I am assessing the effect of Thermotherapy on Citrus Huanglongbing pathogen in root zone. PCR test result is still positive but I am not sure if there are any approach to identify if the pathogen is live or dead.
Hello,
You can use Propidium monoazide (PMA) to differentiate between live and dead cells in PCR. You need to treat the cells with PMA before the DNA extraction, it binds to free DNA or DNA within cells with a compromised membrane i.e. dead cells.
Here is a paper for further information:
http://journal.frontiersin.org/article/10.3389/fmicb.2016.00912/abstract
Hope this is helpful!
PCR amplifies DNA whether the organism from which it is isolated is dead or alive. It cannot differentiate live from dead.
PCR wont works as stated above. if you have specific pathogen try to transformed with GFP plasmid and check the flurescence. live cell will give the flurescence.
I don't know about plants, but I've seen some work that involved upregulation of genes during apoptosis in rat spinal cord cells. As Panduranga suggested, there is no way to determine cell viability from simple DNA, but perhaps you could access the effect of Thermotherapy by looking at changes in gene expression profiles.
Hello,
You can use Propidium monoazide (PMA) to differentiate between live and dead cells in PCR. You need to treat the cells with PMA before the DNA extraction, it binds to free DNA or DNA within cells with a compromised membrane i.e. dead cells.
Here is a paper for further information:
http://journal.frontiersin.org/article/10.3389/fmicb.2016.00912/abstract
Hope this is helpful!
You can use PMA or EMA prior to DNA extraction; PMA treatment results in more accurate outcomes. However, PMA/EMA treatment can distinguish between membrane-intact and -destructed cells. Using specific bacteriophages would also be helpful.
Shahram Hanifian:
Sir, many thanks for your answer. Please can you help me with detail elaboration and also with reading materials.
Method To Detect Only Live Bacteria during PCR Amplification ▿
Takashi Soejima,1,2,* Ken-ichiro Iida,1 Tian Qin,1 Hiroaki Taniai,1 Masanori Seki,1 and Shin-ichi Yoshida1
ABSTRACT
Ethidium monoazide (EMA) is a DNA cross-linking agent and eukaryotic topoisomerase II poison. We previously reported that the treatment of EMA with visible light irradiation (EMA + Light) directly cleaved chromosomal DNA of Escherichia coli (T. Soejima, K. Iida, T. Qin, H. Taniai, M. Seki, A. Takade, and S. Yoshida, Microbiol. Immunol. 51:763-775, 2007). Herein, we report that EMA + Light randomly cleaved chromosomal DNA of heat-treated, but not live, Listeria monocytogenes cells within 10 min of treatment. When PCR amplified DNA that was 894 bp in size, PCR final products from 108 heat-treated L. monocytogenes were completely suppressed by EMA + Light. When target DNA was short (113 bp), like the hly gene of L. monocytogenes, DNA amplification was not completely suppressed by EMA + Light only. Thus, we used DNA gyrase/topoisomerase IV and mammalian topoisomerase poisons (here abbreviated as T-poisons) together with EMA + Light. T-poisons could penetrate heat-treated, but not live, L. monocytogenes cells within 30 min to cleave chromosomal DNA by poisoning activity. The PCR product of the hly gene from 108 heat-treated L. monocytogenes cells was inhibited by a combination of EMA + Light and T-poisons (EMA + Light + T-poisons), but those from live bacteria were not suppressed. As a model for clinical application to bacteremia, we tried to discriminate live and antibiotic-treated L. monocytogenes cells present in human blood. EMA + Light + T-poisons completely suppressed the PCR product from 103 to 107antibiotic-treated L. monocytogenes cells but could detect 102 live bacteria. Considering the prevention and control of food poisoning, this method was applied to discriminate live and heat-treated L. monocytogenescells spiked into pasteurized milk. EMA + Light + T-poisons inhibited the PCR product from 103 to 107heat-treated cells but could detect 101 live L. monocytogenes cells. Our method is useful in clinical as well as food hygiene tests.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2446937/
Sir Ali A R Aldallal,
Thank you so much for your detail comments and the paper. Looking forward for continued support.
Dear Kinley,
I am afraid that your task (as you stated it) cannot be solved by immediate PCR analysis only. PCR and other tests must be conducted for cured plants during long time period to confirm that they are pathogen-free. I think that DNA of dead pathogen will be destroyed during such assay.
Regards
Alex
- Badges
- Science method