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  1. 1. Bioinformatic approach for DNA signature based diagnosticsDinesh KumarB.Sc. Hons Zoo(BHU), M.Sc. Biotechnology(BHU), Ph.D. Biotechnology (BHU), PDF(USA), PGDIPR, PGDTMASenior Scientist(Biotechnology)Centre for Agricultural Bioinformatics (CABin), IASRI, Pusa, New Delhi-110 012
  2. 2. Focus of concept Recognise limitations of “parasitological” diagnosis. Recognise new molecular methods developed. Identify 3 main methods – biochemical, antibody & DNA – based.
  3. 3. Why identify parasites? Treatment. Epidemiology. Control measures. Fundamental research.
  4. 4. Few popular examples of individual DNAdiagnostics * Rajiv Gandhi Assassination Case (Chennai, Tamil Nadu), * Naina Sahni or the Tandoor case (New Delhi * Priyadarshini Mattoo (New Delhi), * Sishu Vihar Child adoption case (Hyderabad, Andhra Pradesh), * Black Buck killing case (Jodhpur, Rajasthan) * Beanth Singh Assassination Case (Punjab) * ND Tiwari – Biological father hood case Aug 7, 2012
  5. 5. Some definitions Diagnostic test: sensitive & specific. – Mathematically defined. Sensitivity: capacity of test to make correct diagnosis in known cases. Specificity: capacity to correctly diagnose uninfected individuals.
  6. 6. A diagnosis problem?Traditionally diagnosis infectionbased on finding parasite.Problems: Some parasites morphologically indistinguishable. Parasites hidden in host tissue. Low sensitivity.
  7. 7. Traditional diagnosis ofMalaria
  8. 8. The solution?Current parasitological diagnostic techniques not satisfactory. Need trained staff, equipment, slow throughput. But “gold standard”. Rapid molecular tests being developed.
  9. 9. Three types of “molecular” tests.1. Biochemical (first generation).2. Immunological (antibodies).3. Nucleic acid.
  10. 10. 1. Biochemical molecular tests: Enzyme patterns.Isoenzymes. Perform same functions BUT different movement on gels. Genetically controlled∴ parasites with different gel patterns genetically distinct. Isoenzymes) are enzymes that differ in amino acid sequence but catalyze the same chemical reaction
  11. 11. Isoenzyme Analysis ofChagas’ Disease
  12. 12. Assessment of enzyme pattern based diagnosisAdvantages: Simple technique. Large number of typing enzymes available. Many samples typed at same time. Power to distinguish morphologically similar parasites.
  13. 13. Enzyme pattern based diagnosis.Disadvantages:Significant tissue needed for analysis → visceral leishmaniasis requires spleen, liver.Technique not rapid → can take days.Sometimes incorrect diagnosis → enzyme labile.Technique simple but equipment expensive.
  14. 14. Iso-enzymes separated bycharge: Isoelectric focusingequipment.
  15. 15. Enzymes separated by size:SDS-PAGE.
  16. 16.  SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique used in biochemistry, genetics and molecular biology to separate proteins according to their electrophoretic mobility
  17. 17. 2. Antibody based diagnosis.Rely on identification of specific antibodies.Advantages: Rapid easy field-based tests. Both individual & mass population screening. Ig subclasses – to improve specificity / sensitivity.
  18. 18. Antibody based diagnosis.Disadvantages:Cannot distinguish past / present infections.Cannot distinguish morphologically similar parasites.Expensive to develop – significant research prior to commercialization.
  19. 19. Classical ab test: Enzyme-LinkedImmunosorbant Assay (ELISA) fordiagnosis. Positive Negative
  20. 20. Basic principles of ELISA. Secondary antibody with label Primary antibody BlockAntigen Ag Ag Ag unbound Microtitre plate well sites
  21. 21. Example of Antibody based moleculardiagnosis. African Sleeping SicknessAnti-trypanosomal IgM detected by simple / rapidCATT (Card Agglutination Test for Trypanosomiasis) Drop of blood ↓ Mixed with fixed parasites on plastic card ↓ Blue granular deposits = infection
  22. 22. CATT Test for African sleepingsickness.
  23. 23. Future for Ab-based diagnosis? Non-invasive sampling? – saliva, excreta? Good results for saliva abs for African trypanosomiasis. But will need new tests. Saliva abs not detected in CATT or Latex.
  24. 24. 3. DNA based molecular diagnosis. DNA probes. Polymerase Chain Reaction (PCR).
  25. 25. PCR in parasite diagnosis.Amplifies target sequences & increases sensitivity.1. Ribosomal DNA/RNA.  Highly sensitive.  No good for closely related species.2. Specific sequences of genomic DNA.  Highly specific for single species - not sensitive.3. Random primer amplification (RADP) PCR.  Very highly sensitive - not specific.
  26. 26. Nucleic acid based molecular diagnosis.Advantages: Genomic DNA constant -parasite & hosts unique DNA sequences . Very sensitive - small biopsy. Probes can be designed with flexibility: → Specific - detect single parasite species. → Less specific - detect group of parasites.
  27. 27. Nucleic acid based diagnosis.Disadvantages: Expensive - especially PCR . Radioactivity needed: newer non-radioactive probes. PCR can fail: - Contamination & false positives. DNA probes do not distinguish between dead & living parasites
  28. 28. Examples of veterinary parasite nucleic acid diagnosis Cryptosporidium parvum protozoa.1. Faecal microscope analysis: limit 50,000 oocysts per g faeces.2. PCR: amplifies ~400 base pair sequence from faeces - sensitivity to 6 oocysts per g faeces.
  29. 29. DNA barcoding of animal species diagnostics cytochrome c oxidase subunit I gene (COI) potential barcode. Mitochondrial DNA has signatureAug 7, 2012
  30. 30. Identifying flowering plants by species DNA bar code  Kress et al. (2005) -COI sequence- not appropriate for most species of plants slower rate of cytochrome c oxidase I gene evolution in higher plants than in animals”.  Chloroplast has DNA signature of plant species .  nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer as a potential DNA barcode for flowering plants. Some reports supports MatK  DNA barcoding, no master key may be a master keyring, with different kingdoms of life requiring different keys. Aug 7, 2012
  31. 31. Microbial species signature Ribotyping -rRNA database-most used eg 16srDNA Designing of primer for gene sequencing. Eg HKG,Topoiso II, Designing of probes for identifications-Eg.real time, microarray In silico development of RFLP test for close species differentiation(our experiences) Aug 7, 2012
  32. 32. rRNA genes - the ideal markers for microbial identification  Small subunit - highest order differences (domains)  Large subunit - medium order differences  ITS - low order differences (species/strains?) Small Sub-Unit rRNA (16S)  ubiquitous  1.6 - 2.0kb  good molecular chronometer.  some areas conserved (for priming/alignment)  some areas variable (for resolving differences) Aug 7, 2012
  33. 33. Plant pathogen signature•Naked Eye: Colony - shape, colour.•Microscopy: Cell - shape, colour, size•Biochemistry:•Molecular Level: Role of bioinformatics Aug 7, 2012
  34. 34. Microsatellite DNA marker in Fungal diagnostics Aug 7, 2012
  35. 35. How fungal microsatellite data gives signature Aug 7, 2012
  36. 36. Why we need molecular & bioinformatics tool?  Case study of Ug99-signature searchStem rust never sleeps- Norman E. Borlaug , 26th April, 2008, New York Times Aug 7, 2012
  37. 37. Can we have DNA based signatures of Ug99?  National Debate!  Global meet at Delhi, Oct, 2008  Action plan? Aug 7, 2012
  38. 38. A case study updates-How to identify Ug99?  Puccinia graminis tritici Ug99  DNA signature is the only answer !  DNA signature of Fungi – Private alleles of STR(rare) – STR allele frequency signatures – SNP based signatures(???)  Where is the signature of Ug99 ?? Aug 7, 2012
  39. 39. What are the available DNAmarkers to identify Ug99 SSR AFLP Mol Plant Path Latest Nov 2008 issue Aug 7, 2012
  40. 40. SSR data- no DNA signature Mol Plant Path Nov 2008 issue Aug 7, 2012
  41. 41. AFLP adapters used to generate signature data Mol Plant Path Nov 2008 issue Aug 7, 2012
  42. 42. AFLP signatures Mol Plant Path Nov 2008 issueAug 7, 2012
  43. 43. No clear signature of Ug55 & 99 by SSR Mol Plant Path Nov 2008 issue Aug 7, 2012
  44. 44. AFLP again poor signature b/w Ug99/55 Mol Plant Path Nov 2008 issue Aug 7, 2012
  45. 45. DNA signature of Ug99 by combining dataof SSR+AFLP Mol Plant Path Nov 2008 issue Aug 7, 2012
  46. 46. Clear DNA signature by Minimum-spanning network analysis Mol Plant Path Nov 2008 issueAug 7, 2012
  47. 47. What bioinformatics can do more inUg99 identification? Allele mining of Puccinia graminis tritici Ug99 – USDA is targeting 400 SNP STR mining from Puccinia graminis genome data base STR based signature searchAug 7, 2012
  48. 48. Our experiences-DNA based genderdiagnostics using bioinformatics tools Aug 7, 2012
  49. 49. Our experiences-DNA based microbial species signature using bioinformaticsHouse Keeping Genes -CLUSTAL-W-signature with spelling mistake- CLEAVER KspAI Bsh1236I Mun I L P P R RR P P R RR P P R RR KEU1 5-AAY ATG ATI ACI GGI GCI GCI CAR ATG GA-3 KEU 2 5-AYR TTI TCI CCI GGC ATI ACC AT-3. KspAI L.paracasei 542,158bp; L.rhamnosa 701bp Bsh1236I L. paracasei 547,153bp; L. rhamnosa 701bp Mun I L. paracasei 594,106bp; L. rhamnosa 701bp New PCR-RFLP test developed for Lactobacillus species differentiation Aug 7, 2012
  50. 50. DNA based signature of domestic species Mitochindrial DNA markers used especially for meat identification, poaching of wild animals, adulteration of dairy milk, dairy products(like cheese) of various domestic animal species.Aug 7, 2012
  51. 51. STR & breed-specific signatures/profiles Pig-In UK, Signer et al. (2000) - minisatellite probe pCMS12 -three breeds of pig viz Chinese, Meishan, Large White and other European breeds. The linear discrimination analysis revealed that the DNA profiles were breed specific. Fish-In Finland, Primmer et al. (2000) - disputed fish to a specific population out of 4 suspected fish populations using 7 microsatellite loci by Bayesian method with confidence limit of 99%. Sheep-In Spain, Arranz et al. (2001) - Bayesian method with 99.63% accuracy among five Spanish sheep breed viz. Churra, Latxa, Castellana, Rasa-Aragonesa and Merino using 18 microsatellite markers. Horse-In Norway, Bjornstad et al. (2001) -26 microsatellite loci in six breeds of horses, Fjord, Nordland/ Lyngen, Dole, Trotter, Icelandic horse and Shetland pony with more than 95% confidence limit. Cattle-In European countries, Canon et al. (2001) -confidence limit of 99% for 18 local breeds of cattle of different countries; Alistana, Astruriana, Asturiana Valles, Sayaguesa, Tudanca, Avilena Negra-Iberica, Bruna del Pirineus, Morucha, Pirenaica, Retinta of Spain; Alentejana, Barrosa, Maronesa, Mertolenga, Mirandesa of Portugal and Aubrac, Gasconne, Salers of France Camel- In Kenya, Mburu et al. (2003) - 4 breeds using 14 microsatellite loci of camel viz. Somali, Turkana, Rendille, Gabbra) using maximum likelihood method up to 48 % confidence limit.-weak genetic differentiation and gene flow between populations. Dog-In2012 Aug 7, Finland, Koskinen (2003) has assigned breeds of domestic dog using
  52. 52. Few examples: Signature applications 1. Application of polymerase chain reaction to detect adulteration of sheeps milk with goats: J Dairy Sci (2005) 88: 3115-20. 2. A novel approach to the quantification of bovine milk in ovine cheeses using a duplex PCR: J Agric Food Chem (2004) 52: 4943-7. 3. Rapid detection of cows milk in sheeps and goats milk by a species- specific polymerase: J Dairy Sci (2004) 87: 2839-45. 4. Identification of cows milk in "buffalo" cheese by duplex polymerase chain reaction. J Food Prot (2002) 65: 362-6. 5. Forensic identification of ungulate species using restriction digests of PCR-amplified mit J Forensic Sci (1995) 40: 943-51. (15 species) 6. Detection of cows milk in goats cheeses inferred from mitochondrial DNA polymorphism Journal of Dairy Research (2001), 68:229-235 7.Application of polymerase chain reaction for detection of goats milk adulteration by milk of cow. Journal of Dairy Research (2001), 68:333-336 Aug 7, 2012
  53. 53. DNA based signature of plant variety, example- Basmati rice Basmati rice -aroma compound -2-acetyl-1- pyrroline. Fraudulent traders to adulterate traditional basmati. PCR-based assay similar to DNA fingerprinting in humans allows for the detection of adulterated and non-basmati strains. Its detection limit for adulteration is from 1% upwards with an error rate of ±1.5%. Exporters of basmati rice use purity certificates based on DNA tests for their basmati rice consignments. It was developed by CDFD, Labindia, Worlds First Single-tube, Multiplex(co-amplify eight microsatellite loci) Microsatellite Assay-based Kit for Basmati Authentication. Aug 7, 2012
  54. 54. Few examples of individual DNA diagnostics * Rajiv Gandhi Assassination Case (Chennai, Tamil Nadu), * Naina Sahni or the Tandoor case (New Delhi * Priyadarshini Mattoo (New Delhi), * Sishu Vihar Child adoption case (Hyderabad, Andhra Pradesh), * Black Buck killing case (Jodhpur, Rajasthan) * Beanth Singh Assassination Case (Punjab) Aug 7, 2012
  55. 55. Acknowledgements• Our team : Prem, Vijay, Nishant, Dhiraj, Vishal, Bhola, Vipin, Udai, Pooja Thank you dineshkumarbhu@gmail.com,