Boiling at 95 degrees will lyse mycobacterial cells and make DNA available for PCR. If my memory is serving me correctly, mycobacterial cells are heat killed by incubating at 80 degrees for 20 min. This heat treatment should be sufficient to coagulate/denature mycobacterial nucleases. Endonucleases and DNA modifying enzymes are routinely heat inactivated by incubating between 65-80 degrees for up to 20 min.

As far as my knowledge is concerned, heating the samples at 95 degrees for 5-10 mins will kill the mtb. This incubation will also inactivate the nuclease (DNase) in your samples. Use the lysozome method for DNA extraction. Please follow the link below for papers, which might help you better. 

SD

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249990/

http://www.ncbi.nlm.nih.gov/pubmed/23825755

Dear Renu Verma, 

Such heat will kill and lyse MTB. After lysis you can centrifuge down cell debris and  use your crude lysate for PCR based reactions. No need to extract or further purify DNA.

as far as nucleases is concern I would strongly recommend you to use TE buffer. TE will prevent your DNA from degradation. although we are usually using PBS and/or DI water without loosing any of our DNA samples.

Yes it kills but that again depends on time of heat killing. I have obtained successful killing by keeping it for 30min at 95 degree celsius. To arrive on tat I have plated the heat killed at different time points on agar growth media. 15 min is sufficient time but still i keep it for 30 min to be extra careful due to the virulent nature of MTB. At that temperature mostly all DNAses get denatured. So it shouldnt be creating a problem. But I suggest u do it immediately to have a better yield and quality of DNA.

For genomic DNA isolation you can firstly store MTB culture pellet (after washing with TE buffer) at -80 for 3 hrs or at -20 for for 4-5 hrs, this results in weakening of the cell wall and will provide more efficient lysis. You will need to perform moderate heat treatment (at 55 degree celsius)  but after adding lysis solution (10% SDS and  lysozyme).  Incubate the samples at 37 degree celsius (12- 16 hrs) after adding lysozyme (followed by SDS and heat treatment)

We have 3 day protocol for the isolation of genomic DNA very efficiently and of good quality. Few months before I isolated gDNA from M. tuberculosis (100 ml culture) that have the purity (A260/A280) of 1.85 and concentration is 1176 nanogram per microliter.

If you need I can forward you the soft copy.

Best,

Hi All,

since you are talking about MTB i want to adress two issues:

- First the DNA extraction, heating at 95C will partially lyse mycobacteria and kill it depending on the incubation time (more than 15 min), using boiling is better than using heat dry block. For better protocol you can use beads, and add lysing solutions . freezing thawing or beatbitter can enhance DNA extraction yield.

- Second Biosafety issue is of great concern, each lab must assess its own protocol and check for MTB inactivation , I have a doubt concerning protocols inactivating MTB at 55C since other protocols based on DNA extration for RFLP have been showm inefficient and MTB is still viable after heating at 80 C + Lysosyme and proteinase K.

So be sure that your protocol is validated for safe DNA extraction.

http://bmcinfectdis.biomedcentral.com/articles/10.1186/1471-2334-5-4

here a study from our lab

http://jcm.asm.org/content/43/6/2996.long

Thank you all. One more clarification, has anyone noticed DNA shearing when the MTB is heat killed at 95 degrees for 30 min and stored at 4 degrees for 4-5 days before DNA isolation is done? I need the DNA for Whole genome sequencing library prep.

Hi Renu,

I second what Hafid has said about biosafety issues. You have to prove that the TB DNA is not viable before you remove it from the CL3 facility, no matter which way you heat-kill. A thorough validation would include a PRE and POST heat-kill set using H37RV and other isolates. The PRE should grow in a MGIT tube after approx 1 week, the POST should be negative in a MGIT tube at 6 weeks. For whole genome sequencing, it is difficult to obtain sufficient DNA in MTBC. You cannot just do a crude extraction, but must also purify your DNA before quantifying and preparing a library. Bear in mind that if the TB culture has originally come from a human specimen, it could contain human and/or nasopharyngeal flora DNA as well as TB DNA. I would recommend reading the paper by Votintseva et al 2015 (mycobacterial DNA extraction for whole-genome sequencing from early positive liquid MGIT cultures).

Emma

http://www.ncbi.nlm.nih.gov/pubmed/25631807

I agree with Hafid and Emma about Biosafety issues. The important thing is that TB is not viable before you remove from CL3 facility. We have validated with TB and found that TB is killed at 90oC for 45 mins. Lower temp we still get growth of TB.We have carried out whole genome sequencing and PCR after using mycobacterial DNA extraction using the Qiagen kit.

Jasvir

Hi Jasvir,

I would really appreciate if you could share the article where you heat killed MTB at 90 c for 45min.

Basically, MTB is so labile for physical methods and hence excessive heat able to destroy the bacilli

As explained above, it may kill the bacilli but the question is if you want to analyse the cell components after lysis?   If yes it is not advisable to use because it will denature the proteins and other biomolecules so you may not be able to analyse it.