Culture-Free ultrasensitive mycobacterium tuberculosis detection ELISA

Culture-Free ultrasensitive mycobacterium tuberculosis detection ELISA

       Tuberculosis (TB) is a disease caused by bacteria that are spread through the air from person to person. According to the reports from Taiwan Centers for Disease Control, there were 11,000 cases TB infection each year and 600 of those cases result in deaths in Taiwan. Clinically, the bacteriology examination is the major testing for tuberculosis diagnosis including acid-fast staining microscopy examination, MGIT culture testing, strip speciation and polymerase chain reaction for genome of mycobacterium. Because the slow growth rate of Mycobacteria tuberculosis, the bacteria culture for TB diagnosis usually take 4-8 weeks incubation period. A quick, safe and correct testing for tuberculosis will help patients receive appropriate treatment and prevention the spread of M. tuberculosis. In this research, Professor Po-Liang Lu in KMU, Professor ITO Etsuro in Waseda University and Japan TAUNS Laboratories Inc. cooperate to develop the culture-free TB detection ELISA method. The enzyme cycling reaction is used to detect MBP64 protein which is secreted by live Mycobacteria tuberculosis. The results showed that the detection limitation of culture-free ultrasensitive TB detection ELISA is 100 CFU/ml.

        Characteristics of ultrasensitive ELISA combined with the enzyme cycling reaction. After proceed the conventional ELISA, the ALP-labeled enzyme will retain in the 96-well plate. And then, the coloring reagent was added into the wells. The 17-OCH₃5b-A3P in the solution will be dephosphorylated by ALP into 17b-Methoxy-5b-androstan-3a-ol. In thr presence of Thio-NAD, 17b-Methoxy-5b-androstan-3a-ol will be converted into 17b-Methoxy-5b-androstan-3a-one by 3a-HSD. Thio-NADH is formed at the same time, which is then measured at 405/660 nm.

Application and Highlights:

1. A novel, rapid (same day) diagnostic method for detecting M. tuberculosis with high sensitivity.

2. The detection limitation of this method is about 100 CFU/ml.

3. A quick, safe and correct testing for tuberculosis will help patients receive appropriate treatment and prevention the spread of M. tuberculosis.

Research Team Members: Po-Liang Lu, Wen-Hung Wang

Representative Department: Division of Infectious Disease, Internal Medicine, Kaohsiung Medicine University Hospital.

Introduction of Research Team:

The research of division of Infectious Diseases Lab involve in development of new diagnosis methods for infectious disease, including tuberculosis, dengue virus and respiratory syndrome coronavirus 2. We also focus on antimicrobial drug resistance analysis, and virulence gene expression and cell viability studies, too.

Contact Email: d830166@gmail.com ; bole0918@gmail.com  

Publication: A novel, rapid (within hours) culture-free diagnostic method for detecting live Mycobacterium tuberculosis with high sensitivity., EBioMedicine, 60, 103007, October 01, 2020

Full-Text Article: https://www.thelancet.com/journals/ebiom/article/PIIS2352-3964(20)30383-2/fulltext