![REFERENCES
• GIBALDI, M. AND PERRIER, D. (1982). PHARMACOKINETICS. NEW YORK: M.
DEKKER.
• HIV. (2002). JAMA, [ONLINE] 288(2), P.253. AVAILABLE AT:
HTTP://DX.DOI.ORG/10.1001/JAMA.288.2.253-JBK0710-5-1 [ACCESSED 28
OCT. 2016].
• TATRO, D. (2012). DRUG INTERACTION FACTS 2013. ST. LOUIS, MO.: WOLTERS
KLUWER HEALTH/FACTS & COMPARISONS.](https://image.slidesharecdn.com/specialinvestigations-201212080501/85/Special-Investigation-Techniques-20-320.jpg)
Special Investigation Techniques
- 2. CONTENTS • PCR • WESTERN BLOT • ELISA • FINGER PRINTING • HAEMAGGLUTINATION INHIBITION
- 3. PCR – POLYMERASE CHAIN REACTION • Polymerase Chain Reaction is used one of the most common methods to identify the micro-organisms • Specific Primers are used here for the amplication of the DNA/RNA material in the specific pathogens • The procedure involves specific DNA primers binding to the DNA/RNA strands 30-40 times • Initially the denaturation of the DNA/RNA strand happens to break the nucleotides and the bonds between them, 94C for a minute • Second step is where the cooling takes place until 54C for a period of 45 seconds to allow the nucleotides to bind with the complementary strands formed and primers bounded. • Final step where the strands are extended with primers, this happens at 72C for a period of 2 minutes.
- 4. END-POINT PCR REAL TIME PCR • This reaction, the results are obtained on the endpoint of the PCR. • This is time consuming and results are not obtained immediately for these tests. • The end point is not fixed for any sample, it varies from sample to sample • The agarose solution used does not provide a proper resolution inside the samples tested and they vary with the use of the gel. • The Real time PCR uses fluorescent primers compared to normal primers used in end point • The PCR monitors the primer action and plots an amplification plot accordingly • These results can be obtained quickly as well. • Can identify several different types of micro- organisms involved • Uses less amount of DNA compared to end point PCR to identify the micro organism.
- 5. • RESULTS – DIFFERENT BANDS FOUND IN DIFFERENT SOLUTIONS FOR DIFFERENT SOLUTIONS. • DISEASES – MENINGITIS, HEPATITIS, PNEUMONIA • ERRORS – MORE THAN ONE COPY OF THE GENE CAN CAUSE RESULTS TO BE ALTERED. WRONG PRIMER SEQUENCES. FALSE NEGATIVES AND FALSE POSITIVE RESULTS CAN BE FOUND USING THE LIKES OF PCR WHICH MAKES IT LESS RELIABLE. • APPLICATIONS – GENOTYPING, SEQUENCING, MUTATION DETECTION. • CONCLUSION – ISOLATION OF DNA FRAGMENTS CAN BE DONE THROUGH SELECTIVE AMPLIFICATION.
- 6. WESTERN BLOT • A METHOD MAINLY USED TO DETECT SPECIFIC PROTEINS BINDED TO THE DNA MATERIAL IN THE ORGANISM. • THIS METHOD INVOLVES SEPARATION OF THE DIFFERENT PROTEINS ON THE POLYACRYLAMIDE GEL. • THE CHARGED PARTICLES MOVE ACROSS TO THE ELECTRODES, BUFFER SYSTEM WITH DIFFERENT PH VALUES ARE APPLIED ONTO THE GEL ELECTROPHORESIS. • THE PROTEINS ARE THEN TRANSFERRED ONTO A NITROCELLULOSE PAPER, NON- FAT DRY MILK IS USED TO WASH OFF THE UNREACTED SITES. • PRIMARY ANTIBODY INCUBATION IS INTRODUCED, THIS BINDS WITH A SPECIFIC PROTEIN WHICH SHOWS THE PROTEIN. • SECONDARY ANTIBODY INTRODUCED WILL HAVE TO WASH OFF THE UNREACTED PRIMARY ANTIBODIES
- 7. RADIO IMMUNE ASSAY IMMUNOBLOT TEST • This method uses an immune reaction, this is used identify the drugs, enzymes and hormones inside the body. • Initially the first antibody is released, and binds with the radioactive antibody. • Unknown antigens are released the same time, • A second antibody is released which binds with the Fc region of the first antibody • Using a metal bounded with the ion the molecule is • Analyzing of proteins takes place here, proteins separated by SDS/PCR. • Transferring to a synthetic membrane using wet blotting methods. • The electric field generated allows the charged particles towards oppositely charged poles. • As they move out they are captured onto nitrocellulose paper
- 8. • Results – The nitrocellulose paper taken can be checked for the specific proteins according to the molecular weight. • Diseases - STD’s, Lyme disease • Errors – Bad protein samples could result in the Western blot pattern being irregular. Mixing of the wells, bubbles on blotting gel • Applications – Protein purification, protein sequencing. • Conclusion – This method is useful for protein detection and it allows the quantification of the protein expressions.
- 9. ELISA • ENZYME LINKED IMMUNO-ABSORBENT ASSAY ALSO KNOWN AS ELISA IS USED TO LINK UP ENZYMES TO ANTIGENS OR ANTIBODIES. • DIFFERENT ENZYMES ARE INVOLVED ARE IN ELISA, GALACTOSIDASE AND PEROXIDASE ARE EXAMPLES. • THIS METHOD IS USED TO IDENTIFY AND MEASURE THE ANTIBODIES IN THE BLOOD.
- 10. • DIRECT ELISA – INVOLVES THE ATTACHMENT OF ANTIGEN ONTO THE SOLID PHASE FOLLOWED BY THE ENZYME LABELLED ANTIBODY. (PRIMARY ANTIBODY) • INDIRECT ELISA – INVOLVES THE ATTACHMENT OF ANTIGEN ONTO THE SOLID PHASE FOLLOWED BY THE ENZYME LABELLED ANTIBODY WHICH IS THE SECONDARY ANTIBODY. • COMPETITIVE ELISA – INVOLVES MULTIPLE ANTIBODIES OR PROTEINS THAT ARE ADDED SIMULTANEOUSLY. • SANDWICH ELISA – ANTIBODIES ATTACH TO THE SOLID PHASE, SAMPLES CONTAINING KNOWN OR UNKNOWN ARE ADDED TO THE BUFFER, ENZYME LABELLED ANTIBODY INTRODUCED FOR DETECTION.
- 12. • Results – a graph of absorbence against protein concentration is drawn, this way the duplicates obtained are calculated. • Diseases – HIV, Pernicious Anemia, Rocky mounted spotted fever, Syphillis • Errors – The errors arised from ELISA techniques vary significantly, the buffer solution used needs to be the proper standard. Callibration needs to be done before the test carried out. • Applications – Food allergen tests, identification of an antibody/antigen.
- 13. PLASMA FINGERPRINTING • A METHOD USED TO IDENTIFY MICROBIAL SPECIES. • USING STRAINS OF THE SPECIES, THE MOLECULAR WEIGHTS ARE COMPARED. • THE PROCESS INVOLVES THE STRAINS OBTAINED, THE CELLS ARE LYSED INTO THE AGAROSE GEL AND SEPARATED BY ELECTROPHORESIS. • THE PLASMIDS ARE SEPARATED AND USING ETHIDIUM BROMIDE THE PLASMIDS ARE COMPARED, ETHIDIUM BROMIDE PROVIDES THE ILLUMINE FOR THE SAMPLES.
- 14. • RESULTS – THE BANDS IN THE STRAINS ARE ANALYZED AND WITH THE PRESENCE OF ETHIDIUM BROMIDE THE STRANDS ARE VISIBLE. • DISEASES – CORONARY SYNDROME • ERRORS – THE STRAINS OBTAINED NEED TO BE SET WELL INTO THE AGAROSE SOLUTION IF NOT THE RESULTS WILL NOT BE OBTAINED EASILY. • APPLICATION – IDENTIFICATION OF DNA AND ITS TYPE.
- 16. HAEMAGGLUTINATION INHIBITION • HAEMAGGLUTINATION IS THE PROPERTY WHERE THE RBC’S ARE AGGLUTINATED BY THE SURFACE PROTEIN OF VIRUSES. • THE PRINCIPLE OF HAI IS TO USE ANTIBODIES OF THAT PARTICULAR VIRUS TO PREVENT THE ATTACHING OF VIRUS TO THE RBC. • THERE ARE TWO POSSIBILITIES WHICH CAN TAKE PLACE, IF SERUM CONTAINS NO ANTIBODIES THEN HAEMAGGLUTINATION WILL TAKE PLACE. • IF THERE’S AN ANTIBODY, HAEMAGGLUTINATION WILL NOT BE OBSERVED.
- 18. • RESULTS – USING THE AGGLUTINATION OR THE CLUMPING THE RESULTS CAN BE DETERMINED. • DISEASES – VIRAL DISEASES, SUCH AS INFLUENZA • ERRORS – THE ANTIBODIES TAKEN NEEDS TO BE SUITABLE FOR THE RBC’S TESTED, CAN LEAD TO FALSE RESULTS. • APPLICATIONS – IDENTIFICATION OF VIRUSES.
- 19. CONCLUSION • PCR IS USED TO INCREASE DNA FRAGMENT AMOUNTS MAINLY. • WESTERN BLOT IS USED TO IDENTIFY THE DIFFERENT TYPES OF PROTEINS IN THE DNA STRAND • ELISA INVOLES AB-AG REACTIONS MAINLY • FINGERPRINTING IS USED TO RECOGNIZE THE DNA BANDS WITHIN SPECIES FOR IDENTIFICATION OF SAMPLES. • HAEMAGGLUTINATION INHIBITION INVOLVES THE RBC AND VIRAL BINDING.
- 20. REFERENCES • GIBALDI, M. AND PERRIER, D. (1982). PHARMACOKINETICS. NEW YORK: M. DEKKER. • HIV. (2002). JAMA, [ONLINE] 288(2), P.253. AVAILABLE AT: HTTP://DX.DOI.ORG/10.1001/JAMA.288.2.253-JBK0710-5-1 [ACCESSED 28 OCT. 2016]. • TATRO, D. (2012). DRUG INTERACTION FACTS 2013. ST. LOUIS, MO.: WOLTERS KLUWER HEALTH/FACTS & COMPARISONS.