Topic J10+11: Molecular-biological methods + Clinical virology I (hepatitis A, B & C, HIV) To study: PCR, ELISA, your own notes from serology reactions Task J10/1: DNA isolation of the etiological agent of Lyme borreliosis from clinical material From the submitted clinical material, the DNA of Borrelia burgdorferi s.l. was isolated with using a commercial set. Actual process depends on the type of the used set. Observe the video clip, read the following text and write to each reagent its function. DNA isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic steps in a DNA extraction: Breaking the cells open, commonly referred to as cell disruption, to expose the DNA within, e.g. by grinding or sonicating the sample. Removing membrane lipids by adding a detergent. Precipitating the DNA with an alcohol usually ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate, forming a pellet upon centrifugation. This step also removes alcohol-soluble salt. Refinements of the technique include adding a chelating agent to sequester divalent cations such as Mg 2+ and Ca 2+. This stops DNAse enzymes from degrading the DNA. Cellular and histone proteins bound to the DNA can be removed either by adding a protease or by having precipitated the proteins with sodium or ammonium acetate, or extracted them with a phenol-chloroform mixture prior to the DNA-precipitation. If desired, the DNA can be resolubilized in a slightly alkaline buffer. (http://en.wikipedia.org/wiki/dna_extraction) detergent ethanol or isopropanol chelating agent Task J10/2: Amplification of the specific DNA sections of the etiologicak agent of Lyme borreliosis by PCR method Watch the video clip illustrating the process of polymerase chain reaction (PCR). Read the following text and explain the terms in the table. The polymerase chain reaction (PCR) is a technique widely used in molecular biology. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary to physically separate the strands (at high temperatures) in a DNA double helix (DNA melting) used as the template during DNA synthesis (at lower temperatures) by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions. (http://en.wikipedia.org/wiki/polymerase_chain_reaction) primer Taq polymerase thermal cycling Name General Medicine Date. 4. 2012 Page 1/5
Task J10/3: The detection of PCR products by gel electrophoresis B. burgdorferi s.l. nucleic acid was isolated from clinical material. The specific part of this microbial DNA was many times amplificated with the help of PCR in the next task. We detected the presence of these specific DNA parts by their separation in 1.5% agarose gel containing ethidiumbromid. This dye bounds to DNA and fluoresces in UV-light. For PCR product detection, the gel with wells for the examined sample is prepared in an electrophoretic bath. The gel is flooded with tris-boric acid buffer solution. Ten petted into the 1st well of the gel. 100 pb ladder are pipetted into the next well. The ladder enables sizing of PCR products. Ten mixture of PCR products is put with gel loading buffer into remaining wells. The electrophoretic bath is connected with an electric source. Correct linkage of electrodes should be checked up. Wells with specimens must be on the side of the cathode so that parts of DNA are able to migrate towards the anode. The instrument gets started for one hour (100 mv). After finishing the electrophoresis, the gel is placed on UV-transiluminator. It is covered with a protective board is and the result is surveyed. The board protects the eyes against UVradiation and rubber gloves are also used at work. Draw the result of the reaction and interprete it (in negative reactions, make a minus from a plus ). Drawing Patient No. Proper reaction Internal contol 1 + + 2 + + 3 + + 4 + + Conclusion Task J10/4: The comparison of results obtained by two methods: specific DNA sequence detection and specific antibody detection 4a) PCR of nucleic acid of Lyme borreliosis agent Write down which patient is positive in the PCR. Notice the positive and negative controls. Positive is No. 4b) Detection of antibodies against ethiological agent of Lyme borreliosis This task is not performed in the double practical session. You have already results in 4c). 4c) Compare the results of specific DNA sequence detection (4a) and specific antibodies detection (4b) of etiological agent of Lyme borreliosis. Patient PCR result ELISA IgM ELISA IgG Interpretation 1 2 3 Name General Medicine Date. 4. 2012 Page 2/5
Task J10/5: Detection of Mycobacterium tuberculosis DNA by means of PCR with the detection of the products by ELISA Except gel electrophoresis, PCR products, so-called amplicons, are detected using ELISA. In this case, PCR proceeds with biotinylyzing primers. Amplicons are immobilized on the walls of the wells of a microtitration plate with specific capture probes. Avidin with enzyme binds firmly to biotin at 5' end of amplicon. After adding substrate the reaction is visualized and it can be read on the spectrophotometer. Using the measurement of optical density of each well by the spectrophotometer, evaluate the results of the reaction in the individual patients. Attention, ELISA technique is exploited here, but otherwise it is very different from ELISA reaction for antigen/antibody detection (J09, task 4a of today s practical). Cut off: Patient No. Product of reaction Internal control Conclusion 1 2 3 4 Task J10/6: Real-time PCR Real-time PCR (RT-PCR) is a variant of PCR, where the reaction product is detected by fluorescence sonds. The amount of reaction product is measured continuously, not only at the end of the reaction. Thus, it is possible to observe the development of product in real time (this is where the name comes from). The method is also called quantitative PCR, as it quantifies the tested DNA sequence. Nevertheless, some main principles remain the same as in other PCR types, especially the role and meaning of the internal control. Evaluate positive and negative control and four printscreens of RT-PCR on your working table. The x-axis shows the number of cycles; the y-axis shows the amount of the product. Valid curve should go beyond the limit line (the horizontal line that does not belong to the scale) before the 42 nd cycle. The full line shows the product of the proper reaction, the dot-and-dash line shows the internal control. Cross out the incorrect information: K+ positive? yes no K+ negative? yes no Patient No. 1 positive negative inhibition Patient No. 3 positive negative inhibition Patient No. 2 positive negative inhibition Patient No. 4 positive negative inhibition To all J11 tasks Always read the results of examination, do not forget to read the controls and try to make a conclusion, if possible. In ELISA tests, refer about patients in the form C2, D4, E5 etc. To shorten the time, cut-off, 90 % cut-off and 110 % cut-off values were already counted for you. Task J11/1: Diagnostics of hepatitis A virus (HAV) a) assessment of IgM: Cut off = (0,706 + 0,707)/2 = 0,707 b) assessment of total antibodies: Cut off = (0,425 + 0,422)/2 = 0,424 0,636 0,778 0,382 0,466 Negative(!) patients: Final conclusion (synthesis of both parts): Name General Medicine Date. 4. 2012 Page 3/5
Task J11/2: Diagnostics of hepatitis B virus (HBV) a) assessment of HBsAg: Cut off = (C1 + D1) /2 Cut off = (0,573 + 0,603)/2 = 0,588 b) assessment of HBeAg: Cut off = C1 + D1 /2 Cut off = (0,473 + 0,481)/2 = 0,477 0,529 0,647 0,429 0,525 c) assessment of anti-hbs: Cut off = C1 + D1 /2. Cut off = (0,352 + 0,343)/2 = 0,347 d) assessment of anti-hbe: Cut off = C1 + D1 /2 Cut off = (0,814 + 0,827)/2 = 0,821 0,312 0,382 0,739 0,903 Notes to individual hepatitis B markers: Name General Medicine Date. 4. 2012 Page 4/5
Task J11/3: Diagnostics of hepatitis C virus (HCV) a) Polymerase chain reaction (PCR) in HCV diagnostics Evaluate the picture of a PCR result (gel electrophoresis), draw it and evaluate the results. Picture: 1 = positive control OK (positive) not OK (negative or inhibition) 2 = patient A result: 3 = patient B result: 4 = patient C result: 5 = negative control OK (negative) not OK (positive or inhibition) b) anti-hcv detection using ELISA reaction Cut off = B1 + C1 + D1 + 0.050 / 3 Conclusion: Cut off = (0,307 + 0,348 + 0,370) + 0.050/3 = 0,392 90 % cut off 110 % cut off 0,353 0,431 Task J11/4: Diagnostics of human immunodeficiency virus (HIV) Borderline or positive sera should be confirmed (= sent to the reference laboratory in Prague for verifying the result) Cut off = C1 + D1 /2 Cut off = (0,592 + 0,601)/2 = 0,597 90 % cut off 110 % cut off 0,537 0,657 Patients whose sera need to be confirmed (positive, borderline): In the Czech Republic, we register HIV-positive patients (to the date of.. 20, including foreigners). Name General Medicine Date. 4. 2012 Page 5/5