Session:

Tuberculosis & Other Mycobacterial Infections

Abstract No.:

47.040

Title:

A novel 146 bp region of Ag 85B mRNA as target in Taqman Real-time PCR for efficient detection of active genital tuberculosis

Author(s):

M. Singhal¹, S. Prasad², S. Kumar³, S. S. Negi⁴, P. Gupta⁵, S. Gupta⁴, D. S. Rawat⁵, L. S. Chauhan⁶, A. Rai⁴; ¹National Centre foe Disease Control, Maulana Azad Medical College, Biochemistry and Biotechnology, Delhi, DELHI/IN, ²Maulana Azad Medical Colllege, Obstetrics and Gynaecology, New Delhi/IN, ³National Centre for Disease Control, Biochemistry & Biotechnology; Microbiology, Delhi, DELHI/IN, ⁴National Centre for Disease Control, Biochemistry and Biotechnology, Delhi/IN, ⁵National Centre foe Disease Control, Biochemistry and Biotechnology, Delhi, DELHI/IN, ⁶National Centre for Disease Control, Biochemistry and Biotechnology & Centre for AIDS and Related Diseases, Delhi, NEW DELHI/IN

Abstract:

Background: Advent of nucleic acid-based methods for genomic detection of M. tuberculosis in clinical specimens is emerging as valuable adjuncts for diagnosis of tuberculosis of deeper anatomical sites. DNA-based methods have proved promising in recent years but are unable to differentiate between viable and non viable bacilli. mRNA-based conventional RT-PCR for detecting 216 bp region of Ag 85B has been used to identify active tuberculosis. The present study was intended to identify smaller but novel target within the Ag 85B mRNA to develop Taqman Real-time PCR for more efficient detection of cases of active genital tuberculosis.
Methods: Four distinct groups of specimens (32 laparoscopy+DNA PCR-positive cases of genital tuberculosis; 32 DNA PCR positive casesof other extrapulmonary tuberculosis;  32 culture-confirmed+ DNA PCR positive cases of pulmonary tuberculosis; and  32 DNA PCR-negative endometrial biopsies from patients with normal laparoscopic findings) were included in the study. Extracted RNA were simultaneously subjected to conventional RT-PCR and nucleotide sequencing to confirm the presence of 216 bp region of Ag85B gene of M. tuberculosis.  mRNA-positive samples were then tested for presence of a novel 146 bp region of Ag 85B mRNA in TaqMan Real-time PCR.
Results: The newly developed 146bp Ag85B mRNA-based TaqMan real-time PCR was able to detect positivity in ~9% more cases compared to conventional RT-PCR positive cases of active genital tuberculosis compared to conventional mRNA-based RT-PCR.
Conclusion: Diagnosis of active cases of extrapulmonary tuberculosis is of paramount importance for efficient management of the disease. The novel 146bp TaqMan Real-time PCR for detection of Ag 85B mRNA seems to be ideal for differentiating between viable and non viable bacilli. This assay may also be very useful in monitoring response during the course of anti-tubercular therapy.