[Analysis of differentially expressed genes between resuscitating and active Mycobacterium tuberculosis].
OBJECTIVE
To screen key genes of dormant M. tuberculosis for resuscitation.


METHODS
M. tuberculosis H37Rv strain cultured for 20 days in 7H9 liquid medium was used as active bacteria. Dormant bacteria were obtained by cultivating active bacteria hermetically at 37 degrees C using methylene blue as the indicator of oxygen free until the blue medium became colorless. Then resuscitation promoting factors were added to the culture and the bacteria were cultivated for 3 days to be resuscitated. RNA was extracted from active bacteria and resuscitating bacteria, disposed of DNA in RNA with DNase I , and mRNA was purified and then were hybridized using suppression subtractive hybridization (SSH) technique. Differentially expressed genes between resuscitating M. tuberculosis and active M. tuberculosis were identified by PCR, cloning, and sequence alignment. Identification of the differentially expressed genes was performed by real-time quantitative PCR.


RESULTS
High or specifically expressed genes as tester had been obtained by SSH in correctitude reaction (active M. tuberculosis as tester) and reverse reaction (dormant M. tuberculosis as tester). These genes were cloned into plasmid PGEM-T Easy, and 78 positive bacteria in correctitude reaction and 46 positive bacteria in reverse reaction were obtained. The positive bacteria were amplified by PCR with T7 and M13 primer, and 66 positive bacteria ( >350 bp) in correctitude reaction and 39 positive bacteria ( > 350 bp) in reverse reaction were obtained. After sequencing, 30 positive sequences in correctitude reaction and 21 positive sequences in reverse reaction were obtained. Twenty and 7 high or specifically expressed genes were finally identified in active and resuscitating M. tuberculosis respectively by searching in Genbank. These genes were classified into 8 categories. Real-time quantitative PCR demonstrated that the quantity of 7 high or specifically expressed genes in resuscitating bacteria was more than 4 times that in active bacteria.


CONCLUSIONS
Differentially expressed genes between resuscitating and active M. tuberculosis were identified using SSH technique and the results may help exploring key genes and mechanisms of dormant M. tuberculosis for resuscitation.
Liu
Zhong-Quan
ZQ
Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China.
Zhang
Zong-De
ZD
Xing
Ai-Ying
AY
Chen
Xi
X
Li
Zi-Hui
ZH
Gu
Shu-Xiang
SX
Jia
Hong-Yan
HY
Du
Bo-Ping
BP
Ma
Yu
Y
D001483:Base Sequence; D020869:Gene Expression Profiling; D005798:Genes, Bacterial; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000502:physiology*; D009693:Nucleic Acid Hybridization / Q000379:methods