health dna

1.  Presented by -
 Name: Neetu Yadav
 B.Sc Hons Biotechnology
 Roll No.: 194300028
 Supervisor
 Dr. Kundan kumar chaubey
 Assistant Professor- Research
 Department of Biotechnology
 GLA University, Mathura
Prevalence of Mycobacterium avium subspecies
paratuberculosis infection in cows and it’s
morphological and molecular characterization

2. OBJECTIVES
1- PREVALENCE OF MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS
(MAP) IN FECAL SAMPLE OF COWS.
2- MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF MAP USING
MICROSCOPY AND PCR.
 Materials required
 Glasswares
 Plastic waves
 Eqipments
 Oligonucliotides
 Miscellaneous items
 Methods
 ZN Staining
 Microscopy
 DNA Isolation
 IS900 PCR
 Agarose Gel
Electrophoresis
 Indigenous ELISA for
MAP infection.

3. ZIEHL-NEELSEN STAINING
Cleaning of slide
Smear preparation
1% Carbol fushin
25% Sulphuric acid
0.1% Methylene blue
Dry and Observation
ZN STAINING AND MICROSCOPY OF MILK SAMPLE

4. SAMPLE COLLECTION AND PROCESSING
Sample Collection
Gaushala near by GLA University, Mathura- 32 Cow Sample

5. PROCESS :

6. - RESULTS -
- Microscopy (ZN staining) -
Cow (n=32)
Positive Samples - 10 (31.25%)
MAP bacilli seen in fecal
smears under the microscope

7. DNA ISOLATION
500 µl milk sample +100 µl milk lysis buffer
Incubation at room temperature for 15 min.
Add 100 µl 0f 24% SDS,10 min incubation at room temperature
Add 20 µl of proteinase K and incubation at 56°C for 2 hours
Add 100 µl of 5M NaCl and 64 µl CTAB-NaCl; incubate at 65°C for 30 minutes
Add equal volume of phenol : Chloroform : Isoamyl alcohol (25:24:1)

8. CONTINUED………
Centrifuge at 10000 rpm for 15 minutes at 4°C
Transfer the aqueous phase to sterilized eppendorf tube
Precipitate the DNA by adding 0.6 volume of chilled iso-propanol
Keep tube at -20° C for overnight
Pellet the DNA by centrifuging at 10000 rpm for 20 min at 4°C
Discard the supernatant and wash the pellet with 1 ml 70 % ethanol
Resuspended the pellet the 30 µl 1X TE Buffer. Store the DNA at -20°C

9. DNA ISOLATION AND PCR
- IS900 PCR -
413 bp
Lane M: 1.0 kb DNA ladder or Marker (#SM0243,
Fermentas)
Lane 1: IS900 PCR product from DNA of MAP ‘S
5’ Indian Bison type (Positive Control)
Lane 2 and 4: IS900 PCR product from DNA
harvested from cow fecal sample showing positive
results.
Lane 2, 3 and 4: Sample negative in PCR
1 2 3 4
Genomic DNA from cow feces
DNA Bands Visualization
Cow (n=32)
Positive Samples - 2
(6.25%)

10. DISCUSSIONS AND CONCLUSION
 Study reported 31.25% and 6.25% bio-load of MAP infection
using microscopy (ZN staining) and IS900 PCR, respectively.
 This may help to estimate the actual status of the disease in
cow which help in control the disease to spread to other
animals.
 Consumption of milk products and sharing habitat with
infected cows spread the infection to humans.
 Vaccine based control may be effective on spread of the
infection to other domestic animals in addition to humans.

11. Thank You