How to use computational tools for diagnostics in broader perspective

1. Bioinformatic approach for DNA signature based diagnostics
Dinesh Kumar
B.Sc. Hons Zoo(BHU), M.Sc. Biotechnology(BHU), Ph.D. Biotechnology (BHU), PDF(USA), PGDIPR, PGDTMA
Senior Scientist(Biotechnology)
Centre for Agricultural Bioinformatics (CABin), IASRI, Pusa, New Delhi-110 012

2. Focus of concept
 Recognise limitations of “parasitological”
diagnosis.
 Recognise new molecular methods
developed.
 Identify 3 main methods – biochemical,
antibody & DNA – based.

3. Why identify parasites?
 Treatment.
 Epidemiology.
 Control measures.
 Fundamental research.

4. Few popular 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)
* ND Tiwari – Biological father hood case
Aug 7, 2012

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. A diagnosis problem?
Traditionally diagnosis infection
based on finding parasite.
Problems:
 Some parasites morphologically
indistinguishable.
 Parasites hidden in host tissue.
 Low sensitivity.

7. Traditional diagnosis of
Malaria

8. The solution?
Current parasitological diagnostic techniques
not satisfactory.
 Need trained staff, equipment, slow
throughput.
But “gold standard”.
 Rapid molecular tests being developed.

9. Three types of “molecular” tests.
1. Biochemical (first generation).
2. Immunological (antibodies).
3. Nucleic acid.

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. Isoenzyme Analysis of
Chagas’ Disease

12. Assessment of enzyme pattern
based diagnosis
Advantages:
 Simple technique.
 Large number of typing enzymes available.
 Many samples typed at same time.
 Power to distinguish morphologically
similar parasites.

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. Iso-enzymes separated by
charge: Isoelectric focusing
equipment.

15. Enzymes separated by size:
SDS-PAGE.

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. 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. Antibody based diagnosis.
Disadvantages:
Cannot distinguish past / present
infections.
Cannot distinguish morphologically
similar parasites.
Expensive to develop – significant
research prior to commercialization.

19. Classical ab test: Enzyme-Linked
Immunosorbant Assay (ELISA) for
diagnosis.
Positive Negative

20. Basic principles of ELISA.
Secondary antibody
with label
Primary antibody
Block
Antigen Ag Ag Ag
unbound
Microtitre plate well sites

21. Example of Antibody based molecular
diagnosis.
African Sleeping Sickness
Anti-trypanosomal IgM detected by simple / rapid
CATT (Card Agglutination Test for Trypanosomiasis)
Drop of blood
↓
Mixed with fixed parasites on plastic card
↓
Blue granular deposits = infection

22. CATT Test for African sleeping
sickness.

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. 3. DNA based molecular diagnosis.
 DNA probes.
 Polymerase Chain Reaction (PCR).

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. 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. 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. 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. DNA barcoding of animal species
diagnostics
 cytochrome c oxidase subunit I gene (COI)
potential 'barcode'.
 Mitochondrial DNA has signature
Aug 7, 2012

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. 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. 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. Plant pathogen signature
•Naked Eye: Colony - shape, colour.
•Microscopy: Cell - shape, colour, size
•Biochemistry:
•Molecular Level: Role of bioinformatics
Aug 7, 2012

34. Microsatellite DNA marker in Fungal diagnostics
Aug 7, 2012

35. How fungal microsatellite data gives signature
Aug 7, 2012

36. Why we need molecular & bioinformatics tool?
 Case study of Ug99-signature search
Stem rust never sleeps- Norman E. Borlaug , 26th April, 2008, New York Times
Aug 7, 2012

37. Can we have DNA based signatures of Ug99?
 National Debate!
 Global meet at Delhi, Oct, 2008
 Action plan?
Aug 7, 2012

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. What are the available DNA
markers to identify Ug99
SSR
AFLP
Mol Plant
Path
Latest Nov
2008 issue
Aug 7, 2012

40. SSR data- no DNA signature
Mol Plant Path Nov 2008 issue
Aug 7, 2012

41. AFLP adapters used to generate signature data
Mol Plant Path
Nov 2008 issue
Aug 7, 2012

42. AFLP signatures
Mol Plant Path Nov 2008 issue
Aug 7, 2012

43. No clear signature of Ug55 & 99 by SSR
Mol Plant Path Nov 2008 issue
Aug 7, 2012

44. AFLP again poor signature b/w Ug99/55
Mol Plant Path Nov 2008 issue
Aug 7, 2012

45. DNA signature of Ug99 by combining data
of SSR+AFLP
Mol Plant Path Nov 2008 issue
Aug 7, 2012

46. Clear DNA signature by
Minimum-spanning network analysis
Mol Plant Path Nov 2008 issue
Aug 7, 2012

47. What bioinformatics can do more in
Ug99 identification?
 Allele mining of Puccinia graminis tritici Ug99
– USDA is targeting 400 SNP
 STR mining from Puccinia graminis genome data
base
 STR based signature search
Aug 7, 2012

48. Our experiences-DNA based gender
diagnostics using bioinformatics tools
Aug 7, 2012

49. Our experiences-DNA based microbial species
signature using bioinformatics
House 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. 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. 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. Few examples: Signature applications
1. Application of polymerase chain reaction to detect adulteration of
sheep's 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 cow's 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. 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,
 World's First Single-tube, Multiplex(co-amplify
eight microsatellite loci) Microsatellite Assay-based
Kit for Basmati Authentication.
Aug 7, 2012

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. Acknowledgements
• Our team : Prem, Vijay, Nishant, Dhiraj, Vishal, Bhola, Vipin, Udai,
Pooja
Thank you dineshkumarbhu@gmail.com,