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PCR

PCR. Why we use molecular diagnosis for parasites. 1. Offer greater sensitivity and specificity over the existing diagnostic tests. 2. Differentiation between similar morphological types.

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PCR

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  1. PCR

  2. Why we use molecular diagnosis for parasites 1. Offer greater sensitivity and specificity over the existing diagnostic tests. 2. Differentiation between similar morphological types. 3. They permit the detection of infections from very low parasitized samples including those from asymptomatic patient’s samples. 4. multiplexed PCR allows for the detection of multiple sequences in the same reaction tube proving useful in the diagnosis of several parasitic infections simultaneously. • (Multiplex polymerase chain reaction (Multiplex PCR) refers to the use of PCR to amplify several different DNA targets (genes) simultaneously (as if performing many separate PCR reactions all together in one reaction). • Detects Salmonella sp, Campylobacter sp, Shigella sp, Yersinia enterocolitica, Aeromonas sp, Giardia sp, Entamoeba histolytica, Dientamoebasp, Blastocystissp and Cryptosporidium sp

  3. Sample collection • Microscopic examination is still considered the "gold standard" for the diagnosis of parasitic diseases. • If an unequivocal identification of the parasite can not be made, the stool specimen can be analyzed using molecular techniques such as polymerase chain reaction (PCR).

  4. QIAmp kit • Sample size QIAamp kit: • For use with 180–220 mg fresh or frozen stool, but can also be used with larger amounts of stool. • Starting with larger amounts of stool is recommended when the target DNA is not distributed homogeneously throughout the stool and/or is at a low concentration; • A larger amount of starting material will increase the likelihood of purifying DNA from low-titer sources in stool samples.

  5. QIAmp kit • QIAmp kit can also be used for samples of less than 180 mg (e.g., forensic samples). • In such cases, follow one of the standard protocols, without reducing the amounts of buffers or InhibitEX matrix used

  6. EXTRACTION EXTRACTION DNA from stool • It is necessary to extract DNA from the stool specimens for PCR detection. • The fast and easy procedure comprises the following 3 steps:

  7. Step 1 • Lyse Break down cells to access DNA in the nucleus Lysis buffer, Enzyme, Heat Lysis of stool samples. • Cells in Buffer ASL Digestion protein under 70c for denaturation and presence of proteinase K enzyme (Proteinase K, produced by the fungus Tritirachium album Limber, is a serine protease that exhibits a very broad cleavage specificity. • It cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids and is useful for general digestion of protein in biological samples.)

  8. STEP 2 Adsorption of impurities to Inhibit EX ( a unique adsorption resin) matrix • STEP3Load sample to column

  9. Principle • Spin column-based nucleic acid purification is a solid phase extraction method to quickly purify nucleic acids. • Nucleic acid solid phase extraction: this method relies on the fact that nucleic acid will bind to the solid phase of silica under certain conditions.

  10. Binding • Under buffering conditions to allow optimal binding of DNA to membrane. • High concentration of salt make selectively binding for DNA rather than other. • This help to remove lysate. • DNA binds to the membrane and remaining lysate discarded. • The centrifuge forces the binding solution through a silica gel membrane that is inside the spin column.

  11. PRICIPLE cont… • The principle of silica matrices purification is based on the high affinity of the negatively charged DNA backbone towards the positively charged silica particles (Esser et al., 2006). • DNA binds to silica through hydrogen- binding interaction with an underivatised hydrophilic matrix provided by silica, under concentrated chaotrophicsalt conditions (usually sodium iodide, sodium perchlorate, guanidinium thiocyanate) (Berensmeier, 2006).

  12. Purification – Bind

  13. DNA is adsorbed onto the QIAamp silica membrane during a brief centrifugation step. Optimized salt concentrations and pH conditions in the lysate ensure that remains of digested proteins and other impurities, which can inhibit PCR and other downstream enzymatic reactions, are not retained on the QIAamp membrane.

  14. Purification • Wash: DNA bound to the QIAamp membrane is washed in two centrifugation steps. • Optimized wash conditions using two wash buffers ensure complete removal of any residual impurities without affecting DNA binding. • Impurities and proteins should now have passed through the column and been discarded. • The membrane, however, is still dirty with residual proteins and salt, if using blood it may be tinted yellow or brown , The column is washed with buffers to remove any residual impurities • There are typically 2 washes with a centrifuge step after each. • Wash 1 will contain a low amount of chaotropic salt to remove any remaining proteins and coloured contaminants • Wash 2 contains a high concentration of ethanol to remove the remaining salts. • Salts MUST be removed for good DNA yields and purity, Wash 2 can be repeated to ensure this.

  15. Elute( elution of pure DNA from the spin column ) • When elution buffer is added the nucleic acids become hydrated and will release from the membrane. • If the column still has ethanol on it, the nucleic acids will not fully rehydrate • Buffer AE in the QIAamp mini kit. • DNA more stable at a slightly basic pH and will dissolve more easily in a basic buffer. • Elution of DNA can be maximized by allowing the buffer to sit in the membrane for a few minutes before centrifugation Can be repeated

  16. Results DNA yield is typically 15–60 μg, but, depending on the individual stool sample and the way it was stored, may range from 5–100 μg. • DNA concentration is typically 75–300 ng/μl. The eluted DNA is up to 20 kb long and is suitable for direct use in PCR and other enzymatic reactions. • If the purified DNA is to be stored for longer periods, storage at –20°C is recommended QIAamp spin columns fit into most standard microcentrifuge tubes. Eluted DNA is collected in standard 1.5 ml microcentrifuge tubes (not provided).

  17. To avoid cross-contamination • Change pipet tips between all liquid transfers. The use of aerosol-barrier pipet tips is recommended. • Avoid touching the membrane with the pipet tip. • After all vortexing steps, to avoid cross- contamination, we recommend briefly centrifuging the microcentrifuge tubes to remove drops from the inside of the lid. • Wear gloves throughout the entire procedure. In case of contact between gloves and sample, change gloves immediately. • Close the spin column before placing it in the microcentrifuge.

  18. Precautions • Remove the spin column and collection tube from the microcentrifuge. Place the spin column in a new collection tube. • Discard the filtrate and the collection tube. Please note that the filtrate may contain hazardous substances and should be disposed of appropriately. • Open only one spin column at a time, and take care to avoid generating aerosols. • Discard used collection tubes containing the filtrate and place the new collection tubes containing the spin columns directly in the microcentrifuge

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