect the presence of common resistance genes before traditional bacterial culture-based susceptibilities can be determined. Some include: -E. faecalis/E. faecium - van A/ van B genes= vancomycin resistance -S. aureus - mec A gene confers resistance to all penicillinase-stable penicillins and beta-lactams -K. pneumoniae – blaKPC =beta lactam resistance including penicillins, cephalosporins, monobactams and carbapenems. -Enterobacteriaceae - blaCTX-M =beta-lactam resistance including penicillins, cephalosporins, monobactams

Other Common Molecular Techniques: Transcription mediated amplification (TMA): Like PCR, TMA involves amplification of a target sequence. However, TMA uses the enzymes RNA polymerase and reverse transcriptase to amplify either target RNA or DNA and generate an amplified RNA product. TMA is an isothermal, single-tube system that produces 100–1000 copies per cycle and enables quick, sensitive, simultaneous detection of multiple pathogenic organisms in a single tube. Fluorescent in situ hybridization (FISH): Molecular technique used to detect the presence of microorganisms. Fluorescently labeled probe oligonucleotides fare used to detect specific regions of DNA/RNA for desired target microbes. This allows for bacterial identification. Peptide nucleic acid (PNA) FISH: Is a modified FISH technique using probes containing a peptide backbone (as opposed to the sugar backbone in traditional FISH) with nucleic acids that allows for easier penetration into bacteria. Next Generation Sequencing: Refers to new, rapid, lower-cost methods for determining the nucleic acid sequence for all of the genome (DNA) of an organism. The sequence is determined by monitoring of the order of incorporation of fluorescently-labeled nucleotides complementary to single-stranded template DNA. Currently being used as a quick method to determine antimicrobial resistance of clinical Mycobacterium tuberculosis samples.

Molecular In My Pocket…

Infectious Diseases PCR Cycle

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Prepared by the Association for Molecular Pathology Training and Education Committee For More Educational Resources: www.amp.org/AMPEducation

“Molecular in My Pocket” reference cards are educational resources created by the Association of Molecular Pathology (AMP) for laboratory and other health care professionals. The content does not constitute medical or legal advice, and is not intended for use in the diagnosis or treatment of individual conditions. See www.amp.org for the full “Limitations of Liability” statement. Revised 9/18