Abstract:
Tuberculosis (TB) remains a global health challenge, necessitating the development of
more sensitive and non-invasive diagnostic tools. This preliminary cross-sectional study
explores the potential of detecting the IS6110 insertion sequence, a unique genomic
marker of Mycobacterium tuberculosis, in serum as a novel diagnostic approach. The
study protocol was approved by the Ethics Review Committee, General Hospital,
Kandy. Blood samples were collected from Active TB (n = 20), Latent TB (n = 22), and
contact tracing (n = 28) patients attending to the Kandy chest clinic. RNA was extracted
from the serum samples using the guanidium thiocyanate-phenol-chloroform extraction
method. Then extracted RNA was quantified and subjected to complementary DNA
(cDNA) synthesis using GoScriptTM Reverse Transcription System according to the
manufacturer’s guidelines. A conventional Polymerase Chain Reaction (PCR) was
carried out using 6 ng of template cDNA with IS6110 insertion sequence-specific
primer pairs Pt8: 5’-GTGCGGATGGTCGCAGAGAT-3’, Pt9: 5’-
CTCGATGCCCTCACGGTTCA-3’. The H37Rv bacterial DNA was used as a positive
control for this study. The amplified PCR products were run on 2% agarose gel along
with 100 bp ladder and the negative control. The positive results obtained from the
conventional PCR amplifications were confirmed with real-time PCR by using the
primer pairs specific for IS6110 insertion gene, INS1, F: 5’-
CGTGAGGGCATCGAGGTGGC-3’ and INS2, R: 5’-GCGTAGGCGTCGGTGA
CAAA-3’. The results revealed that the IS6110 gene was detected in 45% of serum
samples (9 out of 20) collected from active TB patients. Latent TB and contract tracing
patients did not possess the IS6110 gene in their serum samples. In conclusion, the
serum samples in this study did not demonstrate the potential to detect the MTBspecific
mRNA transcript in individuals with latent TB (LTB) and contact tracing (CT).
However, it may suggest detecting the bacterial transcript in active TB patients.
Nevertheless, further improvements are necessary for it to be considered a reliable
biomarker for active TB.
