Ebook Quantitative real-time PCR - Methods and protocols: Part 2

Part 2 book "Quantitative real-time PCR - Methods and protocols" includes content: Real-Time PCR detection of mycoplasma pneumoniae in the diagnosis of community acquired pneumonia, a sensible technique to detect mollicutes impurities in human cells cultured in GMP condition, real time quantification assay to monitor BCR ABl1 transcripts in chronic myeloid leukemia, a reliable assay for rapidly defining transplacental metastasis using quantitative PCR,... and other contents.

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Ebook Quantitative real-time PCR - Methods and protocols: Part 2 Chapter 9 Real-Time PCR Detection of Mycoplasma pneumoniae in the Diagnosis of Community-Acquired Pneumonia Eddi Di Marco Abstract Polymerase chain reaction is a useful technique in microbial diagnostics to detect and quantify DNA or RNA of low abundance. Bacterial and viral nucleic acid can be amplified by PCR upon clinical sample extraction using specific primers for classical qualitative PCR and primers and probes for real-time PCR. Here we describe the Scorpion-probe real-time PCR-based assay that offers thermodynamic advan- tages due to its kinetic reaction and provides faster performances compared to a classical double-labeled probe-based assays. Key words Mycoplasma pneumoniae, Community-acquired pneumonia, Quantitative PCR, Scorpion probe 1 Introduction Mycoplasma pneumoniae is a common cause of upper respiratory tract infection and is one of the etiological agents of community- acquired pneumoniae (CAP) [1]. The direct determination of Mycoplasma pneumoniae DNA using real-time PCR in clinical specimens allows an efficient detec- tion of this etiological agent in all the phases of the infection, avoiding the false-negative serological assay responses during the first 1–2 weeks after the primary infection [2]. In addition the qPCR assay on clinical samples performed better than conventional qualitative PCR in terms of sensitivity and specificity, since it allowed the detection of the specific pathogen in some specimens where the classic qualitative PCR failed. The use of real-time PCR in microbial molecular diagnostics can be clinically relevant also for the short-time results compared to traditional assays [3–5]. Different chemistries are employed to monitor the fluorescence emitted during the reaction as a function of amplicon production Roberto Biassoni and Alessandro Raso (eds.), Quantitative Real-Time PCR: Methods and Protocols, Methods in Molecular Biology, vol. 1160, DOI 10.1007/978-1-4939-0733-5_9, © Springer Science+Business Media New York 2014 99 100 Eddi Di Marco at each PCR cycle [6]. Real-time PCR using Scorpion unimolecu- lar probe gives several important advantages, chief of which is faster reaction kinetics due to its intramolecular probing mechanism that ensures a near proximity between probe and target DNA [7]. In addition Scorpion probes do not need any fluorochrome enzy- matic cleavage that occurred during the double-labeled probe- based assays. This allows a rapid-cycling PCR that reduces the overall procedure time by more than 30 %, ideal for its use in hos- pital settings. 2 Materials 2.1 Specimen 1. Nasopharyngeal aspirates, nasopharyngeal swabs, and bron- Collection, Storage, choalveolar lavage specimens are routinely examined for Transport Mycoplasma pneumoniae infection (see Note 1). All samples collected have to be treated as potentially infectious material. 2. The swabs are stored in 1 ml of sterile saline solution or in UTM Kit universal transport medium (Copan); nasopharyn- geal aspirates and bronchoalveolar lavage are collected in ster- ile tubes for up to 72 h before processing (see Note 2). 3. The clinical samples should be transported at R.T. as fast as possible or refrigerated (2–8 °C) if longer time is required. 4. Pretreatment with Sputasol (Oxoid) is recommended in case of viscose samples (see Note 3). 2.2 DNA Extraction 1. Microcentrifuge. 2. Disposable plastic tubes (0.2 and 1.5 ml). 3. Disposable plastic filter tips (1–30 μl; 1–200 μl; 1–1.000 μl Eppendorf tips). 4. P20, P200, and P1000 micropipettes (see Note 4). 5. Sterile saline solution. 6. DNA extraction reagents (see Note 5). 7. Nucleic acid extraction robot (see Note 6). 8. Gloves (see Note 7). 9. Heating block. 2.3 qPCR Analysis 1. Scorpion probe-modified forward and classical reverse primers (Table 1) (see Notes 8 and 9). 2. Ready to use Master Mix (Invitrogen). 3. Tips and micropipettes for PCR (see Note 10). 4. Plastic tubes for reagent storage and master mix preparation (1.5 ml Eppendorf tubes). Mycoplasma pneumoniae qPCR Assay in Clinical Specimens 101 Table 1 Real-time PCR primers and molecular Scorpion probes for the detection of Mycoplasma ...