The selection and application of ssDNA aptamers against MPT64 protein in Mycobacterium tuberculosis.
BACKGROUND
Tuberculosis (TB) remains a major health problem affecting millions of people worldwide. One-third of the world's population is infected with Mycobacterium tuberculosis, the etiologic agent of TB. A simple and rapid method to diagnose TB is urgently needed to be developed. The procedure of systematic evolution of ligands by exponential enrichment (SELEX) is a method in which single-stranded oligonucleotides (called aptamers) are selected from a wide variety of sequences, based on their interaction with a target molecule. Aptamers have been used in numerous investigations as therapeutic or diagnostic tools.


METHODS
In this study, we apply a SELEX method to develop aptamers against MPT64 protein from M. tuberculosis. On this basis, a sandwich assay scheme with the complex of aptamer-MPT64 was designed and tested the feasibility of detecting M. tuberculosis by detecting MPT64 protein levels in the culture filtrates of 77 samples including M. tuberculosis and other Mycobacterium species.


RESULTS
There was a highly significant difference (p<0.01) between group A (non-TB Mycobacterium, bacille Calmette-Guérin) and group B (M. tuberculosis, M. bovis), when they were diagnosed with the sandwich assay scheme based on aptamer-protein complex to detect MPT64 protein levels in the culture filtrates of samples. When the cut-off point was at the optical density value of 0.58 (95%=0.764-0.946; Z=6.130, p=0.0001), the sandwich assay scheme based on aptamer-protein complex had a high sensitivity (negative ration, 24/27, 86.3%) and specificity (positive ration, 46/52, 88.5%).


CONCLUSIONS
Aptamer of MPT64 as a new detection tool, to a certain extent, is feasible to diagnose Mycobacterium tuberculosis.
Qin
Lianhua
L
Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Affiliated to Tongji University, Shanghai, China.
Zheng
Ruijuan
R
Ma
Zhanzhong
Z
Feng
Yonghong
Y
Liu
Zhonghua
Z
Yang
Hua
H
Wang
Jie
J
Jin
Ruiliang
R
Lu
Junmei
J
Ding
Yuansheng
Y
Hu
Zhongyi
Z
D000942:Antigens, Bacterial / Q000032:analysis*; D052157:Aptamers, Nucleotide / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000302:isolation & purification* / Q000378:metabolism*; D052156:SELEX Aptamer Technique; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology*
D000942:Antigens, Bacterial; D052157:Aptamers, Nucleotide; C510537:MPT64 protein, Mycobacterium tuberculosis