Development and Validation of In-house PCR (hPCR) for the Detection of Mycobacterium tuberculosis

· Biomolecular, Lung Infection

Radita Ning Anggraeny¹, Maelita Ramdani Moeis¹, Lidya Chaidir²
¹Bioteknologi, Sekolah Ilmu dan Teknologi Hayati, ITB
²Unit Penelitian Kesehatan, Universitas Padjajaran


Backgrounds: Detection of tuberculosis (TB) using Acid Fast Bacteria (AFB) method lacks sensitivity, while culture is time consuming. The purpose of this study was to develope and validate hPCR assay as the recommended method for Tb detection in the laboratory that has better sensitivity-specificity compared to AFB method.
Methods: 150 sputum samples were collected in TB section of the Health Laboratory Development Unit (BPLK), West Java. Decontaminated sputum was examined by AFB and culture methods, and these specimens were stored at -20˚C for hPCR. Annealing temperature, MgCl₂, Taq Polymerase, primers and number of cycles were optimized for hPCR of hsp65 gene, then applied to decontaminated sputum samples with an added uracyl-N-glycosilase reaction before PCR to remove carry-over contamination.
Results: The optimized condition for hPCR was annealing temperature 64˚C, 3 mM MgCl₂, 1,5 U Taq Polymerase, 0,25 μM primers and 45 cycles with a limit of detection of 94 pg DNA. Only 138 samples with a positive positive-control were used for validation. Compared with culture, the sensitivity and specificity were 56.25% and 98.89% for hPCR, 79.17% and 92.22% for AFB method.
Conclusions: The developed hPCR was not sensitive enough, therefore it could not be used as refference for routine test in TB suspect patients. (J Respir Indo. 2014; 34: 204-10)
Keywords: Acid Fast Bacteria (AFB), culture, in-house PCR, Mycobacterium tuberculosis, sensitivity-specificity.

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