Clinical Applications of Real-time PCR PDF

C l i n i c a l A p p l i c a t i o n s o f Real-time PCR A s s t. P r o f. D r. S o m p h o t S a o i n School of Allied Health Sciences Universit y of Phayao 12 November 2024 Learning Outcome: อธิบายหลักการของ...

C l i n i c a l A p p l i c a t i o n s o f Real-time PCR A s s t. P r o f. D r. S o m p h o t S a o i n School of Allied Health Sciences Universit y of Phayao 12 November 2024 Learning Outcome: อธิบายหลักการของ Real-time PCR ได ้ เข ้าใจการอ่านผลและแนวทางการแปลผลได ้ เข ้าใจแนวทางการนาประยุกต ์ใช ้ในทางคลินิกได ้ 2  What is real time PCR? ✓ Real-Time PCR a specialized technique that Principle allows a PCR reaction to be visualized “in real time” as the reaction progresses. lens ✓ Links an optical system to a thermal cycler cap ✓ Signals captured at every reaction step Reaction vessel ✓ “quantitative Polymerase Chain Reaction: qPCR” thermal block sample How is fluorescence signal detected? 1. dsDNA Intercalator Dye Based Ethidium bromide (1st Generation) SYBR Green I (2nd Generation) LC Green Plus (3rd Generation) Hybridization Molecular beacons FRET Scorpions 2. Probe Based Hydrolysis TaqMan (5' nuclease assay) TaqMan MGB What is difference between these methods? 5 Real-time PCR result Real-time PCR result 7  Types of Real-Time Quantification 1. Absolute Quantitation: standard curve - Serially diluted standards of known concentrations to generate a standard curve 2. Relative Quantitation - Measured based on either an external standard or a reference sample, also known as a calibrator - Target/reference ratio - Comparative Ct (2 -ΔΔCt) method -Pfaffl model -Q-Gene 8  Clinical applications of Real-Time PCR: Viral infection- Viral load - Real‐time PCR has had the strongest impact on virology - A number of factors have contributed to this impact: (i) in terms of genetic complexity, viruses are the simplest pathogens (ii) isolation of nucleic acids from viruses is not complicated by the need to break up rigid membranes of the agents (iii) the effort of standard microbiological diagnosis by culture is greater than for many other pathogens - Helps to identify co‐infections 9  Clinical applications of Real-Time PCR: Bacteria infection -PCR has long been the preferred method for diagnosis of bacteria that are difficult to cultivate, and many real‐time PCR assays ▪ Listeria monocytogenes ▪ Legionella pneumophila ▪ Mycobacterium spp. ▪ Propionibacterium spp. ▪ Borrelia burgdorferi ▪ Leptospira genospecies - Conventional microbiological diagnosis by culture is too slow to provide relevant information for physician and veterinarian for the initial use of antibiotics in infectious disease. - Real‐time PCR can detect and monitor the antibiotic resistance of clinical isolates of bacteria such as S. aureus, S. epidermitis, Helicobacter pylori, Enterococcus faecalis, 10 and Enterococcus faecium  Clinical applications of Real-Time PCR: Cancer diagnosis - Expression analysis of genes as well as differentiation of gene polymorphisms - Gene duplications, deletions, fusions, and overexpression 11 Clinical applications of Real-Time PCR: Gene expression 12  RT-PCR  Real-Time PCR RT-PCR = Reverse Transcriptase PCR qReal time PCR = qPCR= quantitative Real-Time PCR  Reverse transcription PCR (RT-PCR) ❖ Amplification of interested mRNA. ❖ 2 step are required 1.cDNA generation : mRNA as template for a universal primer (Oligo dT) by the action of Reverse transcriptase. 2.cDNA Amplification : gene specific primer were used in second step using cDNA as template. 14 Reverse transcription PCR (RT-PCR) Oligo dT primer 15  What is RT-qPCR? - Quantitative reverse transcription polymerase chain reaction, also called RT-qPCR - Detect and quantify total RNA or mRNA - Firstly, transcribe into complementary DNA (cDNA) - Secondly, the cDNA is then used as the template for the quantitative PCR or real-time PCR reaction (qPCR) 16 cDNA synthesis Real-time PCR reaction 17 RT-PCR vs qPCR vs RT-qPCR 18  Normalization - Normalization of gene expression data is used to correct sample-to-sample variation - Housekeeping genes (mRNA) GAPDH and -actin 18S Ribosomal RNA (rRNA) 19  Normalization - Normalization of gene expression data is used to correct sample-to-sample variation - Housekeeping genes (mRNA) GAPDH and -actin 18S Ribosomal RNA (rRNA) 20  ΔΔCt method experiment control -[(Cttg-Ctcg)-(Cttg-Ctcg)] 2 Ex!  Ct = target gene– ref gene  Ct = 9.70  Ct = target gene– ref gene  Ct = -1.70 Difference = Ct-Ct = Ct = 9.70-(-1.7) = 11.40 Fold change = 211.40 = 2702 Always in duplicate or triplicate! Thank you For your attention