Components of geneOm MTB PCR kit [Cat. No. 1102]:

No. of tests ->	25	50	100
Component	Volume / Number
Lysis buffer-I	7.5ml	15ml	30ml
Lysis buffer-II	15ml	30ml	60ml
Digestion reagent	125µl	250µl	500µl
Precipitation buffer	5ml	10ml	20ml
DNA suspension buffer	750µl	1.5ml	3ml
Ready Reaction tube-I (nos.)	25	50	100
Ready Reaction tube-II (nos.)	25	50	100
PCR-Check sol.	75µl	150µl	300µl
Taq DNA polymerase	12.5µl	25µl	50µl
+ Control tubes I & 2 (nos.)	5	10	20
- Control tube 1 & 2 (nos.)	5	10	20
Control ladder	25µl	50µl	100µl

STORAGE:
Store at below – 200C. Expiration date: 18 months from manufacturing date]

SPECIMEN:
Sputum, blood, tissue, CSF, urine

PROCEDURE:

1. Pretreatment of specimen [Sputum]
   
   1. Suspend the sputum in 1N NaOH and allow it to dissolve.
   2. Transfer 500 µl into a 1.5 ml tube.
   3. Centrifuge at 12000 rpm for 5 minutes & discard the supernatant. Re-suspend pellet in 800 µl of distilled water.
   4. Repeat step iii and discard the supernatant.

2. Pretreatment of specimen [blood]
   
   1. Suspend 300 µl of blood in Lysis buffer-I and centrifuge at 12000 rpm for 88 min. Discard the supernatant.

3. Pretreatment of specimen [Paraffin embedded tissue]
   
   1. Transfer tissue to 1.5 ml tube and suspend in 1X PBS buffer.
   2. Heat at 750C for 5 min.
   3. Centrifuge at 12000 rpm for 3 min and discard supernatant.

4. Pretreatment of specimen [CSF]
   
   1. Pipet 300-500 µl into a 1.5 ml tube.
   2. Centrifuge at 12000 rpm for 2 min, discard supernatant and suspend in 800 µl of distilled water.
   3. Repeat step ii. Centrifuge at 12000 rpm and discard supernatant.

5. Extraction of DNA:
   
   1. Add 600 µl of ice-cold Lysis buffer-II to the pellet & mix thoroughly.
   2. (Optional) Cool & add 6 µl of digestion reagent & incubate at 550C for 3 hrs.
   3. Cool & add 200 µl of Precipitation reagent, mix thoroughly and vortex for 20 sec.
   4. Pellet by centrifugation at max speed for 3 min at 40C.
   5. Transfer supernatant to fresh tube containing 600 µl of Isopropanol. Mix well and precipitate by centrifugation at max speed for 2 min at RT.
   6. Remove supernatant, add 600 µl of 70% Ethanol, centrifuge at max speed for 1 min at RT.
   7. Carefully remove supernatant, dry the pellet at RT for 15 min.
   8. Re-dissolve in 50 µl water.
      (Solubilization of DNA is facilitated by incubation for 30 min at 600C).

6. Setting up the PCR (Reaction-I):
   
   1. Mix 3.0 µl of extracted DNA to the Ready Reaction (RR) tube-I.
   2. Add 0.5 µl of Taq DNA polymerase, mix well and short-spin.
   3. For controls, add 0.5 µl Taq DNA polymerase to + control (+C) and – control (-C) tubes.
   4. Place the tubes on to the PCR block and run.

7. Programming the PCR (Reaction –I):
   

   	Step	°C	Time
   1	Hold [1]	94	5 min
   2	Cycle [20]	94	2 min
   		65	1 min
   		72	30 sec
   3	Hold [1]	72	2 min
   4	Soak	4	Until use

8. Setting up the PCR (Reaction-II):
   
   1. Mix 1.0 µl of completed step-I PCR mix to the Ready Reaction (RR) tube-II.
   2. Mix 3 µl of PCR-check solution.
   3. Add 0.5 µl of Taq DNA polymerase, mix well and short-spin.
   Place the tubes on to the PCR block and run.

9. Programming the PCR (Reaction –II):
   

   	Step	°C	Time
   1	Hold [1]	94	5 min
   2	Cycle [30]	94	2 min
   		65	1 min
   		72	30 sec
   3	Hold [1]	72	2 min
   4	Soak	4	Until use

10. Generating electrophoretogram:
    
    1. Prepare 2.5-3% agarose gel spiked with EtBr (0.5 µg/ml)
    2. Resolve the PCR products (8-12 µl) / ladder (3 µl) at 100-150 volts for 30 min
    3. Detect bands under UV.

11. Analyzing the result:
    
    1. A 244 bp fragment should be present in all samples & is indicative of a successful PCR.
    2. A 73 bp fragment indicate presence of M. tuberculosis genome in the sample.
    3. Both 244 & 73 bp bands (which ever detected in a sample) should align with the ‘control’ ladder bands.
    4. Occasionally, a 123 bp fragment may be detected.

12. geneOm MTB PCR kit [1102] result:
    

    LEGEND:
    

    Lane	Brief description
    +C	+ Control provided
    -C	- Control provided
    Test	A test sample + for TB
    Control ladder	A control ladder with only two reference bands
    MW marker	100 bp molecular wt marker

    WARNING AND CAUTION:
    

    1. UV rays are harmful to eyes and skin.
    2. Use gloves while handling EtBr mixed gel.

    REFERENCE:
    Diagnostic Molecular Microbiology, Principles and applications. American Society for Microbiology (1993) page 191-196.

Contents of this kit or described procedure may change without notice because of the continuous research and development activity at geneOm Biotech.